JOB Vol.1 - N.2 - 2010 by Paolo Trisi

ISSN: 2036-6795

Journal Journal of of Osteology Osteology and Biomaterials Biomaterials and The official Journal of Biomaterial Clinical and histological Research Association

Volume 1 Number 2 2 0 1 0

The

Laser Microfused Titanium Surface by LEADER

Tixos is a porous surface characterized by

IMPLANTS

Perfect adherence between

interconnected cavities,

the

with predetermined geometry, that enhance fast bone formation*.

MUCOSAL

implant neck and soft tissues

C O N N E C T I V E F I B E R S A D H E R E A N D P E N E T R AT E D E E P I N S I D E T H E S U R FA C E O F

T I XO S

IMPLANT

( B LU E )

* References available upon request.

L E AD E R I

LEADER ITALIA s.r.l. Via Aquileja, 49 - 20092 Cinisello Balsamo (MI) ITALY ph. +39 02 618651 - fax +39 02 61290676 - www.leaderitalia.it - export@leaderitalia.it

The

Laser Microfused Titanium Surface by LEADER

Tixos is a porous surface characterized by interconnected cavities, with predetermined geometry, that enhance

IMPLANTS

Faster Bone Growth inside the cavities of the microfused titanium surface

fast bone formation*.

N E W B O N E F O R M AT I O N

INSIDE THE

IN HUMAN AFTER

I M P L A N T C AV I T I E S 8 WEEKS.

AND PORES

* References available upon request.

L E AD E R I

LEADER ITALIA s.r.l. Via Aquileja, 49 - 20092 Cinisello Balsamo (MI) ITALY ph. +39 02 618651 - fax +39 02 61290676 - www.leaderitalia.it - export@leaderitalia.it

Journal of Osteology and Biomaterials The official journal of BioCRA - Biomaterial Clinical and histological Research Association President Giampiero Massei Deputy-president Alberto Rebaudi Editor in-chief Paolo Trisi, DDS PhD Scientfic director BioCRA, Pescara, Italy Associate Editor Gilberto Sammartino, MD DDS University of Naples Federico II, Naples, Italy Assistant Editor Teocrito Carlesi, DDS Secretary BioCRA, Chieti, Italy Managing Editor Renato C. Barbacane, MD University G. d’Annunzio, Chieti, Italy

www.osteobiom.com

Scientific Director Paolo Trisi Secretary Teocrito Carlesi

Editorial Board Roberto Abundo, Turin, Italy Mario Aimetti, Turin, Italy Moshe Ayalon, Hadera, Israel Luigi Ambrosio, Naples, Italy Massimo Balsamo, Thiene, Italy Francesco Benazzo, Pavia, Italy Ermanno Bonucci, Roma, Italy Mauro Bovi, Rome, Italy Maria Luisa Brandi, Firenze, Italy Paul W. Brown, Pennsylvania, USA Ranieri Cancedda, Genova, Italy Saverio Capodiferro, Bari, Italy Sergio Caputi, Chieti, Italy Francesco Carinci, Ferrara, Italy Chih-Hwa Chen, Keelung, Taiwan Joseph Choukroun, Nice, France Gabriela Ciapetti, Bologna, Italy Giuseppe Corrente, Turin, Italy Massimo Del Fabbro, Milan, Italy Marco Esposito, Manchester, UK Antonello Falco, Pescara, Italy Gianfranco Favia, Bari, Italy Paolo Filipponi, Umbertide, Italy Bruno Frediani, Siena, Italy Sergio Gandolfo, Turin, Italy Zhimon Jacobson, Boston, USA

Richard J. Lazzara, West Palm Beach, USA Lorenzo Lo Muzio, Foggia, Italy Gastone Marotti, Modena, Italy Christian T. Makary, Beirut, Lebanon Gideon Mann, Jerusalem, Israel Ivan Martin, Basel, Switzerland Milena Mastrogiacomo, Genoa, Italy Anthony McGrath, Santmore, UK Carla Palumbo, Modena, Italy Sandro Palla, Zurich, Switzerland Michele Paolantonio, Chieti, Italy Giorgio Perfetti, Chieti, Italy Adriano Piattelli, Chieti, Italy Domenique P. Pioletti, Lausanne, Switzerland Sergio Rosini, Pisa, Italy Ugo Ripamonti, Johannesburg, South Africa, Lucia Savarino, Bologna, Italy Arnaud Scherberich, Basel, Switzerland Nicola Marco Sforza, Bologna, Italy Hiroshi Takayanagi, Tokyo, Japan Tiziano Testori, Milan, Italy Anna Teti, L’Aquila, Italy Oriana Trubiani, Chieti, Italy Alexander Veis, Thessaloniki, Greece Raffaele Volpi, Rome, Italy Giovanni Vozzi, Pisa, Italy Hom-Lay Wang, Michigan, USA Xuejun Wen, South Carolina, USA

Journal of Osteology and Biomaterials (ISSN: 2036-6795; On-line version ISSN 2036-6809) is the official journal of the Biomaterial Clinical and histological Research Association (BioCRA). The Journal is published quaterly, one volume per year, by TRIDENT APS, Via Silvio Pellico 68, 65123 Pescara, Italy. Copyright ©2010 by TRIDENT APS. All rights reserved. No part of this journal may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information and retrieval system, without permission in writing from the publisher. The views expressed herein are those of the publisher or the Biomaterial Clinical and histological Research Association (BioCRA). Information included herein is not professional advice and is not intended to replace the judgment of a practitioner with respect to particular patients, procedures, or practices. To the extent permissible under applicable laws, the publisher and BioCRA disclaim responsibility for any injury and/ or damage to person or property as result of any actual or alleged libellous statements, infringement of intellectual property or other proprietary or privacy rights, or from the use or operation of any ideas, instructions, procedure, products, or methods contained in the material therein. The publisher assumes no responsibility for unsolicited manuscript.

Volume 1 - Number 2 - 2010

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Journal of Osteology and Biomaterials

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slides (35-mm transparencies) must be submitted, plus two sets of prints made from them. When a series of clinical images is submitted, tonal values must be uniform. When instruments and appliances are photographed, a neutral background is best. Electronic Files–must contain all parts of the manuscript including figures and tables Resolution must be at least 600 dpi; files saved in jpeg format are preferred. Legends–Figure legends should be grouped on a separate sheet and typed double-spaced. UNITS OF MEASUREMENT Measurements of length, height, weight, and volume should be reported in metric units or their decimal multiples. Temperatures should be given in degrees Celsius and blood pressure in millimeters of mercury. All hematologic and clinical chemistry measurements should be reported in the metric system in terms of the International System of Units (SI). Description of teeth should use the American Dental Association (i.e., Universal/National) numbering system. COPYRIGHT All manuscripts accepted for publication become the property of TRIDENT APS. A copyright form must be signed by the authors, and returned to the Managing Editor. A file containing this form always accompanies the acceptance e-mail. REPRINTS Corresponding authors may purchase reprints at the time pages are received for proofreading. Reprints can be purchased in 4-color or black and white. Submit manuscripts via JOB’s online submission service: www.osteobiom.com Manuscripts should be written in PC Word (doc) file format with tables preferably embedded at the end of the document. Original figures in pdf, tif or jpg. No paper version is required. Info: info@osteobiom.com

Book reference style: Skalak R. Aspects of biomechanical considerations. In: Brånemark P-l, Zarb GA, Albrektsson T (eds). Tissue-Integrated Prostheses: Osseointegration in Clinical Dentistry. Chicago. Quintessence 1985:117-128. ILLUSTRATIONS AND TABLES - All illustrations must be numbered and cited in the text in order of appearance. - Illustrations and tables should be embedded in a PC Word document. JPEG files are highly recommended. For graphs and charts, do not use patterned fills. Solid tones or colors are recommended instead. - All illustrations and tables should be grouped at the end of the text. Radiographs–Submit the original radiograph as well as two sets of prints. Color–Color is used at the discretion of the publisher. No charge is made for such illustrations. Original

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Journal of Osteology and Biomaterials The official journal of BioCRA - Biomaterial Clinical and histological Research Association

contents

69 81

Review articles

Implant survival in maxillary sinus augmentation. An updated systematic review Massimo Del Fabbro, Monica Bortolin, Silvio Taschieri, Gabriele Rosano, Tiziano Testori

Original articles

ISQ (RFA) vs BIC and torque: a histomorphometric and biomechanical analysis in humans Paolo Trisi, Teocrito Carlesi, Marta Rocci, Antonio Rocci

93 Ultra-short porous implants in the posterior maxilla: 103 a 4-year report from a prospective study

Hydraulic sinus lift: a new method proposal

Mirko Andreasi Bassi, Michele Antonio Lopez

Michele Perelli, Giuseppe Corrente, Roberto Abundo, Luca Savio, Alessandro Bermond des Ambrois

109

Regeneration of soft tissue with the ciprofloxacin incorporated collagen scaffold delivery vehicle

Shanmugam Kirubanandan, Praveen Kumar Sehgal

119

Genetic effects of pulsed electromagnetic fields (PEMF) on human osteoblast-like cells (TE85) in vitro

Vincenzo Sollazzo, Ilaria Zollino, Ambra Girardi, Francesca Farinella, Francesco Carinci

Volume 1 - Number 2 - 2010

BioCRA

Review

Implant survival in maxillary sinus augmentation. An updated systematic review Massimo Del Fabbro,BSc,PhD1*, Monica Bortolin,BMT1, Silvio Taschieri,MD,DDS,1 Gabriele Rosano,DDS,1 Tiziano Testori,MD,DDS1

Maxillary sinus augmentation is one of the most common surgical procedures for increasing bone volume prior to endosseous implant placement in the atrophic posterior maxilla. The aim of the present review was to assess the survival of implants placed in the grafted sinus, taking into consideration the influence of implant surface, graft material, implant placement timing, residual ridge height and use of a covering membrane. A search on the main electronic databases and a hand search on the major international journals were performed. Retrieved articles were screened according to specific inclusion criteria. Fifty-four studies were included. A total of 11746 implants were placed in 3638 patients, with an overall survival of 95.2%. Survival using 100% autogenous bone was lower than that obtained using combined grafts or 100% bone substitutes. Particulation of autogenous bone improved the outcome. Implants with rough surface displayed a higher survival than implants with machined surface, independently of the graft type. Simultaneous implant placement showed better outcomes than delayed placement. Using a membrane to cover the graft resulted in an increased survival of implants. Implant survival was higher when residual ridge height at the time of surgery was greater than 5 mm. Sinus floor elevation is a highly predictable procedure. The choice of the appropriate graft material, implant surface and timing of implant placement may affect the clinical outcome. (J Osteol Biomat 2010; 1:69-79)

Keywords: bone substitutes, dental implants, maxillary sinus, sinus augmentation, systematic review.

IRCCS Galeazzi Orthopaedic Institute, Dental Clinic, CRSO (Centro di Ricerca per la Salute Orale), Department of Health Technologies, University of Milan, Milan, Italy.

Corresponding author:

* Massimo Del Fabbro, University of Milano Istituto Ortopedico Galeazzi Via R. Galeazzi 4- 20161 â&#x20AC;&#x201C; Milano, Italy Phone: +39 02 50319950, Fax: +39 02 50319960 Email: massimo.delfabbro@unimi.it

Introduction Rehabilitation of edentulous patients with oral implants has become a routine treatment modality in the last decades, with reliable long-term results. However, implant placement may become a challenging procedure in the presence of unfavorable local condition of the alveolar ridge. This problem is especially magnified in the posterior maxilla, where progressive ridge resorption and sinus pneumatization, together with a poor bone quality, are often encountered1. One of the preferred and better documented technique for the management of the atrophic posterior maxilla is maxillary sinus floor elevation. The sinus augmentation procedure was first presented by Tatum in 19772. Boyne and James published the first report in 19803. The early technique consisted in a lateral approach to the sinus, in which an access window was created, the Schneiderian membrane elevated, and the newly formed space filled with autogenous bone graft. Since its introduction into clinical practice, the sinus augmentation technique has evolved over time. New graft materials, implant surface configurations and surgical techniques have been introduced in order to simplify the treatment, reduce the morbidity of the pro-

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Del Fabbro M. et al.

cedure and improve the predictability of the outcomes. Various materials have been used for grafting the sinus floor: autogenous bone, allografts, xenografts, alloplastic materials or mixtures of different materials. Autogenous bone has long been considered the gold standard grafting material due to its osteoconductive, osteoinductive and osteogenic properties. Bone substitutes have, in general, no osteoinductive potential, but they provide an optimal scaffold for bone growth and reduce donor site morbidity since they avoid bone harvesting procedure. Published data concerning the use of bone substitutes as grafting materials have rapidly increased in the last years. Thus, one of the aims of the present study was to determine the survival of implants in relation to the different graft materials used. A variable that may affect implant survival is the implant surface configuration. Histologic and clinical evidence have suggested that rough-surfaced implants provide a more favorable outcome than machined-surfaced implants 4-9. Implants can be placed either simultaneously with the graft or after a delayed period to allow for graft maturation. The choice of placing implants simultaneously to the grafting procedure or at a later stage is generally influenced by the amount of residual crestal bone height, which must be sufficient to provide implants with adequate primary stability. In a recent review of the literature 10, Rios et al. aimed to find a correlation between the amount of remaining crestal alveolar bone before sinus augmentation and implant survival in grafted areas. The data retrieved

Journal of Osteology and Biomaterials

suggested a higher implant success predictability as the available bone height increased. Another parameter which can be taken into account is the effect of barrier membrane placement. The rationale of covering the graft with a barrier membrane is to better contain the material, preventing its migration or dispersion into the soft tissues and limiting soft tissue invasion into the site, which would render the latter unsuitable for implant placement 11. The aim of the present review was to assess the survival of implants placed in grafted maxillary sinus, and to determine if it is affected by factors such as implant surface, graft material, implant placement timing, residual ridge height in the posterior maxilla and use of a covering membrane. MATERIALS AND METHODS A search was conducted for articles published up to December 2009 using the following electronic databases: MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials. Search terms such as “maxillary sinus lift”, “sinus augmentation”, “sinus floor elevation”, “sinus graft”, ”bone graft”, “endosseous implants”, “oral implants”, and “dental implants” were used, alone and in combination by means of Boolean operators. The search was limited to studies involving human subjects. No restrictions regarding language and study design were applied. A further hand search was performed on the main international journals in the field of implant dentistry and of oral and maxillofacial surgery (Clinical Oral Implants Research, Implant Dentistry, International Journal of Oral &

Maxillofacial Implants, International Journal of Oral & Maxillofacial Surgery, International Journal of Periodontics & Restorative Dentistry, Journal of Clinical Periodontology, Journal of Oral & Maxillofacial Surgery and Journal of Periodontology) from 1986 to December 2009 issue. Finally, reference lists of the most relevant papers and reviews were checked for possible additional studies. Data extraction The titles and abstracts of articles identified from the electronic searches were screened using the following inclusion criteria: (i) a lateral window approach to the maxillary sinus was used; (ii) a minimum of 20 sinus floor elevations were performed; (iii) “root-form” implants were used; (iv) no multiple interventions (e.g. simultaneous ridge augmentation) were carried out (v) minimum follow-up was no less than 12 months of implant loading, or the follow-up range exceeded 36 months; (vi) the implant survival was clearly specified or calculable from data reported in the paper and (vii) the study was prospective. Publications that did not meet the above inclusion criteria and those that were not dealing with clinical cases (e.g. reviews, technical reports) were excluded. Multiple publications, when recognized, were excluded from the database. When articles from the same group of authors, with very similar databases of patients, materials, methods and outcomes were identified, the authors were contacted for clarifying if the pool of patients was indeed the same. In case of multiple publications relative to consecutive phases of the

Del Fabbro M. et al.

Table 1. Features of included studies Article

Year

Lozada et al.12 Keller et al.13 Zinner & Small14 Blomqvist et al.15 Froum et al.16 Peleg et al.17 Van den Bergh et al.18 Buchmann et al.19 De Leonardis & Pecora20 Olson et al.21 Tarnow et al.22 Van den Bergh et al.23 Wannfors et al.65 Bahat & Fontanessi24 Kahnberg et al.25 Tawil & Mawla26 Hallman et al.27 Engelke et al.28 McCarthy et al.29 Philippart et al.30 Rodriguez et al.31 Stricker et al.32 Hallman et al.33 Hatano et al.34 Iturriaga et al.36 John & Wenz35 Schwartz-Arad et al.37 Shlomi et al.38 Testori et al.39 Boyne et al.40 Simunek et al.41 Ewers42 Butz & Huys43 Wallace at al.44 Scarano et al.45 Peleg et al.46 Karabuda et al.47 Mardinger et al.48 Mangano et al.49 Marchetti et al.50 Galindo-Moreno et al.51 Aguirre-Zorzano et al.52 Minichetti et al.53 Lee et al.55 Kahnberg & Vannas-Lofqvist56 Lee et al.54 Yamamichi et al.57 Meyer et al.58 Ferreira et al.59 Torres et al.60 Triplett et al.61 Chaushu et al.62 Bettega et al.63 Cannizzaro et al.64

1993 1994 1996 1998 1998 1998 1998 1999 1999 2000 2000 2000 2000 2001 2001 2001 2002 2003 2003 2003 2003 2003 2004 2004 2004 2004 2004 2004 2004 2005 2005 2005 2005 2005 2006 2006 2006 2007 2007 2007 2007 2007 2008 2008 2008 2008 2008 2009 2009 2009 2009 2009 2009 2009

Study type CT CS CS CT CT CS CS CT CT RCT RCT CS RCT CS CS CT CS CS CS CS CS CS CS CS CS CT CS CT CT RCT CS CS CS CS CS CT CT CS RCT CS CS CS CS CT CS RCT CS CS CS RCT RCT CS RCT RCT

Graft material ABG/BS ABG ABG+BS ABG ABG+BS/BS ABG+BS ABG ABG BS ABG/ABG+BS/BS ABG/ABG+BS/BS BS ABG ABG+BS/BS ABG BS ABG/ABG+BS/BS ABG+BS ABG ABG BS ABG ABG+BS ABG+BS ABG ABG/ABG+BS/BS ABG/ABG+BS ABG/ABG+BS ABG/ABG+BS ABG/BS BS BS ABG+BS ABG+BS/BS ABG/ABG+BS/BS ABG/ABG+BS/BS BS BS BS ABG+BS ABG+BS ABG+BS BS ABG+BS ABG+BS ABG+BS BS BS BS BS ABG/ABG+BS/BS BS ABG ABG+BS

N° patients 60 20 50 50 78 20 42 50 57 29 12 24 40 62 26 29 21 83 18 18 15 41 20 191 58 38 70 63 22 44 24 118 20 51 94 731 91 109 40 30 70 22 42 52 36 41 69 20 314 87 160 28 18 20

N° N° IS (%) sinuses implants 69 158 91.77 23 66 92.42 66 215 98.60 75 202 84.16 113 215 98.14 20 55 100.00 62 161 100.00 75 167 100.00 65 130 98.46 45 120 97.50 24 55 96.36 30 69 100.00 80 150 84.00 83 313 92.65 39 91 61.54 30 61 85.25 36 111 90.99 118 211 94.79 27 79 79.75 25 58 91.38 24 70 92.86 66 183 99.45 30 108 86.11 191 361 94.18 79 223 100.00 38 103 96.12 81 212 95.75 73 253 90.91 26 63 96.83 88 219 83.11 24 45 86.11 209 614 94.18 22 56 100.00 64 135 96.12 144 362 95.75 731 2132 90.91 >91 259 96.83 129 294 97.78 >40 100 95.60 48 140 100.00 98 263 97.78 22 36 98.34 56 136 97.94 58 130 95.75 47 153 100.00 52 97 100.00 69 159 92.45 33 123 97.56 406 1025 98.15 144 286 97.55 240 492 82.32 29 72 94.44 36 111 100.00 20 44 88.64

Imm / del

RRH (mm)

imm/del imm imm del imm/del imm del imm imm/del imm/del del del imm/del imm/del imm imm/del del imm/del imm/del del imm imm/del del imm del imm/del imm/del imm/del imm/del del imm/del del imm/del del imm imm imm/del imm/del imm imm/del imm/del imm del imm/del del del imm/del del imm/del imm/del del imm del imm

<3 / ≥3 NR NR NR NR 1-2 <4 - 8 <5 1-7 NR NR 4-8 2-7 NR 1 - 5.5 (2.5) 4-8 <5 0.5 - 7 / 2 - 9 <4 / >4 1-3 <5 <5 / >5 <5 4-6 <5 <4 / ≥4 NR 2 - 8.3 <5 / >5 <6 <3 / >3 1 - 5 (3.6) <4 / >4 NR 3-5 1-7 <5 / >5 1-8 3.5 - 5 <4.5 / ≥4.5 <5 ≥5 NR <6 5-6 ≤5 ≤4 / 5 - 8 0 - 4.5 (3.3) ≤7 <4 / 4 - 7 5.44 / 5.51 1 - 4 (2.7) 1.8 - 6.3 / 2 - 5.3 3 - 6 (4.4)

Implant surface M/R M R M M/R R R M/R R R M/R R M M M M M R M R NR R M M R R R R R NR R R R NR NR R R R R M/R R R R R R R M/R NR M/R R NR R NR R

FU, range Mean FU (mo) (mo) up to 60 12.0 12 - 72 12.0 7 - >60 12.0 9 - 48 34.1 0 - 48 12.0 15 - 39 26.4 12 - 72 38.2 >36 - >60 60.0 12 12.0 5 - 71 38.2 0 - 60 12.0 12 - 72 34.6 12 12.0 12 - 96 37.3 12 - >60 39.8 12 - 40 22.4 18 18.0 0 - 60 12.0 17 - 66 37.5 12 - >48 31.5 6 - 36 12.0 15 - 40 27.4 60 60.0 up to 108 27.3 >17 17.0 18 18.0 4 - 84.8 43.6 24 24.0 18 - 60 44.0 36 36.0 12 - 23 16.4 up to 156 12.0 up to 84 12.0 >12 12.0 24 - 87 48.0 up to 108 69.0 8 - 72 36.0 20 20.0 18 18.0 12 - 60 12.0 24 24.0 27 - 53 35.0 27 - 54 27.0 6 - 27 13.0 12 - 60 34.8 12 12.0 36 36.0 24 - 72 48.0 3 - 72 42.0 24 24.0 24 24.0 11 - 46 27.0 12 12.0 12 12.0

CT = clinical trial, CS = case series, RCT = randomized controlled trial, ABG = autogenous bone graft, BS = bone substitute, IS = implant survival, imm = immediate implant placement, del = delayed implant placement, RRH = residual ridge height, M = machined, R = rough, FU = follow-up, mo = months, NR = not reported Volume 1 - Number 2 - 2010

Del Fabbro M. et al.

same study only the most recent data (those with the longer follow-up) were considered. The characteristics of included studies were examined by two reviewers (MDF, MB) and the publications were grouped by study design: randomized controlled trials (RCT), controlled trials (CT), case series (CS). The extracted data were divided according to the following five parameters. 1) Type of graft material: (i) autogenous bone alone, (ii) autogenous bone in combination with one or more bone substitutes and (iii) bone substitutes alone. Data from the selected articles were further divided into five subgroups according to the embryological origin of graft material: (i) autograft, (ii) allograft, (iii) xenograft, (iv) alloplast and (v) combined graft. 2) Type of implant surface: (i) machined and (ii) textured surface. 3) Implant placement timing respect to grafting procedure: (i) simultaneous and (ii) delayed technique. 4) Height of the residual ridge in the posterior maxilla (<5mm or ≥5mm). 5) Use of a covering membrane. Each subgroup was further divided according to the mean follow-up duration: shorter than 36 months or equal to or longer than 36 months. The main outcome considered for the analysis was the implant survival. Data analysis Conventional non parametric tests like the Pearson’s Chi square were used. The significance level was set at P=0.05. For each group and subgroup, data were synthesized using the weighted mean along with 95% confidence intervals (CI). Data analysis was performed

Journal of Osteology and Biomaterials

using a statistical software package (SPSS Inc., Chicago, IL, USA). RESULTS The initial search provided over 2300 articles reporting maxillary sinus lift in combination with dental implant placement. Of these, only 54 12-64 met all inclusion criteria (Table 1). Ten articles were RCT (18.5%), 12 were CT (22.2%) and 32 were CS (59.3%). Articles were published in a 16-year period from 1993 to 2009. Overall, 11746 implants were placed in 3638 patients. A total of 561 implants failed, yielding an overall survival of 95.2% (range: 61.4%-100%, 95% CI: 93.4%, 96.2%). The included articles showed substantial differences in patients’ residual crestal bone height, type of implants placed, graft materials used, success and survival criteria, duration of follow-up, study design and objectives, patient’s inclusion and exclusion criteria, data reporting and methods of statistical analysis. All articles provided outcomes considering the implant as the unit of analysis. The mean follow-up of the included studies ranged from 12 to 69 months. Influence of graft material In the subgroup using combination grafts, a variety of graft materials were mixed with autogenous bone: BioOss®, OsteoGraf/N (hydroxyapatite, HA), DFDBA (demineralized freezedried bone allograft), b-TCP (tricalcium phosphate), BioPlant HTR®, BioGran®, FHA (fluoro-hydroxyapatite), Interpore 200 (porous block hydroxyapatite) + DFDBA, OsteoGraf/N + DFDBA, BioOss® + PRGF® (plasma rich in growth factors), Bio-Oss + PRP (platelet-rich

plasma), Puros (bone allograft) + PRP and DFDBA + HA. In the subgroup using only bone substitutes, many different graft materials were used: Bio-Oss®, Laddec® (bovine bone granules), OsteoGraf/N, DFDBA, HA, ReadiGraft® (freeze-dried bone allograft, FDBA), b-TCP, calcium sulfate, marine algae, OsteoGraf/N + DFDBA, HA + DFDBA, Bio-Oss® + PRP and BioOss® + PRGF. In two RCTs 40, 61 recombinant bone morphogenetic protein-2 (rhBMP-2) embedded in an absorbable collagen sponge (ACS) was also used. In Table 2 are synthesized the results of the analysis concerning graft materials. The overall survival using 100% autogenous bone was lower (92.2%; 95% CI: 86.7%, 97.6%) than the overall survival using combined grafts (95.9%; 95% CI: 94.1%, 97.7%) and 100% bone substitutes (95.8%; 95% CI: 93.6%, 98.0%). In both cases the difference was significant (P<0.001). The data were further split according to type of implant surface. It was not possible to obtain pertinent information from seven papers 22, 35, 38, 40, 44-46 out of 54. These publications (accounting for 1033 patients and 3259 implants) used various kinds of grafting materials, but did not provide separate data concerning each type of graft. With regard to the subdivision of data according to the embryological origin of graft material used, information from all included studies, except fourteen papers 12, 22, 24, 35, 38, 40, 44-47, 53, 55, 56, 61 , could be allocated to at least one of the five subgroups. Those fourteen studies, accounting for 1490 patients and 4595 implants, used various types of graft material but did not provide enough details to allow the data to be divided into the various subgroups

Del Fabbro M. et al.

in the present analysis. The results of this analysis are showed in Table 3. The overall survival for implants placed in autogenous bone was 92.2% (95% CI: 86.7%, 97.6%). Data in the autograft group were further split according to the form of graft used (block, particulate or a mixture of both). In the subgroup using only block grafts the implant survival was lower (82.46%) than the subgroups in which a combination of block and particulate grafts or 100% particulate autograft was used (91.9% and 95.0%, respectively). The difference was significant (P<0.001). Two types of allograft were used: DFDBA and FDBA. In this subgroup the implant survival was 97.5% (95% CI: 93.8%, 101.1%). Two types of xenograft were used: deproteinized bovine bone and marine algae. The implant survival in this subgroup was 97.2% (95% CI: 94.7%, 99.7%). The survival of implants placed in alloplastic materials (calcium sulfate, rhBMP-2 in ACS, HA and b-TCP) was 86.6% (95% CI: 73.1%, 100.0%). Since the cases in which rhBMP-2 was tested displayed an unusually low implant survival, a sensitivity analysis was performed, excluding the two studies from this subgroup 40, 61 . After that, implant survival relative to the remaining studies increased to 97.7% (95% CI: 83.3%, 101.6%). Finally, the survival for implants placed in combined grafts was 96.1% (95% CI: 93.1%, 99.0%). Data of this subgroup were further split into two categories: bone substitutes in combination with autogenous bone (implant survival of 96.0%) or different bone substitutes without an autogenous component (implant survival of 96.7%).

Influence of implant surface The results of the analysis based on the implant surface are synthesized in Table 4. Implants with a machined surface displayed a mean survival of 89.4% (95% CI: 83.0%, 95.8%) for 488 patients and 1970 implants placed, while implants with a rough surface displayed a mean survival of 97.6% (95% CI: 96.7%, 98.5%) for 2330 patients and 8333 implants placed. The difference was significant (P<0.001). This comparison did not take account of the degree of roughness, the type of coating or the procedure adopted to roughen the implant surface. It was not possible to retrieve pertinent data from eight papers 12, 16, 22, 31, 40, 58, 61, 63 (accounting for 488 patients and 1443 implants), either because the implant surface was not specified or because both machined-surface and rough-surface implants were used in the same study, but separated survivals were not reported. Influence of implant placement timing The results of the analysis based on the implant placement timing respect to grafting procedure are synthesized in Table 5. The overall survival for implants placed in a simultaneous procedure (3925 implants in 1006 patients) was 96.3% (95% CI: 93.2%, 99.5%), while the overall survival for implants placed in a delayed procedure (4952 implants in 1630 patients) was 93.6% (95% CI: 91.4%, 95.5%). The difference was significant (P<0.001). Eleven papers 16, 29, 38, 40, 41, 43, 44, 47, 48, 55, 59, together accounting for 933 patients and 2,869 implants, were not classifiable because it was not possible to determine the timing of implant insertion.

Influence of residual ridge height Ten studies 13-16, 21, 22, 24, 37, 44, 53 did not report the height of the residual ridge in the posterior maxilla at the time of surgery. For the studies that could be evaluated for this parameter a threshold height of 5 mm was considered. Nineteen studies12, 18, 20, 23, 26, 29, 34, 35, 38-41, 43, 46-48, 55, 60, 65 reported the ridge height but did not provide enough details to allow the data to be split into the two groups, therefore only 25 studies out of 54 could be included in this analysis. 4678 implants were placed in correspondence with a residual ridge height lower than 5 mm. The survival was 96.7% (95% CI: 92.9%, 100.4%). 1389 implants were placed in correspondence with a residual ridge height equal to or higher than 5 mm. The survival was 92.7% (95% CI: 88.4%, 96.9%). Similar to the analysis relative to allograft materials, implant survival for â&#x2030;Ľ5 mm ridge height was also evaluated after exclusion of the study 61 in which rhBMP-2 embedded in an ACS was used as bone graft. In fact, the outcome of this study was markedly inferior to the others of the same subgroup. With this sensitivity analysis, implant survival increased to 98.3% (95% CI: 95.1%, 101.5%) for 882 implants placed. The difference between the latter and the data relative to <5mm ridge height was significant (P=0.008). Influence of using a covering membrane Seventeen studies reported the placement of a barrier membrane over the lateral window at the time of sinus grafting, while a membrane was not used in 24 of the included articles. Five of the articles provided clinical out-

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Table 2. Overall implant survival according to graft material FU duration N° articles N° patients N° implants tot 15 434 1668 <36 mo 8 256 1026 100% ABG >36 mo 7 178 642 tot 20 902 2702 <36 mo 15 758 2096 ABG+BS >36 mo 5 144 606 tot 20 1204 3866 <36 mo 15 783 2493 100% BS >36 mo 5 421 1373 ABG = autogenous bone graft, BS = bone substitute, FU = follow-up, IS = implant survival, mo = months Table 3. Comparison between graft materials Material N° articles N° patients N° implants Block 3 47 171 Particulate 8 155 645 ABG Block + particulate 6 216 852 Total 15 418 1668 3 58 157 Allograft 11 694 2302 Xenograft 4 98 306 Alloplast With ABG 18 665 2112 Without ABG 4 97 302 Combined Total 22 762 2414 ABG = atogenous bone graft, FU = follow-up, IS = implant survival, mo = months

comes for both a group treated with a covering membrane and a group in which a membrane was not used. Seventeen articles 14, 19-21, 24, 31, 35-37, 40-43, 45, 46, 61, 63 were not classifiable either because it was not specified if a covering membrane was used or because separated survivals were not reported. The overall implant survival was 96.7% (95% CI: 93.9%, 99.4%) when a membrane was used and 93.4% (95% CI: 89.7%, 97.1%) when it was not used. The difference was significant (P<0.001).

Journal of Osteology and Biomaterials

DISCUSSION Published data concerning sinus floor elevation have rapidly increased over time. The aim of the present study was to perform an updated review on the prognosis of this surgical technique. Since the retrospective study design can be affected by a high risk of bias, it was decided to consider only prospective studies for the analysis. In the present review, it was found that the overall survival using 100% autogenous bone was significantly lower than the overall survival using combined grafts or 100% bone substitutes. However, it wouldn’t be proper to state that bone substitutes or combined grafts by

IS (%) 82.5 95.0 91.9 92.2 97.5 97.2 97.4 96.0 96.7 96.1

IS (%) 92.1 92.9 91.0 95.9 96.8 92.7 95.8 94.9 97.5

Weighted mean FU (mo) 16.3 32.7 31.2 30.4 31.5 29.0 27.7 21.7 20.8 21.6

themselves produce better results than 100% autogenous bone. Other variables must be taken into account, such as the influence of implant surface configuration. Thus, data were further split according to the type of implant surface, to investigate if the graft type, the implant surface or a particular combination of these, was mainly correlated to the treatment outcome in terms of implant survival. Machined implants have been used more often in combination with 100% autogenous bone, while rough implants have been used more frequently together with bone substitutes. This might be one of the reasons why bone substitutes

Del Fabbro M. et al.

showed better outcomes than autogenous bone. This review confirmed, in line with other studies, the superiority of rough-surfaced implants over machined-surfaced ones in each type of graft 4-9. The group of studies in which 100% autogenous bone was used showed a higher variability in the outcomes. This may be explained by the fact that lots of factors, among which the donor site and the graft form (block, particulate or both forms), varied in the different studies. Recently, Klijn et al. 66 published a meta-analysis on histomorphometric studies which showed wide differences in total bone volume (TBV) between different sites and methods of autologous bone grafting in sinus floor augmentation procedures. Actually, the consequence of the TBV for implant survival is not known, but differences in the regenerative potential of autografts of different origin and form might explain a quote of the vari-

ability observed in the above studies. Moreover, autogenous bone has often been used in older studies in conjunction with machined implants. On the contrary, bone substitutes have been generally used in more recent studies, in which implants with rough surface have been mostly used. The results of the analysis based on implant placement timing showed that the overall survival for implants placed in a simultaneous procedure was significantly higher as compared to implants placed in a delayed procedure. The choice of placing implants simultaneously to the grafting procedure or at a later stage is generally influenced by the amount of residual crest bone height. If the residual bone is sufficient to provide implants with adequate primary stability, the simultaneous protocol can be recommended, otherwise if residual crestal bone height is extremely reduced, a delayed protocol is suggested. The initial better local might be one of the

Table 4. Overall implant survival according to implant surface FU duration N° articles tot 14 <36 mo 7 Machined >36 mo 7 tot 36 <36 mo 26 Rough >36 mo 10 FU = follow-up, IS = implant survival, mo = months

reasons of the better clinical outcomes observed for implants placed in a simultaneous procedure. Moreover, the presence of an implant with good primary stability, even if not immediately loaded, may serve as stimulus for graft healing and bone remodeling. In a recent review, Rios et al.10 observed a higher implant success predictability as residual ridge height increased. This finding is in agreement with the results of the present review. A significant difference was found in favor of implants placed in ridges of at least 5 mm height as compared to those inserted in less than 5 mm height ridges. In a meta-analysis on the survival of endosseous dental implants, Wallace and Froum concluded that membrane utilization is a useful adjunctive therapy that results in an increased survival of implants in the grafted maxillary sinus67. It was also reported that the increase in implant survival could be explained by the higher percentage of

N° patients 488 351 137 2330 1212 1118

N° implants 1970 1029 941 8333 4023 4310

IS (%) 89.4 89.7 89.1 97.6 97.3 97.9

Table 5. Overall implant survival according to implant placement timing FU N° articles N° patients tot 28 1630 <36 mo 19 647 Simultaneous >36 mo 9 983 tot 30 1006 <36 mo 24 862 Delayed >36 mo 6 144 FU = follow-up, IS = implant survival, mo = months

N° implants 4993 1916 3077 4043 3340 703

IS (%) 96.4 95.5 96.9 93.5 93.3 94.5

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vital bone 22, 67. This finding was confirmed in the present review. In fact, a significantly higher implant survival was found when a covering membrane was used. A matter of concern in studies evaluating implant survival is the follow-up duration. In fact, even though the implant failure risk is generally higher during the healing phase or within the first year of functional loading, later failures may occur. Each subgroup in the present analysis was divided according to the mean follow-up duration (shorter than 36 months or equal to or longer than 36 months). Little difference was found between these two time frames in any subgroup. This would suggest that implant failures are more probably to occur within the first three years of function, and very few failures occur thereafter. However, a more focused time failure analysis should be performed. Sixteen out of 54 studies included in the present review (29.6%) had a mean or minimum follow-up of only 12 months. This may prevent an accurate estimate of the implant survival over time. In order to detect the actual failure risk in the medium-long term, appropriate studies are needed.

Journal of Osteology and Biomaterials

CONCLUSIONS Sinus floor elevation is a highly predictable procedure. Sinus lift may be influenced by many variables, such as graft material, implant surface, timing of implant insertion, residual ridge height in the posterior maxilla and use of a covering membrane. Further studies of a high level of evidence are needed to evaluate the performance of single bone substitutes, and to determine the influence of other factors (i.e. patientsâ&#x20AC;&#x2122; systemic conditions, smoking habits) on the survival of implants placed in grafted maxillary sinus.

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References 1. Woo I, Le BT. Maxillary sinus floor elevation: Review of anatomy and two techniques. Implant Dent 2004;13:28-32. 2. Tatum HJr. Maxillary and sinus implant reconstructions. Dent Clin North Am 1986;30:207-29. 3. Boyne PJ, James RA. Grafting of the maxillary sinus floor with autogenous marrow and bone. J Oral Surg 1980;38:613-6. 4. Buser D, Schenk RK, Steinemann S, Fiorellini JP, Fox CH, Stich H. Influence of surface characteristics on bone integration of titanium implants. A histomorphometric study in miniature pigs. J Biomed Mater Res 1991;25:889-902. 5. Cochran DL, Schenk RK, Lussi A, Higginbottom FL, Buser D. Bone response to unloaded and loaded titanium implants with a sandblasted and acid-etched surface: A histometric study in the canine mandible. J Biomed Mater Res 1998;40:1-11. 6. Lazzara RJ, Testori T, Trisi P, Porter SS, Weinstein RL. A human histologic analysis of osseotite and machined surfaces using implants with 2 opposing surfaces. Int J Periodontics Restorative Dent 1999;19:117-29. 7. Khang W, Feldman S, Hawley CE, Gunsolley J. A multi-center study comparing dual acid-etched and machined-surfaced implants in various bone qualities. J Periodontol 2001;72:1384-90. 8. Kumar A, Jaffin RA, Berman C. The effect of smoking on achieving osseointegration of surface-modified implants: A clinical report. Int J Oral Maxillofac Implants 2002;17:816-9. 9. Stach RM, Kohles SS. A meta-analysis examining the clinical survivability of machined-surfaced and osseotite implants in poor-quality bone. Implant Dent 2003;12:87-96. 10. Rios HF, Avila G, Galindo P, Bratu E, Wang HL. The influence of remaining alveolare bone upon lateral window sinus augmentation implant survival. Implant Dent 2009;18:402-12.

11. McAllister BS, Margolin MD, Cogan AG, Taylor M, Wollins J. Residual lateral wall defects following sinus grafting with recombinant human osteogenic protein-1 or biooss in the chimpanzee. Int J Periodontics Restorative Dent 1998;18:227-39. 12. Lozada JL, Emanuelli S, James RA, Boskovic M, Lindsted K. Root-form implants placed in subantral grafted sites. J Calif Dent Assoc 1993;21:31-5. 13. Keller EE, Eckert SE, Tolman DE. Maxillary antral and nasal one-stage inlay composite bone graft: Preliminary report on 30 recipient sites. J Oral Maxillofac Surg 1994;52:438-47. 14. Zinner ID, Small SA. Sinus-lift graft: Using the maxillary sinuses to support implants. J Am Dent Assoc 1996;127:51-7. 15. Blomqvist JE, Alberius P, Isaksson S. Two-stage maxillary sinus reconstruction with endosseous implants: A prospective study. Int J Oral Maxillofac Implants 1998;13:758-66. 16. Froum SJ, Tarnow DP, Wallace SS, Rohrer MD, Cho SC. Sinus floor elevation using anorganic bovine bone matrix (OsteoGraf/N) with and without autogenous bone: A clinical, histologic, radiographic, and histomorphometric analysis--part 2 of an ongoing prospective study. Int J Periodontics Restorative Dent 1998;18:528-43. 17. Peleg M, Mazor Z, Chaushu G, Garg AK. Sinus floor augmentation with simultaneous implant placement in the severely atrophic maxilla. J Periodontol 1998;69:1397-403. 18. van den Bergh JP, ten Bruggenkate CM, Krekeler G, Tuinzing DB. Sinusfloor elevation and grafting with autogenous iliac crest bone. Clin Oral Implants Res 1998;9:429-35. 19. Buchmann R, Khoury F, Faust C, Lange DE. Peri-implant conditions in periodontally compromised patients following maxillary sinus augmentation. A long-term post-therapy trial. Clin Oral Implants Res 1999;10:103-10. 20. De Leonardis D, Pecora GE. Augmentation of the maxillary sinus with calcium sulfate: One-year clinical report from a pro-

spective longitudinal study. Int J Oral Maxillofac Implants 1999;14:869-78. 21. Olson JW, Dent CD, Morris HF, Ochi S. Long-term assessment (5 to 71 months) of endosseous dental implants placed in the augmented maxillary sinus. Ann Periodontol 2000;5:152-6. 22. Tarnow DP, Wallace SS, Froum SJ, Rohrer MD, Cho SC. Histologic and clinical comparison of bilateral sinus floor elevations with and without barrier membrane placement in 12 patients: Part 3 of an ongoing prospective study. Int J Periodontics Restorative Dent 2000;20:117-25. 23. van den Bergh JP, ten Bruggenkate CM, Krekeler G, Tuinzing DB. Maxillary sinusfloor elevation and grafting with human demineralized freeze dried bone. Clin Oral Implants Res 2000;11:487-93. 24. Bahat O, Fontanessi RV. Efficacy of implant placement after bone grafting for three-dimensional reconstruction of the posterior jaw. Int J Periodontics Restorative Dent 2001;21:220-31. 25. Kahnberg KE, Ekestubbe A, Grondahl K, Nilsson P, Hirsch JM. Sinus lifting procedure. I. one-stage surgery with bone transplant and implants. Clin Oral Implants Res 2001;12:479-87. 26. Tawil G, Mawla M. Sinus floor elevation using a bovine bone mineral (bio-oss) with or without the concomitant use of a bilayered collagen barrier (bio-gide): A clinical report of immediate and delayed implant placement. Int J Oral Maxillofac Implants 2001;16:713-21. 27. Hallman M, Sennerby L, Lundgren S. A clinical and histologic evaluation of implant integration in the posterior maxilla after sinus floor augmentation with autogenous bone, bovine hydroxyapatite, or a 20:80 mixture. Int J Oral Maxillofac Implants 2002;17:635-43. 28. Engelke W, Schwarzwaller W, Behnsen A, Jacobs HG. Subantroscopic laterobasal sinus floor augmentation (SALSA): An upto-5-year clinical study. Int J Oral Maxillofac Implants 2003;18:135-43.

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29. McCarthy C, Patel RR, Wragg PF, Brook IM. Sinus augmentation bone grafts for the provision of dental implants: Report of clinical outcome. Int J Oral Maxillofac Implants 2003;18:377-82. 30. Philippart P, Brasseur M, Hoyaux D, Pochet R. Human recombinant tissue factor, platelet-rich plasma, and tetracycilne induce a high-quality human bone graft: A 5-year survey. Int J Oral Maxillofac Implants 2003;18:411-6. 31. Rodriguez A, Anastassov GE, Lee H, Buchbinder D, Wettan H. Maxillary sinus augmentation with deproteinated bovine bone and platelet rich plasma with simultaneous insertion of endosseous implants. J Oral Maxillofac Surg 2003;61:157-63. 32. Stricker A, Voss PJ, Gutwald R, Schramm A, Schmelzeisen R. Maxillary sinus floor augmention with autogenous bone grafts to enable placement of SLA-surfaced implants: Preliminary results after 15-40 months. Clin Oral Implants Res 2003;14:207-12. 33. Hallman M, Zetterqvist L. A 5-year prospective follow-up study of implant-supported fixed prostheses in patients subjected to maxillary sinus floor augmentation with an 80:20 mixture of bovine hydroxyapatite and autogenous bone. Clin Implant Dent Relat Res 2004;6:82-9. 34. Hatano N, Shimizu Y, Ooya K. A clinical long-term radiographic evaluation of graft height changes after maxillary sinus floor augmentation with a 2:1 autogenous bone/ xenograft mixture and simultaneous placement of dental implants. Clin Oral Implants Res 2004;15:339-45. 35. John HD, Wenz B. Histomorphometric analysis of natural bone mineral for maxillary sinus augmentation. Int J Oral Maxillofac Implants 2004;19:199-207. 36. Iturriaga MT, Ruiz CC. Maxillary sinus reconstruction with calvarium bone grafts and endosseous implants. J Oral Maxillofac Surg 2004;62:344-7. 37. Schwartz-Arad D, Herzberg R, Dolev E. The prevalence of surgical complications

Journal of Osteology and Biomaterials

of the sinus graft procedure and their impact on implant survival. J Periodontol 2004;75:511-6. 38. Shlomi B, Horowitz I, Kahn A, Dobriyan A, Chaushu G. The effect of sinus membrane perforation and repair with lambone on the outcome of maxillary sinus floor augmentation: A radiographic assessment. Int J Oral Maxillofac Implants 2004;19:559-62. 39. Testori T, Del Fabbro M, Weinstein RL, Wallace SS. Maxillary sinus surgery and alternatives in treatment. Quintessence Publishing Co. Ltd. 40. Boyne PJ, Lilly LC, Marx RE, Moy PK, Nevins M, Spagnoli DB, Triplett RG. De novo bone induction by recombinant human bone morphogenetic protein-2 (rhBMP-2) in maxillary sinus floor augmentation. J Oral Maxillofac Surg 2005;63:1693-707. 41. Simunek A, Cierny M, Kopecka D, Kohout A, Bukac J, Vahalova D. The sinus lift with phycogenic bone substitute. A histomorphometric study. Clin Oral Implants Res 2005;16:342-8. 42. Ewers R. Maxilla sinus grafting with marine algae derived bone forming material: A clinical report of long-term results. J Oral Maxillofac Surg 2005;63:1712-23. 43. Butz SJ, Huys LW. Long-term success of sinus augmentation using a synthetic alloplast: A 20 patients, 7 years clinical report. Implant Dent 2005;14:36-42. 44. Wallace SS, Froum SJ, Cho SC, Elian N, Monteiro D, Kim BS, Tarnow DP. Sinus augmentation utilizing anorganic bovine bone (bio-oss) with absorbable and nonabsorbable membranes placed over the lateral window: Histomorphometric and clinical analyses. Int J Periodontics Restorative Dent 2005;25:551-9. 45. Scarano A, Degidi M, Iezzi G, Pecora G, Piattelli M, Orsini G, Caputi S, Perrotti V, Mangano C, Piattelli A. Maxillary sinus augmentation with different biomaterials: A comparative histologic and histomorphometric study in man. Implant Dent 2006;15:197-207.

46. Peleg M, Garg AK, Mazor Z. Healing in smokers versus nonsmokers: Survival rates for sinus floor augmentation with simultaneous implant placement. Int J Oral Maxillofac Implants 2006;21:551-9. 47. Karabuda C, Arisan V, Hakan O. Effects of sinus membrane perforations on the success of dental implants placed in the augmented sinus. J Periodontol 2006;77:1991-7. 48. Mardinger O, Manor I, Mijiritsky E, Hirshberg A. Maxillary sinus augmentation in the presence of antral pseudocyst: A clinical approach. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;103:180-4. 49. Mangano C, Scarano A, Perrotti V, Iezzi G, Piattelli A. Maxillary sinus augmentation with a porous synthetic hydroxyapatite and bovine-derived hydroxyapatite: A comparative clinical and histologic study. Int J Oral Maxillofac Implants 2007;22:980-6. 50. Marchetti C, Pieri F, Trasarti S, Corinaldesi G, Degidi M. Impact of implant surface and grafting protocol on clinical outcomes of endosseous implants. Int J Oral Maxillofac Implants 2007;22:399-407. 51. Galindo-Moreno P, Avila G, FernandezBarbero JE, Aguilar M, Sanchez-Fernandez E, Cutando A, Wang HL. Evaluation of sinus floor elevation using a composite bone graft mixture. Clin Oral Implants Res 2007;18:376-82. 52. Aguirre Zorzano LA, Rodriguez Tojo MJ, Aguirre Urizar JM. Maxillary sinus lift with intraoral autologous bone and B--tricalcium phosphate: Histological and histomorphometric clinical study. Med Oral Patol Oral Cir Bucal 2007;12:532-6. 53. Minichetti JC, Dâ&#x20AC;&#x2122;Amore JC, Hong AY. Three-year analysis of tapered screw vent implants placed into maxillary sinuses grafted with mineralized bone allograft. J Oral Implantol 2008;34:135-41. 54. Lee CY, Rohrer MD, Prasad HS. Immediate loading of the grafted maxillary sinus using platelet rich plasma and autogenous bone: A preliminary study with histologic and histomorphometric analysis. Implant Dent 2008;17:59-73.

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55. Lee JH, Jung UW, Kim CS, Choi SH, Cho KS. Histologic and clinical evaluation for maxillary sinus augmentation using macroporous biphasic calcium phosphate in human. Clin Oral Implants Res 2008;19:767-71. 56. Kahnberg KE, Vannas-Lofqvist L. Sinus lift procedure using a 2-stage surgical technique: I. clinical and radiographic report up to 5 years. Int J Oral Maxillofac Implants 2008;23:876-84. 57. Yamamichi N, Itose T, Neiva R, Wang HL. Long-term evaluation of implant survival in augmented sinuses: A case series. Int J Periodontics Restorative Dent 2008;28:163-9. 58. Meyer C, Chatelain B, Benarroch M, Garnier JF, Ricbourg B, Camponovo T. Massive sinus-lift procedures with beta-tricalcium phosphate: Long-term results. Rev Stomatol Chir Maxillofac 2009;110:69-75. 59. Ferreira CE, Novaes AB, Haraszthy VI, Bittencourt M, Martinelli CB, Luczyszyn SM. A clinical study of 406 sinus augmentations with 100% anorganic bovine bone. J Periodontol 2009;80:1920-7. 60. Torres J, Tamimi F, Martinez PP, Alkhraisat MH, Linares R, Hernandez G, Torres-Macho J, Lopez-Cabarcos E. Effect of platelet-rich plasma on sinus lifting: A randomized-controlled clinical trial. J Clin Periodontol 2009;36:677-87.

trates for bone graft enhancement in sinus lift procedure. Transfusion 2009;49:779-85. 64. Cannizzaro G, Felice P, Leone M, Viola P, Esposito M. Early loading of implants in the atrophic posterior maxilla: Lateral sinus lift with autogenous bone and bio-oss versus crestal mini sinus lift and 8-mm hydroxyapatite-coated implants. A randomised controlled clinical trial. Eur J Oral Implantol 2009;2:25-38. 65. Wannfors K, Johansson B, Hallman M, Strandkvist T. A prospective randomized study of 1- and 2-stage sinus inlay bone grafts: 1-year follow-up. Int J Oral Maxillofac Implants 2000;15:625-32. 66. Klijn RJ, Meijer GJ, Bronkhorst EM, Jansen JA. Sinus floor augmentation surgery using autologous bone grafts from various donor sites: A meta-analysis of the total bone volume. Tissue Eng Part B Rev 2010;16:295-303. 67. Wallace SS, Froum SJ. Effect of maxillary sinus augmentation on the survival of endosseous dental implants. A systematic review. Ann Periodontol 2003;8:328-43.

61. Triplett RG, Nevins M, Marx RE, Spagnoli DB, Oates TW, Moy PK, Boyne PJ. Pivotal, randomized, parallel evaluation of recombinant human bone morphogenetic protein-2/absorbable collagen sponge and autogenous bone graft for maxillary sinus floor augmentation. J Oral Maxillofac Surg 2009;67:1947-60. 62. Chaushu G, Mardinger O, Calderon S, Moses O, Nissan J. The use of cancellous block allograft for sinus floor augmentation with simultaneous implant placement in the posterior atrophic maxilla. J Periodontol 2009;80:422-8. 63. Bettega G, Brun JP, Boutonnat J, Cracowski JL, Quesada JL, Hegelhofer H, Drillat P, Richard MJ. Autologous platelet concen-

Volume 1 - Number 2 - 2010

BioCRA

Original article

ISQ (RFA) vs BIC and torque: a histomorphometric and biomechanical analysis in humans Paolo Trisi DDS PhD1,2, Teocrito Carlesi DDS1,2, Marta Rocci DDS3, Antonio Rocci DDS3

The aim of the present study is to clarify the controversy and to demonstrate in human implants if a statistically significant correlation exists between the ISQ values and measurement of osseointegration (degree of osseointegration), the Torque-Out values and histomorphometric data of bone implant anchorage. Twelve implants were removed from the superior and inferior maxillaries of 12 patients after a healing period of about 60 days, 3 3iTM Osseotite and 9 MK III implant Ti-Unite and machined surface. The Torque-In values were computed during the implant placement. The Torque-Out test was computed at the time of removal. The ISQ values were taken before the Torque-Out test and before retrieval of the implants. Subsequently, histological specimens were prepared, and bone volume (BV), bone-implant-contact (BIC), and the number of threads in contact with the compact bone (n.Tcb) were measured. A lack of statistical significance was found between the values of ISQ and torque-out values (ρ= 0.391552 r2=0.1533 P=0.2081), the percentage of bone-implant-contact (BIC) (ρ= 0.222076 r2=0.0493 P=0.4879) and the peri-implant bone volume (BV/TV %) (ρ= 0.431972 r2=0.1866 P=0,1608). The ISQ values and the number of threads in contact with the compact bone (n.Tcb) ( ρ= 0.634807 r2=0.403 P= 0.0266) were statistically significantly related. After two months healing Torque-in values were still statistically significantly correlated with the values of ISQ (ρ= 0.669018 r2=0.4476 P=0.0174), while there was a lack of statistical significance between the values of ISQ and the clinical bone density (bone type) (ρ=-0.2477 r2=0.06133 P=0.4377). A statistically significant correlation was found between BIC and Torque-out values, (ρ=0.905561 r2=0.82 P=0.0001). Within the limits of the present study, the results suggest that the development of osseointegration after a healing period of two months, measured through the percentage of BIC does not influence the values of ISQ. On the other hand, the ISQ seems to be related to the cortical anchorage of the implants at two months healing. (J Osteol Biomat 2010; 1:81-91)

Key words: dental implant, implant stability, resonance frequency analysis, histomorphometry, torque test. Bio.C.R.A. Biomaterial Clinical and histological Research Association, Pescara, Italy; Private Practice, Pescara, Italy; 3 Private Practice, Chieti, Italy. 1 2

Corresponding author: Teocrito Carlesi DDS; Biomaterial Clinical and histological Research Association, Via Silvio Pellico 68, 65132 Pescara, Italy. Tel:+39 085 28432; Fax: +39 085 28427; e-mail: t.carlesi@email.it

Introduction It is thought that the development of a stable bone-implant interface is an indispensable requirement for the long and short-term correct function of an implant. The development and the characteristics of the bone-implant interface can be influenced by the different geometric shapes of the implant surface and the bone where the implant is anchored. Therefore, it is in the common interest to develop and evaluate methods capable to measure different biomechanical properties of the bone-implant interface, either experimentally or in clinical practice. In the past, histological analysis was considered the gold standard to measure the degree of osseointegration. This method, along with a biomechanical Torque-Out test are still today credited as the most reliable and objective methods available. Nevertheless, both methods are limited since they have been used only in experimental studies and since the samples are destroyed after the experimentation, they cannot be followed-up or monitored in time. Recently, new methods were developed to measure the non-destructive implant stability, there are literature reports on percussion tests, radiographs (BMD), cutting resistance, torque-in, impact hammer method (Periotest) and even resonance frequency analysis (RFA).

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Table 1. The values of each implant at the time of the placement. N° 1 Osseotite

Torque- in (N cm) 40

N° 2 Osseotite

2.0

Mand. ant.

N° 3 Osseotite

3.5

Maxilla post.

N° 4 Mk III Ti Unite

2.0

Mand. ant.

N° 5 MKIII Ti Unite

3.5

Maxilla post.

N° 6 MKIII Ti Unite

4.0

Mand. post.

N° 7 MKIII Ti Unite

2.0

Mand. post.

N° 8 MKIII Ti Unite

2.0

Mand. post.

N° 9 MKIII Ti Unite

3.0

Mand. post.

N°10 MkIII standard

2.5

Mand. post.

N°11 MkIII standard

3.0

Mand. post.

N°12 MkIII standard

2.5

Mand. post.

35 ± 9.7

2.79 ± 0.72

Implant and surface

Mean and SD

Among these, the RFA is the most used in experimental studies, as well as in clinical practice, but many aspects still need to be clarified. Resonance Frequency Analysis developed by Meredith et al.1 uses specified resonance characteristics of acoustically excited implants and utilises a small Lshaped transducer which is screwed onto an implant fixture or abutment. The transducer comprises 2 piezoceramic elements, one of which vibrates by a sinusoidal signal (5 to 15 kHz). The other serves as a receptor for the signal. Resonance peaks from the received signal indicate the first flexural (bending) resonance frequency of the measured object. The specific value that indicates the implant stability of a given situation is called the resonance frequency.1,2 In vitro and in vivo studies have suggested that this resonance peak may be used to assess implant stability in a quantitative manner.3,4 Currently, 2 RFA machines are in clinical use: the Osstelltm device (Integration Diagnostics AB, Göteborg, Sweden) and Im-

Journal of Osteology and Biomaterials

Bone-Type

Position inthe Jaws 3.5

Mand. post.

plomates (Bio Tech One,Taipei,Taiwan). Osstell combines the transducer, computerized analysis and the excitation source into one machine closely resembling the model used by Meredith. In the early studies, the Hertz signal was used as a measurement unit.1-5 Later, Osstell created the implant stability quotient (ISQ) as a measurement unit in place of Hertz. Resonance frequency values ranging from 3500 to 8500 Hz are translated into an ISQ of 0 to 100. A high value indicates greater stability, whereas a low value implies instability. The manufacturer’s guidelines suggest that a successful implant typically has an ISQ greater than 65. An ISQ < 50 may indicate potential failure or increased risk of failure.6 However, until today there are no literature reports describing any exact values on long-term implant osseointegration, or any value that can identify an implant failure. Only very wide ranges are hypothesized since there are many variables that come into play. More frequently there are literature data that help identify the numerous factors that

can influence such measurements, as for example, the characteristics of the bone tissue (density and quality), mono and bicortical anchoring of the implant,7 the inclination of the transducer,8 the effective length of the implant above the bone crest, the diameter of the implant, the micro and macro geometry of the implant9. Not only, but in a recent study, some authors explain how the simplicity of the algorithm used from Osstell’s software to determine values of resonance frequency could produce erratic results, consequently the authors suggest using more suitable and precise software.10 In the first European Osseointegration Association Consensus Conference held in 2006 11, some authors sustained that a single measure using RFA does not define the characteristics of the bone-implant interface and does not offer any reliable quantitative evaluation of the degree of osseointegration. Not only that, but the RFA would not have any prognostic validity on the development of the instability. These authors assert that the validity and reliability of RFA, from a clinical point of view, still remain to be demonstrated, for every implant system, such as the ISQ values which indicate the stability or the risk of loss of stability of the precise implant system. Research evidence demonstrates that elevated values of ISQ in a specific implant indicates that the implant is stable, and if in a follow-up the ISQ values remain high, it would indicate that the stability is maintained; while low values of ISQ, or a lowering of the values with time would indicate risk of instability of the implant.12 These evidences could prove to be valid, however, there are still many aspects that need clarification. First of all, there are no literature reports that demon-

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Table 2. The values of each implant after about 2 months of healing. N° 1 Osseotite

Torque-out (N cm) 32

30.42

50.76

N° 2 Osseotite

16.27

26.58

N° 3 Osseotite

17.91

25.95

N° 4 Mk III Ti Unite

31.78

60.23

N° 5 MKIII Ti Unite

32.54

42.52

N° 6 MKIII Ti Unite

44.81

37.33

N° 7 MKIII Ti Unite

55.18

52.26

Implant and surface

ISQ

BIC (%)

BV (%)

n. Tcb

N° 8 MKIII Ti Unite

65.36

62.70

N° 9 MKIII Ti Unite

63.58

33.18

N°10 MkIII standard

11.52

45.37

N°11 MkIII standard

53.93

60.05

N°12 MkIII standard

47.38

69.04

70.75 ± 6.68

42.67 ± 15.89

39.22 ± 18.51

47.16 ± 14.42

5 ± 3.86

Mean and SD

strate a correlation between RFA and the micro-movements of an implant inserted in the bone. If such a measurement was demonstrated to indicate the rigidity of the bone-implant interface, it could be expected that the results could be correlated with the density of the peri-implant bone volume, or the percent bone-implant contact, or at least with the necessary force to insert or remove the implant from the bone site. In the literature, this non destructive measuring method (RFA measuring) has been compared to radiograms, mechanical tests and histomorphometric tests giving contradictory results. 13-27 The aim of the present study is to clarify the controversy and to demonstrate in human implants if a statistically significant correlation exists between the RFA values and measurement of osseointegration (BIC %), the Torque-Out values and histomorphometric data of bone implant anchorage, the percentage bone volume round the implant (BV) and the number of threads in contact to the compact bone (n.Tcb).

Materials and methods Subjects and dental implant history From 2002 to 2007, 12 patients (8 males and 4 females) have had necessity of implants removal for different reasons: pain, psychological and prosthetic difficulties. The subjects had no specific documented medical problems. The patients agreed to participate in the study and gave their informed written consent after receiving a thorough explanation of the surgical procedures. The study was performed in accordance with the Helsinki Declaration of 1975. Twelve implants were removed from the superior and inferior jaws of 12 patients after about two months unloaded healing. (n=3 3iTM Osseotite [Implant Innovations Inc., Palm Beach Gardens, FL, USA], (n=3 MK III Standard and n=6 MK III TiUnite [Nobel Biocare AB, Goteborg, Sweden]). All the implants had a diameter of 3.75 mm and a length of 8.5 mm. The implants were inserted in the maxillary bones of subjects without the addition of any bone regeneration. The bone quality of each implant was measured by

the operator during the standard surgical procedure, basing themselves on the manual perception of the cutting resistance and classifying them according to Misch system described in 1999.28 The bone types were from the most dense (D 1) to the least dense (D 4). For the statistical analysis, the intermediate types (1-2, 2-3, 3-4) were classified as type 1.5 - 2.5 - 3.5. The preparation of the implant site was carried out with a conventional method. All the implants were submerged up to the crest and for each implant, a maximum torque measurement using a manual surgical torque wrench was made (Torque-In). The cover screws were placed and all the implants were submerged. The implant site placement into the jaws (mandible or maxillae, anterior or posterior) is shown in table 1. Before removal ISQ measurement was performed according to Osstell. Subsequently, the Torque-Out test was measured using a dynamometric wrench. After the implants were all repositioned in the same position before the Torque-Out test, all implants were extracted using a trephine cutter of 4.5 mm and immersed in 10% formalin for histological studies. Torque test To measure the maximum resistance of insertion (Torque-In) a manual surgical torque wrench (NobelReplace™ Manual Torque Wrench – Surgical, [Nobel Biocare AB, Goteborg, Sweden]) was used. The Torque-In values were computed during the positioning of all the 12 implants. To measure the maximum removal torque (Torque-Out) a customized digital hand torque wrench was used to measure the peak reverse torque. In addition, electronic equipment consisting of a digital hand operated torque wrench,

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Fig. 1.

Fig. 2.

Figure 1. Scatter plot compares data points for values BIC vs Torque-out, Test-1. Positive liner trend line show that correlation could be found between bone implant contact percentage values and Torque-out values (Pearson coefficient of correlation ρ=0.905561; r2=0.82) P=0.0001, is statistically significant Fig. 3.

Fig. 4.

Figure 3. Scatter plot compares data points for values BIC vs ISQ, Test-3. Positive liner trend line show that no correlation could be found between BIC values and ISQ values (Pearson coefficient of correlation ρ= 0.222076; r2=0.0493) P=0.4879, is not statistically significant.

equipped with a calibrated strain gauge and connected to a PC reading the peak insertion torque value every 0.5 ms, was customized for this study. To obtain the peak extrusion torque, the signal was subsequently evaluated by the MECODAREC software (ATech s.r.l., Bergamo, Italy). When an implant was unscrewed, the peak torque value fell quickly when the rupture between bone and implant occurred; up to this moment no macroscopic movement of the implant was evident. After interface breakage the implant was repositioned as precisely

Journal of Osteology and Biomaterials

Figure 2. Scatter plot compares data points for values Torque-out vs ISQ, Test-2. Positive liner trend line show that no correlation could be found between torque-out values and ISQ values (Pearson coefficient of correlation ρ= 0.391552; r2=0.1533) P=0.2081, is not statistically significant.

Figure 4. Scatter plot compares data points for values BV vs ISQ, Test-4. Positive liner trend line show that no correlation could be found between bone volume percentage values and ISQ values (Pearson coefficient of correlation ρ= 0.431972; r2=0.1866). P=0.1608, is not statistically significant.

as possible into the initial position. This tissue was fixed and processed to obtain a readable interface under histomorphometric analysis. In the past a similar procedure to study the morphology of the bone-metal rupture was used by Sennerby et al.29 For both tests the peak values were taken to the closest numerical unity and calculated as Ncm. Resonance frequency analysis Resonance Frequency Analysis was calculated using Osstell (Integration Diagnostics, Göteborg, Sweden). The RFA

measurements were performed on all 12 removed implants, and the values were registered in ISQ units (Implant Stability Quotient). This test was performed before the Torque-Out test and before removal of the implants. The transducers were specific for the implants and were screwed in the implants without the specific abutment. The screw-fixation of the transducer was obtained with 10 Ncm torque, even if some authors have demonstrated that the degree of screw-in transducer torque does not influence the measurement.30 RFA values were meas-

Trisi P. et al.

Fig. 5.

Fig. 6.

Figure 5. Scatter plot compares data points for values n.TCB vs ISQ, Test-5. Positive liner trend line show that correlation could be found between number of the threads in contact to the compact bone (n.Tcb) and ISQ values (Pearson coefficient of correlation ρ= 0.634807; r2=0.403). P=0.0266, is statistically significant.

Fig. 7.

Figure 6. Scatter plot compares data points for values Torque-in vs ISQ, Test-6. Positive liner trend line show that correlation could be found between Torque-in values and ISQ values (Pearson coefficient of correlation ρ= 0.669018; r2=0.4476). P=0.0174, is statistically significant. Figure 7. Scatter plot compares data points for values Bone Type vs ISQ, Test-7. Negative liner trend line show that no correlation could be found between Bone type (clinical density) and ISQ values. (Pearson coefficient of correlation ρ=-0.2477; r2=0.06133). P=0.4377, is not statistically significant.

ured in each implant. Histologic and Histomorphometric Procedure The evaluated specimens were infiltrated in methacrylate resin from a starting solution of 50% ethanol resin and subsequently 100% resin. Each step in this process required a 24-hour period. Photopolymerization was obtained using a 48-hour blue-light exposure, and the implants were oriented longitudinally to display the opposing surfaces. After polymerization, the blocks were ground to remove excess resin and expose the tissue and then glued on plastic slides using a methacrylate-based glue. A Micromet high-speed rotating- blade microtome (Remet, Bologna, Italy) was used to separate the section from the block to obtain a 250-µm-thick section. The section was

ground down to about 40 µm using an LS-2 grinding machine (Remet, Bologna, Italy) equipped with waterproof grinding paper. After grinding, each section was polished with polishing paper and a 3-µm polishing cream. Two different staining procedures were used for these sections. Toluidine blue was used to analyze the different ages and remodeling patterns of the bone, and basic fuchsin was used to distinguish the fibrous tissue and for better contrast. The histomorphometric analysis was performed by digitizing the images from the microscope via a JVC TK-C1380 color video camera (JVC Victor, Yokohama, Japan) and a frame grabber. Subsequently, the digitized images were analyzed by image-analysis software (IAS 2000, Delta Sistemi, Rome, Italy). The images were acquired with a 50 magnification of the

implant and surrounding bone. For each implant the two most central sections were analyzed. The BIC percentage was expressed by considering the total length of the implant interface. The parameters calculated using the IAS 2000 software were: - Bone volume % (BV/TV): the amount of bone matrix measured over the entire microscopic field. This measurement was accomplished by outlining the bone islands and surfaces to determine the surface area of bone in each particular microscopic field, representing in clinical terms “bone quality”. - Bone-to-implant contact % (BIC): the linear surface of the implant directly contacted by the bone matrix and expressed as a percentage of the total implant surface. - n.Tcb: the number of threads in contact

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Figures 8. (a: original magnification X 100; b: original magnification X 75) A space (arrow) is present between the implant surface and tissue, which indicates that the rupture has occurred between the implant surface and the bone. No fractures are visibile in the bone.

to the compact bone, both in crest and along the whole bone-implant interface. At times the compact bone was finded to the apex or along the implant surface. Stastistical analysis All 12 samples were incuded in the study. The linear Pearson coeficient of correlation (ρ) was used for the 7 items each one composed of 2 variables. For the first hypothesis the BIC % vs Torque-out values were included (Test-1); for the second hypothesis Torque-Out vs ISQ values were included (Test-2); for the third hypothesis BIC % vs ISQ values were evaluated (Test-3); the fourth hypothesis BV % vs ISQ values were included (Test 4); for the five hypotheses the number of threads in contact to the compact bone (n.Tcb) vs ISQ values were included (Test5); for the six hypotheses Torque-In vs ISQ values were included (Test-6), and finally clinical bone density (bone type) vs ISQ values were included (Test-7). For each function an r2 value was also calcu-

Journal of Osteology and Biomaterials

lated and graphed (scatter plots) where the linear trend line was shown to better explain the relevance of such a correlation. Significance testing was also made for each correlation (P-value). Results All implants were regarded as clinically osseointegrated and stable, not one was mobile. Radiographically there was no appreciable peri-implant bone resorption. Table 1 shows, the torque-in (N cm), the clinical bone density (bone type) and the position in the jaws, at the time of the placement (Table 1). Table 2 shows for each implant, the ISQ values, the torque-out (N cm), the bone-implant contact (BIC %), the bone volume (BV/TV %), the number of threads in contact to the compact bone (n.Tcb) (Table 2). The linear Pearson Coeficient of correlation (ρ) and (r2) values for each items (Test1-7) was: Test-1 (BIC vs Torque-out) ρ=0.905561 r2=0.82 P=0.0001, the variables are correlated and test is statistically

significant (Fig.1); Test-2 (Torque-Out vs ISQ) ρ= 0.391552 r2=0.1533 P=0.2081, the variables are not correlated and the test is not statistically significant (Fig.2); Test-3 (BIC vs ISQ) ρ= 0.222076 r2=0.0493 P=0.4879, the variables are not correlated and the test is not statistically significant (Fig.3); Test-4 (BV vs ISQ) ρ= 0.431972 r2=0.1866 P=0,1608, the variables are not correlated and the test is not statistically significant (Fig.4); Test5 (n.Tcb vs ISQ) ρ= 0.634807 r2=0.403 P= 0.0266 the variables are correlated and test is statistically significant (Fig.5); Test-6 (Torque-In vs ISQ) ρ= 0.669018 r2=0.4476 P=0.0174 the variables are correlated and test is statistically significant (Fig.6); Test-7 (Bone Type vs ISQ) ρ=-0.2477 r2=0.06133 P=0.4377 the variables are not correlated and the test is not statistically significant (Fig.7). Histologic Results Ground sections of this samples, clearly showed that the rupture occurred close to the implant surface and not within the bone. Sometimes, a gap was present between the implant surface and the bone and no tissue remained on the implant surface. (Figs. 8) - Osseotite implants The osseotite implants showed a certain degree of osseoconduction. Since the three most coronal threads are machined surface few degree of bone implant contact was found in this region of the implant. Conversely, the more apical threads show higher level of bone conduction in the typical flowing pattern. Nevertheless in cancellous bone the new trabeculae formed on the titanium surface were very thin and made of woven bone. In the crest bone was still incompletely remodelled and microc-

Trisi P. et al.

Figures 9. The osseotite implants showed a certain degree of osseoconduction. (a: original magnification 8 X; toluidine blue-basic fuchsine) Since the three most coronal threads are machined surface few degree of bone implant contact was found in this region of the implant. Conversely the more apical threads show higher level of bone conduction in the typical flowing pattern. (b: original magnification X 25) Nevertheless in cancellous bone the new trabeculae formed on the titanium surface were very thin and made of woven bone. In the crest bone was still incompletely remodelled and microcracks, lamellar delamination and bone debris were still visible. (c: original magnification X 100) Large osteoid bands coupled with osteoclastic resorption lacunae testify the active on going remodeling.

racks, lamellar delamination and bone debris were still visible. Large osteoid bands coupled with osteoclastic resorption lacunae testify the active on going remodelingÂ. (Figs.9) - Ti Unite implants Ti unite implants showed a high level of osteoconduttivity. Both in cortical crestal regions and in cancellous areas these implants had large amount of implants surface covered by new bone. The trabeculae facing the implant surface were quite thick made of composite bone. Remodeling fenomena were evident in all samples. (Figs.10) - MkIII standard implants These implants showed the typical pattern of machined implant. The bone contacted implant surface in dense bone while in cancellous bone few contact points were evident. Dense fibrous tis-

sue was facing the titanium-machined surface in marrow areas. This could represent the initial stage of bone formation due to the early stage of implant retrieval. (Figs.11) Discussion The results of the present study showed that the ISQ is not statistically related to the percentage of osseointegration (BIC%), contrarily a statistically significant correlation was found between BIC and Torque-out values. The calculation of the percentage of the bone-implant-contact is considered one of the best methods to analyze the bone-implant interface and therefore infers the biomechanical properties of the interface. Osseointegration may only be measured by destructive methods such as histomorphometric analysis of per-

centage bone-implant contact (BIC) or by mechanical measurements of torque removal.31 Also in the present study the values of the percentage of bone-implant contact and the values of torque-out were directly correlated and showed an elevated statistical significance (Fig.1 Ď =0.905561 r2=0.82 P=0.0001). No statistically relation between the values of ISQ and torque-out values was found (Fig.2 Ď = 0.391552 r2=0.1533 P=0.2081). To our knowledge, there are no studies that are similar to the present one. Akkocaoglu et al. in a human cadaver study, show that there is no correlation between the values of ISQ and the values of torque removal for implants placed into freshly prepared extraction sockets without healing.19 In the present study the removal torque was performed after about two months of unloaded healing.

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10a

10b

10c

Figures 10. Ti unite implants showed a high level of osteoconduttivity. (a: original magnification 8 X; toluidine blue-basic fuchsine) Both in cortical crestal regions and in cancellous areas these implants had large amount of implants surface covered by new bone. The trabeculae facing the implant surface were quite thick made of composite bone. (b: original magnification X 25) Remodeling fenomena were evident in all samples. (c: original magnification X 25)

No correlation between the values of ISQ and the percentage of bone-implant-contact was found (% BIC) (Fig.3 Ď = 0.222076 r2=0.0493 P=0.4879) and the peri-implant bone volume (% BV/TV) (Fig.4. Ď = 0.431972 r2=0.1866 P=0.1608), but only between the values ISQ and the number of threads in contact with the compact bone (n.Tcb) (Fig.5 Ď = 0.634807 r2=0.403 P=0.0266, the correlation was statistically significant). Rocci et al. in a human study, could not establish a correlation between ISQ values and BIC % for implant placed in the posterior mandible and retrieved after 2 months healing and 5-7 months of prosthetic load.23 The results of the present study are also confirmed by studies performed on living dogs22 and in pigs24 at different times of healing. No correlation was found between the percentage of BIC and the values of ISQ in both the studies, analyzed

Journal of Osteology and Biomaterials

implants after a healing period of 1 and 3 months in dogs, and of 1, 2 and 4 weeks in pigs. However, in pig tibias Ito et al. 24 underline that BIC vs ISQ values are not correlated, but if the percentage of the bone-implant contact is analyzed at the level of the implant crest the correlation increases. The present study demonstrates that there is a correlation between the values of ISQ and the number of threads in contact with the compact bone, both in the crest and along the entire bone-implant interface; in some samples compact bone was found at the apex or along the lateral surface. Similar results were found in a experimental study on cadaver jaws18 in which the histomorphometric analysis was performed at the time of implant placement. The authors found that the BMD values, TBPf (trabecular bone pattern factor),

BV/TV (density of trabecular bone) were not related to ISQ, while the BIC measured on the lingual aspect of the implants gave a positive correlation with ISQ values; this correlation increased only when implants were in contact with the cortical bone. A similar positive correlation (the height of the cortical passage implants vs ISQ) was found by Miyamoto et al.25, who digitally measured (Computed Tomography) the thickness of the cortical bone at the implant sites (mesial and distal). In another human study a micro-CT was used for measurements of the BVD (bone volume density) and BCT (bone trabecular connettivity) of the implant site before the insertion of the implant, no significant correlation was found with values of ISQ.26 These data lead us to hypothesize that RFA measures the rigidity of the boneimplant complex only within the com-

Trisi P. et al.

11a

11b

Figures 11. MkIII standard implants showed the typical pattern of machined implant. The bone contacted implant surface in dense bone (a) while in cancellous bone few contact points were evident (b). Dense fibrous tissue was facing the titanium machined surface in marrow areas. This could represent the initial stage of bone formation due to the early stage of implant retrieval. (a,b: original magnification 8 X; toluidine blue-basic fuchsine)

pact bone, while it does not account for osseointegration in cancellous bone. The insertion torque measures, in Ncm, the maximum torque of insertion obtained during implant placement until it is totally lodged in its site. Such a procedure may be influenced by the preparation of most bone sites, the bone density and the type of implant (self-tapping or not, cone-shaped or cylindrical). High insertion torque values correspond to a high degree of primary stability of an implant 32. It has also been demonstrated that insertion torque values is correlated with bone mineral density (BMD) of the receiving bone site, obtained by measuring TC or micro TC33-35, or by the sensitivity of the operator during the preparation of the surgical site36. Such measurements have therefore been considered valid instruments for the determination of the quality of the implant site, and can fore-

see good primary stability of the implant. With regard to measuring RFA, there are human studies that assert a correlation between the values of RFA (ISQ) measured at the time of implant placement and the values of insertion torque13-15. In other studies this correlation is confirmed for Hounsfield values of the implant site calculated using TC16,17. In the present study the ISQ value was measured about 60 days after implant positioning, while torque-in test and clinical bone density (bone type) was executed during implant placement. It was found that torque-in values were still statistically related to the values of ISQ (Fig.13: Ď = 0.669018 r2=0.4476 P=0.0174, statistically significant correlation), and this data could lead to hypothesize that such correlation could have been found at the time of implant placement similarly to the previously mentioned stud-

13-15 ies 10c . There are, however, literature reports that demonstrate a lack of correlation between the Torque-In test and ISQ values measured at implant insertion on cadavers18,19, in humans15,20,21 and in dogs22. In the first studies15 finding a correlation between RFA and cutting-torque, only the cutting-torque at the crest (first third of implant insertion) was correlated, while the overall insertion torque values were not related to the ISQ. Unlike the torque-in, the clinical bone density was not correlated to the values of ISQ measured two months later (Fig.7 Ď =-0.2477 r2=0.06133 P=0.4377); this may be questionable, but at the time of ISQ measurement a correlation between the values of ISQ and the percentage of bone volume (% BV/BT) was not found. The only humans study in the literature which found a correlation between the ISQ and the %BIC is from Scarano et al. 27 In this study27 seven implants were evaluated while in the present study 12 implants were analized. The reason for the different results from the present study could be due to the particular statistical analysis performed, or to the different healing time and number of implants. In that humans study 27 the implants were unloaded and retrieved after a healing period of six months. It is also possible that a longer healing period may allow for a more reliable results of the ISQ values accounting for the BIC %. In conclusion, the degree of osseointegration can be measured only through mechanical test of removal torque or through the histomorphometric analysis of the % of BIC, which are highly statistically related. The ISQ values were statistically significant related to the number of threads in contact to the compact bone

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and after two months from the implant placement such values were related to the torque-in values. Within the limits of the present study , these results can imply that the development of the osseointegration after a healing period of two months, as measured through the percentage of BIC, does not influence the values of ISQ. The ISQ depends, strongly on the quantity of cortical bone and does not account for the osseointegration in cancellous bone, it does not measure the % of osseointegration, but only the cortical anchorage. Further studies in humans implants are need to better clarify these aspects of the ISQ values. Acknowledgments This study was supported by grants from the no-profit foundation “Bio.C.R.A.” (Biomaterial Clinical and histological Research Association, Pescara, Italy). The authors would like to thank Giorgia Duranti for her help in the statistical calculations. There are no conflicts of interest among authors and commercial companies mentioned.

REFERENCE 1. Meredith N, Alleyne D, Cawley P. Quantitative determination of the stability of the implant-tissue interface using resonance frequency analysis. Clin Oral Implants Res 1996;7:261–267. 2. Glauser R, Meredith N. Diagnostic possibility for the evaluation of the implant stability (in German). Implantologie 2001;9:147– 159. 3. Meredith N, Book K, Friberg B, Jemt T, Sennerby L. Resonance frequency measurements of implant stability in vivo. A cross-sectional and longitudinal study of resonance frequency measurements on implants in the edentulous and partially dentate maxilla. Clin Oral Implant Res 1997;8:226-233. 4. Meredith N, Shagaldi F, Alleyne D, Sennerby L, Cawley P. The application of resonance frequency measurements to study the stability of titanium implants during healing in the rabbit tibia. Clin Oral Implant Res 1997;8:234–243. 5. Meredith N. Assesment of implant stability as a prognostic determinant. Int J Prosthodont 1998;11:491-501. 6. Gahleitner A, Monov G. Assessment of bone quality: techniques, procedures and limitations. In: Watzek G, editors. Implants in Qualitatively Compromised Bone, Chicago: Quintessence; 2004:55-66. 7. Pattijn V, Van Lierde C, Van der Perre G, Naert L, Vander Sloten J. The resonance frequency and mode shapes of dental implants: rigid body behaviour versus bending behaviour. A numerical approach. J Biomech 2006;39:939–947. 8. Veltri M, Balleri P, Ferrari M. Influence of transducer orientation on Osstell stability measurements of osseointegrated implants. Clin Implant Dent Relat Res 2007;9:60-4. 9. Atsumi M, Park SH, Wang HL. Methods used to assess implant stability: current status. Int J Oral Maxillofac Implants 2007;22:743-754.

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10. Pattijn V, Jaecques SVN, De Smet E, Muraru L, Van Lierde C, Van der Perre G, Naert I, Vander Sloten J. Resonance frequency analysis of implants in the guinea pig model: Influence of boundary conditions and orientation of the transducer. Med Eng Phys 2007;29:182-190. 11. Aparicio C, Lang NP, Rangert B. Validity and clinical significance of biomechanical testing of implant/bone interface. Clin Oral Impl Res 2006;17 (supp.):2–7. 12. Glauser R, Sennerby L, Meredith N, Rée A, Lundgren AK, Gottlow J, Hämmerle CH. Resonance frequency analysis of implants subjected to immediate or early functional occlusal loading. Successful vs. failing implants. Clin Oral Impl Res 2004;15:428– 434. 13. Turkyilmaz I. A comparison between insertion torque and resonance frequency in the assessment of torque capacity and primary stability of Branemark system implants. J Oral Rehabil 2006;33:754-759. 14. Alsaadi G, Quirynen M, Michiels K, Jacobs R, van Steenberghe D. A biomechanical assessment of the relation between the oral implant stability at insertion and subjective bone quality assessment. J Clin Periodontol 2007;34:359–366. 15. Friberg B, Sennerby L, Meredith N, Lekholm U. A comparison between cutting torque and resonance frequency measurements of maxillary implants. A 20- months clinical study. Int J Oral Maxillofac Surg 1999;28:297–303. 16. Turkyilmaz I, Tözüm TF, Tumer C, Ozbek EN. Assessment of correlation between computerized tomography values of the bone, and maximum torque and resonance frequency values at dental implant placement. J Oral Rehabil 2006;33:881-888. 17. Turkyilmaz I, Tumer C, Ozbek EN, Tozum TF. Relations between the bone density values from computerized tomography, and implant stability parameters: a clinical study of 230 regular platform implants. J Clin Periodontol 2007;34:716–722.

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18. Nkenke E, Hahn M, Weinzierl K, Radespiel-Troger M, Neukam FW, Engelke K. Implant stability and histomorphometry: a correlation study in human cadavers using stepped cylinder implants. Clin Oral Implants Res 2003 ;14:601–609.

26. Huwiler MA, Pjetursson BE, Bosshardt DD, Salvi GE, Lang NP. Resonance frequency analysis in relation to jawbone characteristics and during early healing of implant installation. Clin Oral Impl Res 2007;18(3):275-280.

19. Akkocaoglu M, Uysal S, Tekdemir I, Akca K, Cehreli MC. Implant design and intraosseous stability of immediately placed implants: a human cadaver study. Clin Oral Implants Res 2005;16:202-209.

27. Scarano A, Degidi M, Iezzi G, Petrone G, Piattelli A. Correlation between implant stability quotient and bone-implant contact: a retrospective histological and histomorphometrical study of seven titanium implants retrieved from humans. Clin Implant Dent Relat Res 2006;8:218-222.

20. Rabel A, Köhler SG, Schmidt-Westhausen AM. Clinical study on the primary stability of two dental implant systems with resonance frequency analysis. Clin Oral Invest 2007;11:257-265. 21. Cunha HA, Francischone CE, Filho HN, de Oliveira RC. A comparison between cutting torque and resonance frequency in the assessment of primary stability and final torque capacity of standard and TiUnite single-tooth implants under immediate loading. Int J Oral Maxillofac Implants 2004;19:578-85 22. Schliephake H, Sewing A, Aref A. Resonance frequency measurements of implant stability in the dog mandible: experimental comparison with histomorphometric data. Int J Oral Maxillofac Surg 2006;35:941-946. 23. Rocci A, Martignoni M, Burgos PM, Gottlow J, Sennerby L. Histology of retrieved immediately and early loaded oxidized implants:light microscopic observations after 5 to 9 months of loading in the posterior mandibole. Clin Implant Dent Relat Res 2003;5(suppl.1):88-98. 24. Ito Y, Sato D, Yoneda S, Ito D, Kondo H, Kasugai S. Relevance of resonance frequency analysis to evaluate dental implant stability: simulation and histomorphometrical animal experiments. Clin Oral Implants Res 2008;19:9-14. 25. Miyamoto I, Tsuboi Y, Wada E, Suwa H, Iizuka T. Influence of cortical bone thickness and implant length on implant stability at the time of surgery-clinical, prospective, biomechanical, and imaging study. Bone 2005;37:776–780.

28. Misch CE. Density of bone: effect of treatment planning, surgical approach, and healing. In Misch CE, editors. Contemporary Implant Dentistry, St Louis: Mosby Year-Book, 1993:469-485.

34. Homolka P, Beer A, Birkfellner W, Nowotny R, Gahleitner A, Tschabitscher. M, Bergmann H. Bone mineral density measurement with dental quantitative CT prior to dental implant placement in cadaver mandibles: pilot study. Radiology 2002;224:247-252. 35. Rebaudi A, Koller B, Laib A, Trisi P. Microcomputed tomographic analysis of the peri-implant bone. Int J Periodontics Restorative Dent 2004;24:316-325. 36. Trisi P, Rao W. Bone classification: clinical-histomorphometric comparison. Clin Oral Implants Res 1999;10:1-7.

29. Sennerby L, Thomsen P, Ericson LE. A morphometric and biomechanic comparison of titanium implants inserted in rabbit cortical and cancellous bone. Int J Oral Maxillofac Implants 1992;7(1):62-71 30. Lachmann S, Jäger B, Axmann D, Gomez-Roman G, Groten M, Weber H. Resonance frequency analysis and camping capacity assessment. Part 1: an in vitro study on measurement reliability and a method of comparison in the determination of primary dental implant stability. Clin Oral Implants Res 2006;17(1):75-79. 31. Branemark R, Ohrnell LO, Nilsson P, Thomsen P. Biomechanical characterization of osseointegration during healing: an experimental in vivo study in the rat. Biomaterials 1997;18:969-978. 32. Trisi P, Todisco M, Consolo U, Travaglino D. High vs. low implant insertion torque. A histologic and biomechanical in vivo study. (In press) 33. Beer A, Gahleitner A, Holm A, Tschabitscher M, Homolka P. Correlation of insertion torques with bone mineral density from dental quantitative CT in the mandible. Clin Oral Implants Res 2003;14:616620.

Volume 1 - Number 2 - 2010

BioCRA

Original article

Hydraulic sinus lift: a new method proposal Mirko Andreasi Bassi DDS PhD 1* Michele Antonio Lopez MDS1

In the present article the authors propose a new method for the hydraulic Shneiderian membrane elevation useful in the maxillary sinus lift with crestal approach. Some purposely realized instruments, a syringe equipped with a micrometric piston control and a dispenser, were used for injecting in the sub-shneiderian space, paste-like graft material (ultramicronized hydroxyapatite dispersed in watery matrix) with calibrated way and known volumes. The advantages of the proposed method are represented by the conspicuous membrane elevations obtainable with operating time reduction because the hydraulic detachment of the Shneiderian and the sub-antral graft filling occur simultaneously. This technique, which requires a brief learning period, can be placed side by side with the most common methods of maxillary sinus lift with crestal approach. (J Osteol Biomat 2010; 1:93-101)

Key words: maxillary sinus lift, hydraulic sinus lift, sinus floor augmentation.

Private practice, Rome,Italy

Corresponding authors: *Mirko Andreasi Bassi Via Lucio Elio Seiano, 15-Rome, Italy Tel. 0039-06-7480736 Fax. 0039-06-45426826 e-mail: m.andreasi@tiscali.it

INTRODUCTION Techniques of the maxillary sinus floor elevation, by lateral window approach or crestal approach, are widely employed in clinical outpatient management of lateral-posterior maxillary atrophy. The substantial difference between lateral window and crestal approaches, is that the first being a direct technique allows the visualization of the maxillary sinus membrane when its elevation occur, therefore it facilitates management of complications, such as membrane lacerations1. Conversely this method requires a lateral antrostomy which, while it improves both optical and instrumental access to the preternatural sub-shneiderian space, on the other hand, discontinuing the lateral wall of the maxillary sinus, compromises its osteogenic contribution to graft consolidation and therefore the membraneâ&#x20AC;&#x2122;s detachment must necessarily be extended to the medial sinus wall2,3. Moreover the lateral antrostomy creates a prerequisite for a competitive colonization between bone and connective tissue of the sub-antral space, which could be countered only through the use of barrier membrane4,5. Although this technique is widely documented in the literature and supported by several longitudinal studies which attest to an average implant survival

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Figure 1. The Hydro-mab device provided of a micrometric piston control.

Figure 2. The dispenser ML Injector available in 2 shapes and 4 sizes.

Figure 3. The surgical steel needle, provided of Luer-Lock link complementary to the disposable syringe, which allows to connect Hydro-mab to ML Injector.

Figure 4. The lateral holes of the ML Injector allow an uniform and radial distribution of the grafting material (Ostim) into the sub-shneiderian space.

rate close to 92%, as a counterpart, it has greater morbidity than techniques using the crestal approach 6,7,8. Moreover the lateral window sinus lift implies executions of a large mucous-periostal flap that inevitably affects post-operative recovery of the patients, while in the crestal approach method the bone surface involved is minimal, enough to allow in selected cases to proceed flapless9. The crestal approach instead is a closed sinus elevation generally implies a partial lifting of the maxillary sinus floor and could also be multiple, depending on the extent of edentulous ridge and the number of implants that have to be placed 10. The partial elevation of the mucous membrane through the crestal approach limits optical and instrumental access and requires a more prudent

operatorâ&#x20AC;&#x2122;s approach, thus limiting the extension of membranes elevation and the length of the implants used 11,12. Over the years several authors have proposed variations of this method and have contributed to understanding and spreading this method among the operators13,14,15. The partial maxillary sinus lift through a crestal approach is a very predictable technique, when the height of residual bone ridge is at least 5mm, in order to achieve primary stability of the implant 16, for the lower amount of residual bone ridge there are various techniques of maxillary sinus floor elevation that prepare the implant site for deferred placement 15,17. While in the past the indications for these two techniques were distinct, and the lateral window approach was

Journal of Osteology and Biomaterials

preferred in the majority of circumstances 18, over the years, we are assisting to a gradual increase of indications for maxillary sinus lift via the crestal approach, with overlapping areas of competence of these two techniques, through the development of new operational methods and new implant surfaces 12,14-16. In this paper a new method for sinus lift via a crestal approach is proposed. Such technique utilizes a controlled mechanical movement and hydraulic pressure together to elevate the Shneiderian membrane. Hydraulic detachment of the sinus membrane We can distinguish three main phases in the operating procedure of partial elevation of the maxillary sinus floor: 1. Preparation of the implant tunnel 2. Discontinuity of the sinus floor 3. Detachment of the Shneiderian membrane These phases were largely discussed by various authors over the years and modified to create numerous techniques available today. The first and the second phase are feasible using osteotomes, drills or piezoelectric surgery unit (ultrasonic bone surgery). Osteotomic techniques are widely described in the literature and should be preferred for reduced bone density cases (D3, D4); on the other hand these techniques are particularly traumatic for the patient because of the pounding procedure3,10,11,14-17,19. The procedures that use drills or ultrasound have been proposed recently and have the advantage of being much less traumatic 13,20. The cortical sinus floor bone fracture or consumption by piezoelectric surgery

Andreasi Bassi M. et al.

Figure 5. Edentulous site on 2.6, occlusal view.

Figure 6. Pre-operatory radiograph of the case.

Figure 7. According to the flapless technique the gingival punch excision is performed, and the gingival punch is removed, uncovering the bony surface.

Figure 8. After drill preparation of the implant site the sinus floor is abraded by means piezoelectric surgery.

Figure 9. A collagen sponge wetted with saline solution is applied into the bony tunnel and gently condensed.

Figure 10. The first detecting of sinus membrane is carried out by means collagen sponge.

and the subsequent displacement of the sinus membrane are the key steps of the crestal sinus lift approach10,12 because they are unsupported by direct vision; apart from those procedures which could be performed under endoscopic control. Endoscopic procedure, despite the undeniable operation advantages, requires favourable anatomic conditions (pneumatised maxillary sinus, edentulism distal to bicuspid teeth) and the presence of a second

operator, which severely limit, its use in clinical practice 3,9,21,24. The detachment of the Shneiderian membrane can be performed ether by hand instruments3 or indirectly through the graft material; most of the techniques use this latter approach 11,12,17,19. The reason for this is that the graft material is usually soaked in blood, an incompressible fluid. Pascalâ&#x20AC;&#x2122;s principle states that in fact the pressure exerted on a portion of a liquid is transmitted

unaltered in any portion of the liquid itself 22. In 1994 Summers stated that the pressure exerted by osteotomes on the graft material mixed with blood resulted in the indirect detachment of the sinus mucosa 11,19. It is therefore conceptually correct to assert that the majority of the methods of sinus lift via crestal approach (although not mentioned in the literature) use hydraulic pressure. On the other hand it should be mentioned that the force exerted by graft material compaction can not be easily controlled bringing sometimes detrimental effects to the integrity of the sinus membrane 3. If the shape of elevation obtained in a single membrane elevation via crestal approach is analyzed, it has a domed appearance with prevalent vertical to horizontal diameter, whereas in the case of multiple contiguous sinus lift this discrepancy between these two diameters is much smaller 10. This phenomena is attributable to the use of bone compactors that are forced into sub-shneiderian space subjecting the sinus mucous membrane to dangerous mechanical stretching by vertical forces, its effects being less evident in the multiple contiguous lift because the force is distributed over the larger area. Also for this reason it is believed that the presence of oblique sinus floors would facilitate sinus floor elevation, not only vertically but also laterally 10. The methods currently in use are therefore not predictable and reproducible if elevations greater than 5mm are needed and often better results reported in the literature are operator-dependant 10. The membraneâ&#x20AC;&#x2122;s integrity could also be compromised by placement of graft

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Figure 11. The ML Injector is screwed into the bony tunnel.

Figure 12. Control radiograph for evaluating the position of the ML Injector, the hemispherical head and the radial holes must to be placed into the sub-antral space.

Figure 13. 0,7 ml of grafting material (Ostim) is injected, in a controlled manner, into the subshneiderian space.

Figure 14. Control radiograph for evaluating the obtained elevation of the sinus membrane.

Figure 15. After grafting material placement the ML injector is removed.

Figure 16. The bony tunnel is enlarged.

Figure 17. The implant is positioned .

Figure 18. The healing screw is applied, occlusal view.

Journal of Osteology and Biomaterials

material of rough consistency. The roughness of the particles can in fact cause lesions to the Shneiderian membrane during graft insertion, especially if it occurs abruptly 3, thatâ&#x20AC;&#x2122;s why some operators suggest the membrane detachment through the use of collagen sponges 12. Some authors have recently proposed techniques of sinus lift that utilize hydraulic pressure for the relocation of the sinus membrane and these techniques have alternative approaches to those currently in use. Among these Sotirakis and Goushor, in 2005, proposed injection of saline solution with a disposable syringe in the sub-shneiderian space. When the desired elevation is obtained the syringe is removed from the bone, the fluid comes spontaneously through the osteotomy site into the oral cavity25. Kfir et. Al. also proposed in 2006 the use of an instrument specifically designed for the mucosal detachment of the maxillary sinus membrane through the use of a catheter inflated in the sub-shneiderian space, using a radiopaque liquid26. Both methods proposed include preliminary detachment of Shneiderâ&#x20AC;&#x2122;s membrane by liquid injection in the sub-shneiderian space, followed by itâ&#x20AC;&#x2122;s spontaneous expulsion or aspiration, and finally placement of graft material in the space thus created. Although effective, these methods imply elongation of the operating procedure, and it is conceptually easier to use graft material in a fluid state in order to elevate the sinus membrane, by exerting hydraulic pressure on it, and filling the sub-antral space in just one step. Moreover, the methods described

Andreasi Bassi M. et al.

Figure 19. Radiograph immediately after surgical procedure.

Figure 20. The prosthetic finalization of the case, 6 months post-operatively, vestibular view.

Figure 21. Radiographic control image 1,5 years post-operatively.

above use conventional disposable syringes lacking fine control on the piston progression which is dependant on individual operator’s sensitivity. The Description of Methods It is important to say that this technique, working just on the phase of the elevation of the sinus mucous membrane and on the insertion of graft material, can be used together with the common methods adopted for the preparation of the implant tunnel and the discontinuation of the sinus floor, therefore the operator will not have to change his surgical protocol but rather will increase his know-how. The authors called this technique Hydraulic Sinus Lift (HySiLift). The surgical instruments appositely used for this technique are mainly 3: a titanium syringe (Hydro-mab, FMD, Rome, Italy) (fig.1) with a micrometric control piston on which is possible to apply 5 ml LuerLock disposable plastic syringes (Pick Indolor, Artsana, Grandate (CO), Italy); a threaded surgical steel dispenser (ML Injector, FMD, Rome, Italy) available in 2 shapes (cylindrical and conical) and 4 measures (2 cylindrical: ø 3,2 and 4,0mm; 2 conical: ø 2,8-4,0 and ø 3,54,6mm) (fig. 2); a surgical steel needle

with a Luer-Lock link which allows to connect the two instruments described above (fig. 3). The disposable syringes can be filled with the desired quantity of grafting material which, basing on our experience, is represented by nanocrystal hydroxiapatite in an aqueous matrix (Ostim, Heraeus-Kulzer, Hanau, Germany) 27-29. The ML injector’s semi spherical extremity allows the instrument to proceed for just 3 mm in the sub-shneiderian space without damaging the upper mucosa, while the lateral open sides allow a radial, uniform distribution of Ostim (fig. 4) which, thanks to its pasty consistency, generates a dome shape space in the future implant site. The threaded portion of the dispenser has a 6mm length, therefore its use is advised for crests with a thickness between 3 and 6mm, in order to guarantee a sufficient stability of the instruments during the process of injection. Although it is theoretically possible to stabilize the dispenser also on residual crests with a 2mm thickness, provided that the bone has a good density (≥ D2) 6. The initial membrane detachment, before the injection of Ostim, can be obtained directly with the semi spherical apex portion of the ML injector, or indirectly with the use of a collagen sponge

wetted with physiological solution (Condress, Abiogen Pharma, Ospedaletto (PI), Italy) and condensed smoothly with the use of a concave chisel, with the same diameter of the implant tunnel and with a 2mm shorter depth stop in comparison with the crest thickness (FAL-06-001, FMD, Rome, Italy )12. The authors used this technique together with the most commonly used methods of crestal sinus lifting, preferring the less traumatic approaches with the use of drills or ultrasound 13,20. The choice of the dispenser depended on the diameter and the shape of the implant that we want to insert after the sinus elevation. Although, where the crestal thickness is less than 4mm, this technique can be adopted without the subsequent insertion of the implant, as the primary stability could not be guaranteed. In this case, the technique foresees the injection of the biomaterial and after 6-8 months, when the graft has consolidated, it is possible to proceed with the implant insertion. The implant tunnel will be 0,5mm smaller in case of cylindrical ML Injector, while with the use of a conical injector the diameter of the implant tunnel must be as large as the apical diameter of the chosen instrument (ø2,8 or 3,5mm).

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After the implant tunnel preparation involved the sinus floor, the ML Injector can be screwed manually or by contraangle handpiece, letting its apical portion proceed inside the sub-shneiderian space (3mm) so that the threaded part of the instrument will stop the dispenser on the implant tunnel walls. At this step a radiography is advised, in order to ensure that the apical portion of the dispenser has gone through the implant tunnel. In case of a single elevation, it is advised to inject Ostim inside the dispenser before, in order to avoid the risk of introducing air into the sub-antral space. This is not necessary for multiple contiguous elevations, as the implant tunnels preparation and the dispenser positioning will be done simultaneously; therefore, as the injection of biomaterial will be done with the use of one ML Injector per time, air and blood will go through the one left free. After the positioning of ML Injector, the needle will be connected, together with the syringe and the material will be injected progressively. The link between needle and dispenser is ensured by a TeflonÂŽ o-ring positioned on its distal extremity. The time necessary to inject 1 ml of biomaterial is generally 5 minutes, but in most of the operative situations, which foresee the treatment of a single site, the injected volume is between 0,5 and 0,7 ml. One complete revolution of the micrometric screw is equivalent to a 0,5mm progression of the piston; usually it is possible to proceed with rotations of Âź of revolution of the micrometric screw (125Îźm) followed by a pause of 3 seconds. After the injection, the Hydro-mab can be unhooked and it is

Journal of Osteology and Biomaterials

possible to have a radiography to check the elevation obtained. If the elevation is not sufficient, there is the possibility to increase it by the injection of more biomaterial, with the same method described above. Once the ML Injector is unhooked, if the residual crest has a height of at least 4mm, it is possible to insert the implant which should be 0,40,5mm larger than the diameter of the dispenser employed. Compatibly with the horizontal dimensions of the osseous crest, the implant tunnel can be modified, if the operator reckons it is required, with osteotomes or drills, in order to make it more suitable for the implant chosen. If the implant is not inserted at the same time, the space left by the dispenser can be fulfilled with Ostim or with calcium sulphate (Surgiplaster P30, Class Implant, Rome, Italy) reminding to cover the crestal antrostomy with a bio-absorbable membrane. For a better understanding of the above described method a clinical case is described. (fig.5-21).

Andreasi Bassi M. et al.

DISCUSSION The present method is versatile, as it can be used in both flap and flapless operations, and it allows to obtain an increase of the sub-shneiderian volume in the 3 dimensions of space. Besides, the micrometric control on the progression of the piston, enables a precise and progressive insertion of the biomaterial, which simultaneously elevates the sinus mucosa and fills the sub-shneiderian space. Based on our over two years old clinical experiences, there have been no intra-operatory lacerations of the sinus mucosa, and there was no dispersion of biomaterial inside the maxillary sinus and, consequently, no the failure in the osseous regeneration around the implant apex. However the pasty consistency of the Ostim enables theoretically to drain dispersed biomaterial by mucus-ciliary transport mechanism. In addition we can assert that the graft is directly inserted into the implant site, therefore there is less biomaterial manipulation, thus reducing the working time and, most of all, the risks of contamination. HySiLift technique has the advantage of being user friendly, with a short learning curve. During its insertion, Ostim reveals to be a weakly radio-opaque material while, with the passing of the weeks, there is a progressive increase in the radio-opacity, most likely due to bone tissue mineralization. Also the volume of the graft does not undergo substantial modifications during bone rearrangement. On the other hand, during the insertion of the biomaterial, the Shneiderianâ&#x20AC;&#x2122;s membrane detachment follows the directions of less resistance, in pres-

ence of adhesions due to previous tooth extractions or septum, changing the detachment pattern. In addition the membrane detachment of oblique sinus walls, will follow both a vertical and a horizontal direction, along the portion of the wall which is more declivous. Anyway, the situation described above is already known and documented in the traditional techniques used for the maxillary sinus lift with crestal approach10,23,24. In our clinical experience we studied 20 cases in all (14 women and 6 men; age range: 32â&#x20AC;&#x201C;67 years; average age: 49 years). The average preoperative residual alveolar ridge height was 5 mm, and the average postoperative height elevation into the sinus was 8 mm. There were 26 implants placed. In 14 cases there was a single implant placed, with 6 cases having 2 implants in the grafted site. All cases have been loaded from 6 months to as long as 8 months. There were no implant loss in any of the cases.

CONCLUSION The HySiLyft allows the hydraulic elevation of the maxillary sinusÂ mucosa and at the same time the filling of the subshneiderian space with the grafting material. Such a method can be used with the most common implant tunnel preparation techniques adopted in the management of maxillary sinus floor elevation, thus offering the clinician the advantages of the maxillary sinus lift with lateral window approach, together with the simplicity of the crestal approach. The employment of the above method requires: a short learning curve; a reduced invasivity; a higher precision and consequent reproducibility. At present a longitudinal clinical study is being developed as well as a study finalized to the histomorphometric evaluation of the newly grown bone tissue by this technique, in the case of future sites development. In the near future it is advisable to employ such a technique also with osteoinductive inject able graft materials. ACKNOWLEDGMENTS Thanks to Dr. Micael German (MDS) and to Mr. Massimo Maiolini for their invaluable contribution.

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REFERENCE 1. Jensen OT, Shulman LB, Block MS, Iacono VJ. Report of the Sinus Consensus Conference of 1996. Int J Oral Maxillofac Implants 1998;Suppl:11-45. 2. Tatum OH Jr. Maxillary and sinus implant reconstructions. Dent Clin North Am 1986; 30: 207-229. 3. Nkenke E, Schlegel A, Schultze-Mosgay S, Neukam FW, Witfang J. The endoscopically controlled osteotome sinus floor elevation:a preliminary prospective study. Int J Oral Maxillofac Implants 2002;17:557-566. 4. Tarnow DP, Wallace SS, Froum SJ, Rohrer MD, Cho SC. Histologic and clinical comparison of bilateral sinus floor elevations with and without barrier membrane placement in 12 patients: Part 3 and ongoing prospective study. Int J Periodontics Restorative Dent 2000;20:117-25. 5. Tawil G, Mawla M. Sinus floor elevation using a bovine bone mineral (Bio-Oss) with or without the concomitant use of a bilayered collagen barrier (Bio-Gide): a clinical report of immediate and delayed implant placement. Int J Oral Maxillofac Implants 2001;16: 713-21. 6. Del Fabbro M, Testori T, Francetti L, Weinstein R. Systematic review of survival rates for implants placed in the grafted maxillary sinus. Int J Periodontics Restorative Dent 2004;24:565-577. 7. Zitzmann NU, Sch채rer P. Sinus elevation procedures in the resorbed posterior maxilla. Comparison of the crestal and lateral approaches. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998; 85:8-17. 8. Ziccardi VB, Betts NJ. Complications of maxillary sinus augmentation. In: Jensen OT. The Sinus Bone Graft. Chicago. Quintessence Books 1999: 201-208. 9. Engelke W, Capobianco M. Flapless sinus floor augmentation using endoscopy combined with CT scan-designed surgical templates: method and report of 6 consecutive cases. Int J Oral Maxillofac Implants 2005;20(6):891-7.

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10. Cavicchia F, Bravi F, Petrelli G. Localized augmentation of the maxillary sinus floor through a coronal approach for the placement of implants. Int. J Periodontics Restorative Dent 2001;21:75-485. 11. Summers RB. The osteotome technique. Part 3-Less invasive methods of elevating the sinus floor. Compendium 1994;15: 698-708. 12. Deporter D, Todescan R, Caudry S. Simplifying management of the posterior maxilla using short, porous-surfaces dental implants and simultaneous indirect sinus elevation. Int J Periodontics Restorative Dent. 2000;20:477-485. 13. Cosci F, Luccioli M. A new sinus lift technique in conjunction with placement of 256 implants: A 6-year retrospective study. Imp Dent 2000;9: 363-368. 14. Fugazzotto PA. Sinus floor augmentation at the time of maxillary molar extraction: technique and report of preliminary results. Int J Oral Maxillofac Implants 1999;14:536-542. 15. Fugazzotto PA. The modified trephine/osteotome sinus augmentation technique: technical considerations and discussion of indications. Imp Dent 2001;110:259-264. 16. Rosen PS, Summers R, Mellado JR, Saikin LM, Shanaman RH, Marks MH, Fugazzotto PA. The bone-added osteotome sinus floor elevation procedure: multicenter retrospective report of consecutively treated patients. Int J Oral Maxillofac Implants 1999; 14:853-858. 17. Summers RB. The osteotome technique: Part 4 Future site development. Compendium 1995;16: 1090-09. 18. Misch CE. Maxilllary sinus augmentation for endosteal implants: organized alternative treatment plans. Int Oral Implantol 1987;4:49-58. 19. Summers RB. A new concept in maxillary implant surgery: the osteotome technique. Compendium 1994;15:152-160.

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20. González-García A, Diniz-Freitas M, Somoza-Martín M, García-García A. Ultrasonic osteotomy in oral surgery and implantology. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;108(3):360-7. 21. Timmenga NM, Raghoebar GM, Van Weissenbruch R, Vissink A. Maxillary sinus floor elevation surgery. A clinical, radiographic and endoscopic evaluation. Clin Oral Implant Res 2003;14:322-28.

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29. Smeets R, Grosjean MB, Jelitte G, Heiland M, Kasaj A, Riediger D, Yildirim M, Spiekermann H, Maciejewski O. Hydroxyapatite bone substitute (Ostim) in sinus floor elevation. Maxillary sinus floor augmentation: bone regeneration by means of a nanocrystalline in-phase hydroxyapatite (Ostim). Schweiz Monatsschr Zahnmed 2008;118(3):203-12.

22. Wang DL, Li MJ, He T, Zheng Z, Duan X, Zheng YJ. Development of the remote hydraulic pressure control injection. Chinese journal of medical instrumentation- Zhongguo Yi Liao Qi Xie Za Zhi 2009;33(1):34-5. 23. Reiser GM, Rabinowitz D, Bruno J, Damoulis PD, Griffin TJ. Evaluation of maxillary sinus membrane response following elevation with the crestal osteotome technique in human cadavers. Int J Oral Maxillofac Implants 2001;16:833-840. 24. Berengo M, Sivolella S, Majzoub Z, Cordioli GP. Endoscopic evaluation of the bone-added osteotome sinus floor elevation procedure. Int J Oral Maxillofac Surg 2004; 33(2):189-94. 25. Sotirakis EG, Gonshor A. Elevation of the maxillary sinus floor with hydraulic pressure. J Oral Implantol. 2005;31(4):197204. 26. Kfir E, Kfir V, Mijiritsky E, Rafaeloff R, Kaluski E. Minimally invasive antral membrane balloon elevation followed by maxillary bone augmentation and implant fixation. J Oral Implantol 2006;32(1):26-33. 27. Busenlechner D, Huber CD, Vasak C, Dobsak A, Gruber R, Watzek G. Sinus augmentation analysis revised: the gradient of graft consolidation. Clin Oral Implants Res 2009;20(10):1078-83. 28. Carmagnola D, Abati S, Celestino S, Chiapasco M, Bosshardt D, Lang NP. Oral implants placed in bone defects treated with Bio-Oss, Ostim-Paste or PerioGlas: an experimental study in the rabbit tibiae. Clin Oral Implants Res 2008;19(12):1246-53.

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Original article

Ultra-short porous implants in the posterior maxilla: a 4-year report from a prospective study Michele Perelli DDS1* Giuseppe Corrente MD DDS1,2 Roberto Abundo MD DDS1,2 Luca Savio DDS1 Alessandro Bermond des Ambrois MD1 Introduction: This ongoing prospective study provides data concerning a 48-month survival rate of ultra-short ( 5-mm-long) sintered porous-surfaced (SPS) implants placed in the posterior maxilla with 2-6 mm of initial bone height in 27 patients. Materials and Methods: 27 implants were inserted to replace maxillary premolar and molar teeth. 23 were inserted with an available bone height less than 4 mm and they required a sinus elevation by means of osteotome with the addition of a xenograft. After a submerged primary healing period, all implants were loaded, 19 with single crowns and 8 splinted to the closer adjacent fixture. Results: At the end of the follow-up period the cumulative survival rate was 92.1 %. Two implants failed. Conclusion: the results of this case series report suggest that the use of ultrashort porous surfaced press-fit implants showed high predictability and safety in the treatment of resorbed posterior maxilla. (J Osteol Biomat 2010; 1:103-107)

Key Words: short implants, posterior maxilla, osteotome technique.

Private practice, Torino, Italy. Adjunct professor, Department of Periodontics (Chair: Dr. J.P.Fiorellini), University of Pennsylvania, Philadelphia (USA)

1 2

Corresponding author: *Michele Perelli Corso Sicilia 51 – 10133 Torino (Italy) Phone:+39 011 6615452 Fax: +39 011 6618378 e-mail: micperelli@yahoo.it, info@sicor-corsi.com

Introduction Replacement of teeth lost from trauma or periodontitis with implant-supported prosthesis has been well documented and widely accepted and is often the treatment of choice in partially edentulous patients.1-3 In the posterior maxilla managing available bone height and width is the limiting factor influencing implant diameter and length. To solve these problems, different bone augmentation techniques have been described and related implant survival rates investigated.4,5 Among these techniques maxillary sinus floor elevation with lateral approach shows positive long–term results6-8, but an increased morbidity due to complications, infection risks, longer treatment duration and costs9,10. Emmerich et al. reviewed the literature concerning a less invasive technique, the osteotome sinus floor elevation with a crestal approach and reported similar clinical outcomes.11 The use of short implants has been proposed by several authors as an alternative option to restore posterior jaws with good predictability and safety for the patient. Renouard and Nisand redefined “short implant” as an implant with the designed intrabony length less 8 mm. Their systematic review shows that short implants perform better when

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Table 1. Implants sites.

a roughened surface is present and a modified surgical approach is applied to increase initial implant stability.12 The development of new implant surfaces and designs allows to increase the bone to implant contact area, providing successful long-term results.13 Sintered porous surface press-fit implants (Innova, Lifesciences, Toronto, Ontario, Canada) demonstrated to perform well with short lengths because a threedimensional bone interlock is achieved whereby bony ingrowth occurs among the macroporous surface layer.14 As shown in various clinical studies, these implants produced a positive results both in the short term and the long term periods.15-18 The objective of this preliminary clinical study is to evaluate the use of ultra-short sintered porous surface implants in the prosthetic rehabilitation of edentulous sites in the posterior maxilla associated, if required, with a local indirect sinus floor elevation by means of osteotomes according to the technique described by Deporter et al.19 MATERIALS AND METHODS 27 patients took part in the study ( 14 males and 13 females). None of them

Journal of Osteology and Biomaterials

Table 2. Pre-operative basal bone height.

presented with any systemic disease or contraindications to implant therapy. Non smokers and moderate smokers (less than 10 cigarettes/day) were included in the study. 27 press-fit sintered porous-surfaced implants (5 mm in length and 5 mm in diameter) were placed by four different surgeons. All implants were placed in the maxillary premolar/molar region and where required a localized sinus lift with osteotomes and addition of de-proteinized bovine bone (Bio-Oss, Geistlich Pharma, Wolhusen, Switzerland) was applied. The initial measurement of available bone (residual crestal height) were recorded with digital radiography taken with a paralleling technique by means of Rinn’s holder. Prior to implant placement a full-thickness muco-periosteal flap was elevated. The osteotomy sites were prepared according to the type of bone found: where drills were used in type 2 and 3 and osteotomes hand instruments in type 4 20. Care was taken when preparing the site with rotary instruments in order to achieve a good primary stability considering that on such a short length a little discrepancy between the osteotomy and the implant would lead to a

loss of primary stability. After preparing the osteotomy a “trial fit” gauge was used to verify the osteotomy site17,21. Once the gauge was fully submerged with a tight press-fit the surgeon had the certainty that the implant would achieve the desired primary stability. All implants but one where placed following a two-stage approach with the 1 mm smooth collar placed at the crestal level. The healing period was 4 months for routine surgery and at least 6 months in cases where a localized sinus lift was carried out. The implants were then loaded with single crowns, cemented or directly screwed depending on the angulation of the fixture/ crown present or splinted with other adjacent implants. Clinical and radiographic examinations where scheduled at 1,6,12 months after crown insertion, then yearly. The data collected at the end of this observation period was statistically analyzed by means of Kaplan-Meier’s life table analysis in order to provide a cumulative survival rate.22

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Table 3. Universal life table analisys.

Interval Start Time 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48

Number Entering Interval 27 27 27 27 26 26 19 17 16 16 13 9 8 7 6 3 2

Number Withdrawing during Interval 0 0 0 0 0 6 2 1 0 3 4 1 1 1 3 1 2

Number Exposed to Risk 27 27 27 27 26 23 18 16,5 16 14,5 11 8,5 7,5 6,5 4,5 2,5 1

RESULTS There were no surgical complications and all implants integrated successfully. Two implants failed after loading, respectively 11 and 16 months after the prosthetic rehabilitation. Both the patients were moderate smokers (less than 10 cigarettes/day) and showed a progressive bone loss around implants. Tables 1 to 3 show the implants sites (sequentially numbered), the pre-operative basal bone height present at each site and the survival rate table respectively. (Tab. 1-3) The mean loading period for the implants reported in the study was 28.7 months and more than 60% of implants were observed for a longer period. 6 implants were placed in the second bicuspid site, 16 implants were used to replace the first molar and 5 the second molar (Figs. 1-2). The pre-surgical basal bone height was less 4 mm in 23 cases and consequently a localized sinus lift with addition of demineralized bovine bone was required at the time of implant placement. The overall mean height was 3.5 mm and all implants were inserted with their smooth collar

Number of Terminal Events 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0

Proportion Terminating 0 0 0 0,037 0 0,043 0 0 0 0 0 0 0 0 0 0 0

Proportion Surviving 1 1 1 0,963 1 0,957 1 1 1 1 1 1 1 1 1 1 1

Cumulative Proportion Surviving at End of Interval 1 1 1 0,963 0,963 0,921 0,921 0,921 0,921 0,921 0,921 0,921 0,921 0,921 0,921 0,921 0,921

Std. Error of Cumulative Proportion Surviving at End of Interval 0 0 0 0,036 0,036 0,054 0,054 0,054 0,054 0,054 0,054 0,054 0,054 0,054 0,054 0,054 0,054

Probability Density 0 0 0 0,012 0 0,014 0 0 0 0 0 0 0 0 0 0 0

Std. Error of Std. Error of Probability Hazard Rate Hazard Rate Density 0 0 0 0 0 0 0 0 0 0,012 0,013 0,013 0 0 0 0,014 0,015 0,015 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Case 1.

Figure 1a. Tooth 1.6 presents with periodontal problems and furcations involvement. An extraction with a socket preservation with de-proteinized bovine bone and fibrin sealant is planned.

Figure 1b. 6 months following the regenerative procedure healing: a reduced crestal height is present.

Figure 1c. A porous implant of 5 mm in length and 5 mm in diameter is placed with a localized sinus lift with crestal approach by means of osteotomes with de-proteinized bovine bone. A two stage surgical approach is chosen.

Figure 1d. Following a 6 month healing period, the implant integrated and was loaded with temporary crown. After 3 years a well-manteined crestal profile can be noticed.

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at the crestal level. From the statistical analysis at 48 month SR of 92.1 % (95 % confidence interval between 0.81 â&#x20AC;&#x201C; 0.98) was shown (Fig. 3). DISCUSSION In this ongoing prospective study an ultra-short press-fit implant with a porous surface of 4 mm and a smooth collar of 1 mm was used. Among various options, the sintered-porous surface allows for a mechanical bone interlocking enabling to resist tensile and axial forces distributed along the implant. Due to such bony ingrowth between the pores, more binding between implant and the bone is provided compared to that of conventional implant surfaces.23-27 This occurs also in poor bone quality, as in the posterior maxilla. Evaluating the clinical and anatomical situations described in this study, where most sites had pre-surgical basal bone height less than 4 mm, conventionally there is an indication for a sinus lift with lateral approach when using threaded implants. Reviews of the literature on this surgical approach have shown a cumulative survival rate of 91.8 % / 91.49 % for implants placed in the augmented sinus4,5, while other authors refer unacceptably high failure rates when placing threaded implants with such pre-operative basal bone height with an osteotome technique31,32. CONCLUSION Taking into account that the observation period of this study is 48 months, the 92.1 % cumulative survival rate of sintered porous surface ultra short implants placed in the posterior maxilla shows a possible valid alternative to the above mentioned surgical approach.

Journal of Osteology and Biomaterials

Case 2.

Figure 2a. Pre-operative radiograph: following severe periodontal disease an extensive bone defect is present after extraction of tooth 1.6.

Figure 2b. Bone regeneration with de-proteinized bovine bone and fibrin sealant is performed.

Figure 2c. 8 months after later, an implant of 5 mm in length and 5 mm in diameter can be placed in the augmented site. A localized sinus lift with no added biomaterial is performed and the implant is left to heal submerged.

Figure 2d. 2 year radiographic control. The complete recorticalization of the sinus floor can be observed; an abutment platform switching technique was used.

The osteotome crestal approach technique allows for a less invasive surgery, a reduced surgical healing period and a reduced post-surgical discomfort for the patients as well.11,28,29 In all cases in the study no vertigo syndrome was observed or reported by the patients.30 The results of this 48 month report from a prospective study were consistent with those of a meta-analysis of osteotome sinus floor elevation and with those of the 1 to 8 years study by Deporter et al. on the same type of implant 20. A longer observation period and a

greater number of patients are required to confirm the encouraging results in terms of predictability and safety of these 5 mm x 5 mm porous surfaced press-fit implants positioned in the posterior maxilla. ACKNOWLEDGEMENTS The authors wish to thank Prof. Giuseppe Migliaretti â&#x20AC;&#x201C; Department of Public Health and Microbiology, University of Torino- for the statistical analysis. The authors report no conflicts of interest related to this study.

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REFERENCE 1. Jemt T, Lekholm U, Adell R. Osseointegrated implants in the treatment of partially edentulous patients: A preliminary study on 876 consecutively placed fixtures. Int J Oral Maxillofac Implants 1989;4:211-217. 2. Branemark PI, Svensson B, van Steenberghe D. Ten-year survival of fixed prostheses on four or six implants ad modum Branemark in full edentulism. Clin Oral Implants Res 1995;6:227-231. 3. Buser D, Ingimarsson S, Dula K, Lussi A, Hirt HP, Belser UC. Long-term stability of osseointegrated implants in augmented bone: A 5-year prospective study in partially edentulous patients. Int J Periodontics Restorative Dent 2002;22:109-117. 4. Wallace SS, Froum SJ. Effect of maxillary sinus augmentation on the survival of endosseous dental implants. A systematic review. Ann Periodontol 2003;8:328-343. 5. Del Fabbro M, Rosano G, Taschieri S. Implant survival rates after maxillary sinus augmentation. Eur J Oral Sci 2008;116(6):497-506. 6. Raghoebar GM,Timmenga NM, Reintsema H, Stegenga B, Vissink A. Maxillary bone grafting for insertion of endosseous implants: result after 12 to 124 months. Clin Oral Implants Res 2001;12:279-286. 7. Hurzeler MB, Kirsh A, Ackermann K-L, Quinones CR. Reconstruction of severely resorbed maxilla with dental implants in the augmented maxillary sinus: A 5-years clinical investigation. Int J Oral Maxillofac Implants 1996;11: 466475. 8. Bahat O. Branemark System implants in the posterior maxilla: Clinical study of 660 implants followed for 5 to 12 years. Int J Oral Maxillofac Implants 2000;15:646-653. 9. Regev E, Smith RA, Perrot DH, Pogrel MA. Maxillary sinus complications related to endosseous implants. Int J Oral Maxillofac Implants 1995;10:451–456. 10. Schwartz–Arad D, Herzberg R, Dolev E. The prevalence of surgical complications of the sinus graft procedure and their impact on implant survival. J Periodontol 2004;75:511–516.

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11. Emmerich D, Att W, Stappert C. Sinus floor elevation using osteotomes: a systematic review and meta-analysis. J Periodontol 2005;76(08):1237-51.

rior mandible of partially edentulous patients with short, porous-surfaced dental implants: early data from a clinical trial. Int J Oral Maxillofac Implants 2001,16:653-658.

12. Renouard F, Nisand D. Impact of implant length and diameter on survival rates. Clin Oral Implants Res 2006;17(2):35-51.

22. Kaplan E, Meier P. Non parametric estimation from incomplete observations. J Am Stat Assoc 1958;53:457-462.

13. Cochran DL. A comparison of endosseous dental implant surfaces. J. Periodontol 1999;70:1523-1539.

23. Pilliar RM. Overview of surface variability of metallic endosseous dental implants: textured and porous–surfaced designs. Implant Dent 1998;7:305-314.

14. Deporter DA, Watson PA, Pilliar RM, Chipman M, Valiquette N. A histological comparison in the dog of porous-surfaced vs threaded dental implants. J Dent Res 1990;69:1138-1145. 15. Deporter DA, Todescan R, Watson PA, Pharoah M, Pilliar RM, Tomlinson GA. A prospective human clinical trial of Endopore dental implants in restoring the partially edentulous maxilla using fixed prostheses. Int J Oral Maxillofac Implants 2001;16:527–536. 16. Deporter DA, Watson PA, Pharoah M, Todescan R, Tomlinson GA. Ten-year results of prospective study using porous-surfaced dental implants and a mandibular overdenture. Clin Implant Dent Realt Res 2002;4:183-189. 17. Corrente G, Abundo R, des Ambrois AB, Savio L, Perelli M. Short porous implants in the posterior maxilla : a 3-year report of a prospective study. Int J Periodontics Restorative Dent. 2009;29(1):23-9. 18. Deporter DA, Pilliar RM, todescan R, Watson PA, Pharoah M. Managing the posterior mandible of partially edentulous patients with short, porous-surfaced dental implants: early data from a clinical trial. Int J Oral Maxillofac Implants 2001,16:653-658. 19. Deporter DA, Todescan R, Caudry S. Simplifying management of the posterior maxilla using short, porous – surfaced dental implants and simultaneous indirect sinus elevation. Int J Periodontics Restorative Dent 2000;20:477-485. 20. Deporter D, Ogiso B, Sohn DS, Ruljancich K, Pharoah M. Ulltrashort sintered poroussurfaced dental implants used to replace posterior teeth. J Periodontol 2008;79(7):1280-6. 21. Deporter DA, Pilliar RM, todescan R, Watson PA, Pharoah M. Managing the poste-

24. Pilliar RM, Simmons CA. Mechanical factors and osseointegration: Influence of implant design. In: Zarb G, Osteoporosis and Dental Implants. Chicago: Quintessence Publishing Co.2001:35-44. 25. Oyonarte R, Deporter DA, Pilliar RM, Woodside DW. Peri-implant bone response to orthodontic loading: Implant surface geometry and its impact on regional bone remodelling. Am J Orthod 2004;2005:182-189. 26. Pilliar RM, Sagals G, Meguid SA, Oyonarte R, Deporter DA. Threaded versus poroussurfaced implants as anchorage units for orthodontic treatment :three-dimensional finite element analysis of peri-implant bone tissue stresses. Int J Oral Maxillofac Implants 2006;21:879-889. 27. Vaillancourt H, Pilliar RM, McCammond D. Factors affecting crestal bone loss with dental implants partially covered with a porous coating: A finite element analysis. Int J Oral Maxillofac Implants 1996;11:351-359. 28. Davarpanah M, Martinez H, Tecucianu JF, Hage G, Lazzara R. The modified osteotome technique. Int J Periodontics Restorative Dent 2001;21:599-607. 29. Zitzmann NU, Schaerer P. Sinus elevation procedures in the resorbed posterior maxilla. Comparison of the crestal and lateral approaches. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:8–17. 30. Peñarrocha M, Pérez H, Garciá A, Guarinos J. Benign paroxysmal positional vertigo as a complication of osteotome expansion of the maxillary alveolar ridge. J Oral Maxillofac Surg 2001;59:106-107.

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Original article

Regeneration of soft tissue with the ciprofloxacin incorporated collagen scaffold delivery vehicle S. Kirubanandan 1* P.K. Sehgal 2

The objective of this study is to investigate the use of ciprofloxacin- incorporated collagen based scaffold in the dermal wound healing process. The Type 1 collagen was extracted from bovine tendons. The 1% collagen solution was prepared in acidified water using acetic acid and to this, 400 μl of Triton X100, non-ionic wetting agent was added, agitated for homogenization for few minutes and poured into a trough. This preparation was allowed to dry in a dust free chamber. This preparation was incorporated with ciprofloxacin by physical entrapment method. The release of ciprofloxacin from ciprofloxacin incorporated collagen scaffold was studied. Antimicrobial efficacy of ciprofloxacin incorporated collagen scaffold (11 mm diameter) was tested on Mueller-Hinton agar (MHA) against wound pathogens following Kirby-Bauer disk diffusion test. An animal experiment was performed for testing the biomaterial according to the Institute’s ethical committee approval and guidelines (466/01/a/CPCSEA). Full thickness wounds (1.5 X 1.5 cm) were created on the shaved dorsal side of rats using a sterile surgical blade. All surgical procedures were carried out under anesthesia using thiopentone sodium (40 mg/ kg body weight, intramuscular).The percentage of wound closure was calculated using the initial and final area drawn on glass slides during the experiments. A porous collagen sponge impregnated with ciprofloxacin showed a sustained release of ciprofloxacin with 37% of drug burst release within 5 hours followed by controlled release up to 24 hrs. The in vitro antimicrobial efficiency of ciprofloxacin incorporated collagen scaffold against wound pathogens showed the zone of inhibition 38 ± 2mm whereas nil zone of inhibition for plain collagen scaffold. The in vivo studies shows that animals treated by drug-incorporated collagen scaffold provides 90% wound contraction at the end of 16th day whereas plain collagen scaffold treated groups shows 65 % wound closure and open wound groups shows 50% wound closure at the end of 16th day. The antimicrobialagents incorporated collagen-based scaffolds shows the ability to regenerate tissue better than the scaffold without ciprofloxacin. (J Osteol Biomat 2010; 1:109-117)

Key Words: regenerate tissue, ciprofloxacin, collagen scaffold.

Department of Biotechnology, Sri Venkateswara Collage of Engineering, Sriperumbudur-602105, Tamilnadu, India. 2 Bioproducts Laboratory, Central Leather Research Institute, Adyar, Chennai-20, India. 1

Corresponding author: *S.Kirubanandan, M.Tech. Lecturer,Faculty of Biotechnology, Sri Venkateswara College of Engineering, Pennalur, Sriperumbudur -602105 Tamilnadu, India. Cell:+919444325196 e-mail: skirubanandan@svce.ac.in, skirubanandan80@gmail.com

Introduction The skin, the outer protective organ, protects against toxins and microorganisms that are present in the environment, and prevents dehydration of all the inner organs. It has self renewal capacity. Skin tissue repair normally involves systematic and coordinated process, resulting from vascular connective tissue genesis and epithelial cells generation. Interleukins and growth factors play a major role in the regulation of cellular processes in wound repair1. Wound caused by physical, chemical or biological factors leads to the loss of epidermis which can regenerate but, the loss of dermis does not regenerate. The loss of skin integrity due to injury or illness may result in substantial physiologic imbalance and ultimately leads to disability or even death. The most urgent need for skin regeneration is the quick reconstruction of epidermis and dermis at the wounded site2. Improved wound care products are needed to regenerate tissues with both the structural and functional properties in the wounded tissue. By providing a scaffold at the wound site, formation of extra cellular matrix and dermal regeneration are influenced at a faster wound healing rate. The scaffolds made from biomaterials function

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as primary closure in wounds. In addition, wound dressings should cover the wound surface and create and maintain a moist healing environment3. The infection at the site of injury delays wound healing and causes a strong inflammatory response. Moreover, infected pathogens secrete pathogenic enzymes such as collagenase and hyaluronidase that degrade extra cellular matrix. The infected dermal wound become a chronic wound and therefore wound healing is challenged4, 5. The wound healing biomaterial scaffold should mimic extra cellular matrix (ECM) which supports the wound healing processes. Scaffolds prepared from collagen, an important component of extra cellular matrix (ECM), may provide biological stimuli to support tissue growth. Collagen influences wound healing by regenerating a tissue by processes such as cell proliferation, cell migration and cell differentiation. Interactions between the different tissue components and collagen form an essential substrate for cellular adhesion6. Moreover, collagenâ&#x20AC;&#x201C;based biomaterials have several other advantages such as biocompatible and nontoxic materials to tissues. In addition, it enhances the deposition of oriented and organized, newly synthesized collagen fibers in the remodeling phase of wound healing. Furthermore, collagen functions as a substrate for haemostatisis of blood cells by chemotaxis that promotes wound maturation by providing a scaffold for more rapid transition to normality at the injured site7, 8. Collagen-based materials formed into three dimensional scaffolds serve as a wound dressing since they contain large pores which enhance wound tis-

Journal of Osteology and Biomaterials

Figure 1. The porous collagen scaffold contains the pore size varies from 500 - 800Âľm.

sue infiltration in vivo. This wound dressing with burst release and followed with sustained release of antibiotics to pathogens can cause rapid eradication of pathogens and subsequent prevention of bacterial infection. The sustained release of antimicrobial agents enhances healing of wounds rapidly9,10. In addition, the incorporation of antimicrobial agents to collagen dressings may help to maintain stability of collagen from microbial degradation of collagen at the infected wound site. A possible method to deliver antibiotics in a controlled manner is by physically entrapping it in the collagen polymer matrix. Ciprofloxacin (1-cyclopropyl-6-fluro-1, 4-dihydro-4-oxo-7- (1-pipera Zinyl)-3-

quinoline carboxylic acid) entrapped collagen scaffold was used in this study. The ciprofloxacin was used due to its broad spectrum antimicrobial activity as well as having good tissue penetration property. In this present work, we evaluated the tissue regeneration capacity of ciprofloxacin-incorporated collagen biomaterials using in vivo studies. MATERIALS AND METHODS Extraction of Type I Collagen from Bovine Tendons The preparation of collagen from bovine tendon was done11,12 according to the method developed by Bioproducts lab, Central Leather Research Institute, Chennai, India.

Kirubanandan S. et al.

Incorporation of ciprofloxacin to porous collagen scaffold After homogenized collagen was preparated, a known amount of ciprofloxacin (0.5 mg) was added and stirred well and poured in the trough. Since the minimum inhibitory concentration of ciprofloxacin against S. aureus ATCC 29213 is 0.12 – 0.5 μg/mL and P. aeruginosa ATCC 27853 is 0.25 – 1.0 μg/mL, the amount of drug to be added in the collagen scaffold is 10 times of MIC value of drug per cm2 of the scaffold. The thickness of prepared collagen scaffold is 2mm. Sterilization of Plain collagen dressings and ciprofloxacin incorporated collagen dressings The sterilization of collagen biomaterial dressings is done by the ethylene oxide sterilization method 13. Scanning Electron Microscopy studies of collagen scaffold The SEM analysis was prepared by sprinkling collagen scaffold with gold material one side of double adhesive stub. The stub was then coated with gold using Jeol JFC 1100 sputter coater. The SEM analysis of the Collagen scaffold was carried out by using Jeol JSM 5300, Japan. The scaffold was viewed at an accelerating voltage of 15–20 kV.

Drug Release Profile Cumulative drug release %

Preparation of collagen scaffolds from Type 1 collagen One percent collagen solution was prepared in acidified water using acetic acid. To this, 400 μl of Triton X-100 non-ionic surfactant was added and agitated for a few minutes to attain homogeneity. It was poured into a trough and allowed to dry in air in a dust free chamber.

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60 50 40 30 20 10 0 1

Time in hrs

Figure 2. Release Profile of the Drug.

In vitro release of ciprofloxacin from ciprofloxacin-incorporated Collagen Scaffold In vitro release of ciprofloxacin from ciprofloxacin incorporated collagen scaffold was carried out at 37.1°C in phosphate buffer saline (PBS) (50 ml) pH 7.4. The release medium PBS was collected at predetermined time intervals, and replaced with a fresh PBS (1 ml) each time. The collected samples were filtered through a 0.45 µm Millipore filter. The amount of ciprofloxacin released was then measured at 278nm using a shimadzu UV2100S spectrophotometer. In Vitro Antimicrobial Activity Antibacterial efficacy of ciprofloxacinincorporated collagen scaffold (11 mm diameter) was tested on Mueller-Hinton agar (MHA) for the growth of Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853 12. In vivo studies Male Wister albino rats weighing 150 to 200 g were used in this study. The ani-

mals were fed a commercial pellet diet (Hindustan Lever, Bangalore, India) and had free access to water. The animal experiment was performed according to the Institute’s ethical committee approval and guidelines (466/01/a/CPCSEA). For the study, they were housed individually in standardized environmental conditions. A total of 72 animals were taken in three groups (n = 6 per group) (treatment and two controls for this study). Group 1 – Open wound covered with gauze dressing Group 2 – Plain Collagen Scaffold Group 3 – Ciprofloxacin - incorporated collagen scaffold The animals were rehabilitated following experimentation. Full thickness wounds (1.5 x 1.5 cm) were created on the shaved dorsal side of rats using sterile surgical blade. Wounds were inoculated with the test organisms at 106 CFU (0.1 mL) between thin skin muscle and paraspinus muscle and allowed to infect for 24 h. All surgical procedures were carried out

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Wound Healing Rate The percentage of wound closure was calculated as follows by using the initial and final area drawn on glass slides during the experiments: % of wound contraction = Wound area day 0 – wound area day (n) = Wound Area day 0

n _ number of days (4th, 8th, 12th, and 16th day). Collection of Granulated Tissues The granulated tissues from both treatment and control groups were excised on day 4, 8, 12, and 16 using sterile scissors and forceps. Gelatin Zymography The presence of matrix metalloproteinases (MMPs) in the granulated tissues was analyzed by gelatin zymography. Histological Analysis Tissues collected at different intervals were transferred to 10% neutral buffered formalin for 24 h at 4°C. The formalin fixed tissues were dehydrated through grades of alcohol and cleared in xylene and then embedded in paraffin wax (58 to 60° mp). The molds were labeled and stored until use. The deparaffinized sections were stained with hematoxylin following counterstained with eosin. Masson’s trichrome staining was done for all the samples of all

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Percentage of Wound contraction

under thiopentone (40 mg/kg body weight, intramuscular). The infected wounds were covered with collagen dressings and outer covered with gauze dressings. An infected animal without dressings and an animal with dressings without drugs were also maintained in individual cages.

80 OW

ACD

20 0

4th Day

8th Day 12th Day 16th Day

Figure 3. Wound Contraction in Animals.

the time points to observe collagen deposit in the granulated tissue.14 Statistical Analysis Statistical evaluations were performed using Origin Version 6 and the data were expressed as mean of six samples with standard deviation. The difference between groups was analyzed using one way ANOVA. RESULTS Properties of Ciprofloxacin incorporated collagen scaffold A known amount of ciprofloxacin was added to the collagen solution in the preparation of collagen scaffold, the ciprofloxacin bound to the collagen scaffold by physical entrapment. The FTIR confirms no interaction between ciprofloxacin and collagen. Scanning Electron Microcopy The SEM was done to determine the pore size of ciprofloxacin incorporated collagen scaffold. The Fig. 1 shows SEM image of ciprofloxacin collagen scaffold.(Fig. 1) The scaffold contains the pores size vary from 500 -600 microns.

In vitro release of ciprofloxacin from ciprofloxacin incorporated collagen scaffold In the present study, the collagen scaffold acts as a drug reservoir. Fig 2 shows the release profile of the ciprofloxacin from porous collagen scaffold.(Fig. 2) It releases the ciprofloxacin immediately and also in a sustained fashion as soon as it is exposed to the wound. The ciprofloxacin incorporated porous collagen scaffold showed a 37% of drug burst release within 5 hrs followed by controlled release of the remaining ciprofloxacin up to 24 hrs. Agar diffusion test for in vitro antimicrobial activity The activity of the released drug from the scaffold shows a clear zone of inhibition controlling the growth of both staphylococcus aureus and pseudomonas aeroginosa inoculated separately. Ciprofloxacin-incorporated collagen scaffold showed a bacterial free zone of 38 ± 2 mm.

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Open Wound Group

4th Day

8th Day

12th Day

16th Day

Plain Collagen Scaffold E

Drug Incorporated collagen scaffold I

Figures 4. Hematoxylin and eosin stained sections of the granulation tissue at different time intervals. A, B, C, D are control group on day 4, 8, 12, and 16 respectively. E, F, G and H are treated group by plain collagen scaffold on day 4, 8, 12, and 16 respectively. I, J, K and L are treated group by drug incorporated collagen scaffold on day 4, 8, 12, and 16 respectively. A, B, C, D, E, F and G are at similar magnification (150x) and H, I, J and L are at 400x.

In vivo studies: wound contraction Fig. 3 shows wound contraction in the animals treated by ciprofloxacin incorporated collagen biomaterial. (Fig. 3) In the group of ciprofloxacin incorporated collagen scaffold, wound contracted 40 %, 75 %, 82.5 %, 90 % at 4,8,12 and 16th day of the treatment, respectively, whereas, plain collagen scaffold and open wound group showed wound contraction of 65% wound closure and 50% wound closure at the end of 16th day of the treatment, respectively.

Histological analysis: H & E staining The following histology was observed in the Open wound group. On the 4th day and the 8th day, neutrophils found and bacterial colonies found in the injured area. Complete loss of epithelium with inflammatory infiltrates was observed. On the 12th day, bacterial colonies were seen with angiogenesis occurring. On 16th day, a slight formation of epidermis with angiogenesis, but complete healing did not occurr. In the group treated with collagen scaffold, in the 4th and the 8th day,

well formed epidermis and absence of bacterial colonies were observed. On the 12th and 16th days, complete formation of epidermis and dermis was observed. In the group treated with ciprofloxacin incorporated collagen scaffold, epithilization with moderate extra cellular matrix was observed on the 4th day and the 8th day. On the 12th and 16th days, marked epithelialization with moderate amount of extra cellular matrix synthesis and new blood vessel formation were seen.

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Histological analysis of wound healing by H&E staining In the tissue samples of day 4 of ciprofloxacin incorporated collagen scaffold treated group, minimum neutrophilic infiltration was seen on the surface of the wound. Fig. 4I shows well formed epithilization in the tissue whereas in day 4 of open wound group (Fig. 4A), heavy neutrophilic infiltration was observed in the surface of the wound. A fewer macrophages were also seen. Bacterial colonies were found over the granulated tissue. In the plain collagen scaffold group, mild neutrophilic infiltration was seen on the surface of the wound (Fig. 4E). Partial epithilization has also formed in this group. On the 8th day of ciprofloxacin-incorporated collagen scaffold group, uniform granulation was formed in the tissue. In addition, angiogenesis was seen (Fig. 4J) in this tissue. Maturing fibroblast was also seen in the dermal region with collagen deposition, whereas in day 8th of the open wound group, bacterial infection was still persistent along with heavy neutrophilic infiltration (Fig. 4B). In the plain collagen scaffold group, mild angiogenesis was observed (Fig. 4F). On the 12th day of ciprofloxacin-incorporated collagen scaffold group, epithelialization was seen (Fig. 4K). The dermal region was seen to have good angiogenesis along with mature fibroblasts and collagen deposition, whereas in day 12th of the open wound group, bacterial infection was still prominent along with invading neutrophils in the granulation tissue (Fig. 4 C). Macrophages were also seen along with fibroblastic cells below the neutrophilic infiltration. Extra cellular matrix (ECM)

Journal of Osteology and Biomaterials

formation at the dermal region was seen. Bridging of the wound surface was observed with plain collagen scaffold. Epithelial cell proliferation was also seen (Fig. 4G) in this group. Bacterial infection was lesser. The granulation tissue was well formed with collagen bundles. In the 16th day of ciprofloxacin-incorporated collagen scaffold group, complete healing was seen with good epithelialization (Fig. 4L). Dermal layer was seen to have mature fibroblasts with deposited collagen. Whereas in the 16th day of open wound, tissue remodeling was seen (Fig. 4D) and epithelial proliferation with well-formed collagen bundles was seen but healing was incomplete. Dermal region has mature fibroblasts i.e. surface of the wound was not covered by epithelium. In the plain collagen scaffold, complete epithelialization was seen (Fig. 4H). Mature fibroblastic cells were seen in the dermal region with collagen deposition. Collagen analysis in the tissue by Massonâ&#x20AC;&#x2122;s Trichrome Staining In the open wound group, on the 4th day and the 8th day, (Fig. 5A and 5B) neutrophils were found and bacterial colonies formed in the injured area. Complete loss of epithelium with inflammatory infiltrates was observed and less amount of collagen was seen due to infection. In 12th day and 16th day (Fig. 5C and 5D), partial epidermis formed and a loose collagen fiber was observed. In the group treated with collagen scaffold, on the 4th day (Fig. 5E), neutrophils were found and bacterial colonies formed in the injured area. Complete

loss of epithelium and no collagen content in tissues were observed. On the 8th day (Fig,. 5F), a bundle of collagen was seen. On the 12th day and 16th day (Fig. 5G and 5H), well formed epidermis and dermis was seen in the tissues. A loose collagen bundle in the tissues was observed. In the ciprofloxacin-incorporated collagen scaffold group, on the 4th day Fig 5I, neutrophils were found and bacterial colonies formed in the injured area. Complete loss of epithelium with inflammatory infiltrates was observed and less amount of collagen was seen due to infection. On the 8th day Fig. 5J, partially epidermis formed and a stretched bundles collagen fiber in the dermis was observed. On the 12th day (Fig. 5K) shows well formed epidermis and dermis was seen and stretched collagen bundles formed in the tissue. In Massonâ&#x20AC;&#x2122;s Trichrome stained histological sections of tissue of 12th and 16th days of groups treated by ciprofloxacin incorporated collagen scaffold, bluish violet color indicates staining of well stretched and deposited collagen bundles formed in the tissue. (Fig 5) In the case of open wound at the end of the 12 and 16th days, loose collagenous matrix was also seen with proliferating fibroblast. In case of collagen dressing treated group at the end of 12 and 16th days, mature collagen bundles along with fibroblastic cells were found. Biochemical Studies: Role of MMPs in Tissue remodeling Fig. 6 shows gelatin zymography of MMP taken from tissues of all the groups. In the present study, MMP 2 and MMP 9 were detected in granu-

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Open Wound Group

4th Day

8th Day

12th Day

16th Day

Plain Collagen Scaffold E

Drug Incorporated collagen scaffold I

Figures 5. Massonâ&#x20AC;&#x2122;s Trichrome stained sections of the granulation tissue at different time intervals. A, B, C, D are control group on day 4, 8, 12, and 16 respectively. E, F, G and H are treated group by plain collagen scaffold on day 4, 8, 12, and 16 respectively. I, J, K and L are treated group by drug incorporated collagen scaffold on day 4, 8, 12, and 16 respectively. A, B, C, D, E, F, G,I and K are at similar magnification (150x) and H,J,L are at 400x.

lated tissue of all the groups on days 4, 8, 12, 16. MMPs were detectable in very low amount by gelatin zymography in the treated group than in the control groups. In the infected dermal wound, MMP -9 (gelatinase â&#x20AC;&#x201C;B) is highly expressed in inflammatory period of wound healing .The MMPs secreted by inflammatory cells is highly excreted in the control groups than in the treated groups. The expression of MMP9 decreased in the treatment groups than in the control groups.

DISCUSSION The healing of infected dermal wound is exigent. Wound pathogens secrete enzymes which degrade extra cellular matrix at the site of injury and form a biofilm which delays wound healing. 1- 3 Biomaterials with antimicrobial agents enhance regeneration of dermal tissues and eradicate wound pathogens at the wound site. Generally protein based biomaterials mimic extra cellular matrix of the site of injury and help skin regeneration. Among all the pro-

tein biomaterials, collagen is a potentially useful biomaterial as it is a major constituent of connective tissues. Its characteristics offer several advantages such as biocompatibility and non-toxicity in most tissues15,16, cellular mobility and growth while having a porous nature. These properties allow a highly vascularized granulation bed formation on the wound. In addition, collagen enhances keratinocytes and fibroblast proliferation which are important in wound healing. The cost of synthetic

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biomaterial such as Poly Lactic Glycolic Acid (PLGA), Poly Lactic Acid (PLA) and Poly Caprolactone (PCL) are very high and also cause inflammatory response to host tissue7. Therefore, natural biomaterials are studied as alternative materials to synthetic biomaterials. Type 1 collagen is cheap and it is present in higher order animals especially in the skin, tendon and bone, and is prepared from slaughter houses. Scaffolds used as wound dressings for soft tissue repair should be reabsorbed into the body after successful tissue regeneration. Collagenâ&#x20AC;&#x201C;based scaffold degrade in physiological pathway without induction of inflammatory response. The porosity of collagen scaffold directly influences cellular ingrowth. The porous nature of collagen scaffold helps to encapsulate the drug effectively. The modern tissue engineering task is to develop three-dimensional scaffolds of appropriate biological and biomechanical properties, at the same time mimicking the natural extra cellular matrix (ECM) and promoting tissue regeneration. The scaffold should permit cell adhesion, infiltration, and proliferation for ECM synthesis. Fur-

thermore, it should be biodegradable, bioresorbable and non-inflammatory, should provide sufficient nutrient supply and have appropriate viscoelasticity and strength. Attributed to collagen features mentioned above, collagen fibers represent an obvious appropriate material for tissue engineering scaffolds. Scaffold constructed from naturally occurring proteins in the extra cellular matrix (ECM) such as collagen allows much better infiltration of cells into the scaffold.8, 9 The porous collagen scaffold has the ability to absorb large quantities of wound fluid and also maintains moist environment at the wound site. The macro porosity present in collagen scaffold helps to encapsulate drugs efficiently and to enhance the cell fate process. The SEM observation shows that the pore size of the scaffold varies from 500 - 800 microns and these pores helps to encapsulate the drug effectively. Normally, collagen biomaterial is not suitable for infected dermal wounds, because wound pathogens utilize collagen as a substrate for their growth and increase infection rate at the site

of injury. The incorporation of antimicrobial agents to the collagen biomaterials prevents the above character. An ideal wound care system is the one that delivers sufficient quantity of drug at the site of action, decrease further bacterial proliferation and better dermal regeneration. The sustained release system facilitates it.9 In the ciprofloxacin-incorporated collagen scaffold, the same percentage of the drug is burst-released as soon as it comes in contact with the wound. Moreover, the degradation of scaffold at the site of injury also causes sustained release of bound drug. The drug release during burst-release showed much higher MIC value that can overcome the growth of the wound pathogens. This is confirmed by in vitro antibacterial test with a significant zone of inhibition. Wound contraction is also an element of wound healing, which occurs through the centripetal growth of tissues surrounding the wound1. In vivo studies show that antibiotic incorporated collagen scaffold group have better wound closure than plain collagen scaffold groups and open wound groups.

MMP9 Pre MMP8 Active MMP8 Pro MMP2 Active MMP2

Figures 6. Matrix Metalloproteinase in wound healing. Gelatin Zymography shows Matrix Metalloproteinase expression in granulated tissue. Lane I-4th Day, Lane II-8th Day, Lane III -12th Day, Lane IV -16th Day. In open wound Group, Gels shows high level of expression of MMPs due to high level inflammation and infections. In treated group, MMP 2 and MMP 9 expression is very less due to faster healing.

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Kirubanandan S. et al.

In the histological studies, ciprofloxacin-incorporated collagen scaffold treated group shows epithelialization with moderate extra cellular matrix on the day 8 and the day 12 whereas in the control group, incomplete epithelialization with less extra-cellular matrix synthesis and persistence of inflammatory exudates in the upper dermis with loss of epidermis were observed up to day 16. In Masson’s trichrome staining, in ciprofloxacin incorporated collagen scaffold treated group has shown wellformed collagen bundles and fibroblast proliferation. Significant reduction of MMP 9 and MMP 8 in ciprofloxacin-incorporated collagen scaffold treated group supports the reduction of inflammatory phase. Significant reduction of inflammatory cells in the early phase of healing in ciprofloxacin incorporated collagen scaffold treated group indirectly shows the reduction of bacterial population, thereby enhancing healing18, 19. The ciprofloxacin incorporated porous collagen scaffold for treating infected dermal wound application was developed and the wound healing was observed in a shorter time compared with other control scaffold that lacks ciprofloxacin.

REFERENCE 1. Singer AJ, Richard Clark MD. Cutaneous wound healing. New England Journal of Medicine 1999: 2:738-746. 2. Clark RAF. Wound repairs Overview and general considerations. In: R.A.F. Clark, Editor, The Molecular Biology of Wound Repair, Plenum Publishing, New York, 1996,pp.195248. 3. Bowlerl PG, Duerden BI, Armstrong DG. Wound Microbiology and Associated Approaches to Wound Management. Clinical Microbiology Review 2001:14:244- 269. 4. Schmidtchen A, Holst E, Tapper H et al. Elastase-producing pseudomonas aeruginosa degrade plasma proteins and extracellular products of human skin and fibroblasts, and inhibit fibroblast growth. Microbial Pathogenesis 2003: 34: 47 – 55.

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12. Hsiu-O H, Lun-Huei L, Ming-Thau S. Characterization of collagen isolation and application of collagen gel as a drug carrier. J Cont Release 1997:44:103-112. 13. Gorham SD, Srivastava S, French DA, Scott R. The effect of gamma-ray and ethylene oxide sterilization on collagen-based wound-repair materials. Journal Mater Sci: Materials in Medicine 1993:4:40-49. 14. Luna L et al. Manual of Histological Staining Methods. In 3rd edition. 1968 McGraw hill publishers. 15. Gelse k, et al. Collagens-structure, function, and biosynthesis. Adv Drug Del Rev 2003:55:1531-1546. 16. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson J. Molecular biology of the cell. In 4th Ed. New York: Garland Publishing; 2002.

5. Pachence JM, Collagen-based Devices for Soft Tissue Repair. J Biomed Mater Res Part B: Appl Biomaterials 1996: 33:35 -40.

17. Rao KP. Recent developments of collagenbased materials for medical and drug delivery systems. J Biomater Sci 1995:7:623-645.

6. Lee CH, et al. Biomedical Application of collagen-Review. Int J Pharm 2001: 221:1 -22.

18. David G, Armstrong DPM, Edward B, Jude MD. The Role of Matrix Metalloproteinases in Wound Healing. J Am Pediatr Med Assoc 2002:92:12

7. Ruszczak Z. Effect of collagen matrices on dermal wound healing. Adv Drug Del Rev 2003: 55(12):1595-1611. 8. Chvapil M. Collagen sponge: Theory and practice of medical applications. J Biomed Mater Res 1977:11:721-741.

19. Salo M, Makela M, Kylmaniemi H. Autio-Harmainen and H. Larjava, expression of matrix metalloproteinase-2 and-9 during early human wound healing. Lab Invest.1994:70:176.

9. Loke WK, Lau SK, Yong LL, Khor E, Sum Ck. Wound dressing with sustained antimicrobial capability. J Biomed Mater Res 2000:53:8-17. 10. Ruszczak Z and Friess W. Collagen as a carrier for on-site delivery of antibacterial drugs. Adv Drug Del Rev 2003:55:16791698. 11. Sripriya R, Rafiuddin Ahmed Md, Sehgal PK, Jayakumar R. Influence of laboratory ware related changes in conformational and mechanical properties of collagen. J Appl Polym Sci 2003:87:2186-2192.

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BioCRA

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Original article

Genetic effects of pulsed electromagnetic fields (PEMF) on human osteoblast-like cells (TE85) in vitro Vincenzo Sollazzo,1 Ilaria Zollino,2 Ambra Girardi,3 Francesca Farinella,2 Francesco Carinci,2*

Pulsed electromagnetic fields (PEMF) have been widely accepted in the clinical community in the treatment of several pathologies of the bone and recently of the cartilage. Although therapeutic properties of PEMF are well known, the sequence of events by which electromagnetic stimulation can lead its desirable effects on bone healing and cartilage are not completely understood. Here we are testing the effect of PEMF on osteoblast-like cells (TE85) by using DNA microarrays containing 20,000 genes. We identified several genes covering a broad range of functional activities whose expression was significantly up- or down-regulated. PEMF seem to exert an anabolic effect and act on cell behavior setting the cell in a proliferative way and inducing both osteoblastogenesis and differentiation of osteoblasts. Moreover, PEMF promote extra-cellular matrix apposition and mineralization while at the same time decrease the degradation and absorption processes of extra-cellular matrix. The data come out from this study, constitute the first genetic portrait of PEMF effects on human osteoblast-like cells in vitro. They permit a detailed description of the effects of electromagnetic stimulation and give a better explanation of the observed clinical effect suggesting the possibility of using them in other fields like regenerative medicine. (J Osteol Biomat 2010; 1:119-126)

Key Words: pulsed electromagnetic fields, osteoblast-like cells.

Orthopedic Clinic, University of Ferrara, Ferrara, Italy; Department of Maxillofacial Surgery, University of Ferrara, Ferrara, Italy; 3 Institute of Histology, Embryology and Applied Biology, University of Bologna, Bologna, Italy; 1 2

Corresponding author: Prof. Francesco Carinci, MD, Dept. of D.M.C.C.C. University of Ferrara Corso Giovecca, 203- 44100 Ferrara,Italy Tel/Fax :0039-0532-455582 e-mail:crc@unife.it ; web:www.carinci.org

Introduction Pulsed electromagnetic fields (PEMF) have been widely accepted in the clinical community since over 30 years. PEMF are effective in the treatment of several pathologies in which enhancement of bone healing is needed such as non union 1-3, delayed union 1,4,5, osteotomies6, avascular necrosis of the femoral head7, bone grafts and spinal fusion8. Although therapeutic properties of PEMF are well known, the sequence of events by which electromagnetic stimulation can lead its desirable effects on bone healing and cartilage are not completely understood. PEMF are known to modify some important physiological parameters of cells cultured in vitro such as proliferation, transduction, transcription, synthesis and secretion of growth factors9. PEMF are able to induce cell proliferation in mitogen stimulated lymphocytes3 and to improve IL-2 receptor expression and IL-2 utilization in lymphocytes from aged donors, which are characterized from defective production and utilization of this growth factor3. PEMF exposure induces cell proliferation in human osteoblasts and chondrocytes cultured in vitro5,10,11. Moreover, normal human osteoblasts require minimal exposure times to PEMF to increase their cell proliferation, similar

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to the time needed to stimulate bone formation in vivo. Electromagnetic fields determine signal transduction by means of intracellular release of Ca2+ leading to an increase in cytosolic Ca2+ and an increase in activated cytoskeletal calmodulin12. PEMF induce a dosedependent increase in bone13 and cartilage differentiation and the up-regulation of mRNA expression of extracellular matrix molecules, proteoglycan, and type II collagen14. The acceleration of chondrogenic differentiation is associated with increased expression of transforming growth factor b1 (TGFb1) mRNA and protein15 suggesting that the stimulation of TGFb1 may be a mechanism through which PEMF affect complex tissue behavior such as cell differentiation and through which the effects of PEMF may be amplified 15 . PEMF are also postulated to act at a membrane level influencing signal transduction of several hormones or growth factors like PTH, IGFII and Adenosine A2a producing the amplification of their transmembrane receptors1,16,17. In the present study the genetic effect of PEMF on human osteoblast-like cells cultured in vitro was investigated throughout a microarray approach. DNA microarray molecular technology enables the analysis of gene expression in parallel on a very large number of genes, spanning a significant fraction of the human genome. Gene expression is performed by a process of (i) RNA extraction, (ii) reverse transcription, and (iii) labeling of cDNA. Reference (i.e. cells cultured without PEMF) and investigated (i.e. cells cultured with PEMF) cDNA are labeled with different dyes and then hybridized on slides

Journal of Osteology and Biomaterials

containing cDNA fragments. Then the slides are scanned with a laser system, and two false color images are generated for each hybridization, with cDNA from the investigated and reference cells. The overall result is the generation of a so-called genetic portrait .It corresponds to up- or down-regulated genes in the investigated cell system. In the present study we have defined the genetic effect of PEMF on cells by using an osteoblast-like cell line (TE85) and microarray slides containing 20,000 different oligonucleotides. MATERIALS AND METHODS Cell culture Osteoblast-like cell (TE85) were cultured in sterile Falcon wells (Becton Dickinson, New Jersey, USA) containing Eagle’s minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS) (Sigma, Chemical Co., St Louis, Mo, USA) and antibiotics (Penicillin 100 U/ml and Streptomycin 100 micrograms/ml - Sigma, Chemical Co., St Louis, Mo, USA). Cultures were maintained in a 5% CO2 humidified atmosphere at 37 °C. TE85 cells were collected and seeded at a density of 1x105 cells/ml into 9 cm2 (3ml) wells by using 0.1% trypsin, 0.02%EDTA in Ca++ - and Mg – free Eagle’s buffer for cell release. Cells were exposed to PEMFs (pulsed electromagnetic fields) for 18 hours using PEMFs generator system (Igea, Carpi, Italy) (Fig. 1). The solenoids were powered using a Biostim pulse generator (Igea, Carpi, Italy), a pulsed electromagnetic field (PEMF) generator. The electromagnetic bioreactor applied a PEMF to the cells on the scaffold sur-

face with the following characteristics: intensity of the magnetic field 2±0.2 mT, amplitude of the induced electric tension 5±1 mV, signal frequency 75±2 Hz, and pulse duration 1.3 ms. Control cultures were maintained in the same incubator at a distance where no electromagnetic field was detectable. After 18 hours, when cultures were sub-confluent, cells were processed for RNA extraction. DNA microarrays screening and analysis The protocol was the same of previous experiments (18-22). RNA was extracted from cells by using RNAzol. Ten micrograms of total RNA were used for each sample. cDNA was synthesized by using Superscript II (Life Technologies, Invitrogen, Milano, Italy) and amino-allyl dUTP (Sigma, St. Louis, MO, USA). Mono-reactive Cy3 and Cy5 esters (Amersham Pharmacia, Little Chalfont, UK) were used for indirect cDNA labeling. RNA extracted from untreated cells was labeled with Cy3 and utilized as control against the Cy5 labeled treated (PG) cDNA in the first experiment and then switched. For 20 K human DNA microarrays slides (MWG Biotech AG, Ebersberg, Germany) hundred microliters of the sample and control cDNAs in DIG Easy hybridization solution (Roche, Basel, Switzerland) were used in a sandwich hybridization of the two slides constituting the 20 K set at 37 degrees Celsius overnight. Washing was performed three times for 10 minutes with 1 x saline sodium citrate (SSC), 0.1% sodium dodecyl sulfate (SDS) at 42 degrees Celsius, and three

Sollazzo V. et al.

times for 5 minutes with 0.1 x SSC at room temperature. Slides were dried by centrifugation for 2 minutes at 2000 rpm. The experiment was repeated twice and the dyes switched. A GenePix 4000a DNA microarrays scanner (Axon, Union City, CA, USA) was used to scan the slides, and data were extracted with GenePix Pro. Genes with expression levels, after removing local background, of less than 1000 were not included in the analysis, since ratios are not reliable at that detection level. RESULTS The hybridization of cDNA (derived from TE85 exposed to PEMF) to cDNA microarrays allowed us to perform systemic analysis of expression profiles for thousands of genes simultaneously and to provide primary information on transcriptional changes related to PEMF. The genes differentially ex-

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Figure 1: PEMFs generator system (Igea, Carpi, Italy).

pressed in cells treated with PEMF are reported Table 1 and 2, whereas in Figure 1 is reported the SAM plot (Fig. 2). We briefly analyzed some of those with better known functions. DISCUSSION The improvement of osteogenesis is an important topic for the researchers

because of the wide clinical applications it may have. PEMF are an effective method to restart osteogenesis in pathologies in which it has stopped and in pathologies in which osteogenesis needs to be enhanced6. Although extensive research has been done on PEMF, their mechanism(s) of action is not completely clear. Moreover, stud-

Table I. Up-regulated genes GenBank H30869 BQ052715 R50700 H02682 R97966 BM793706 H52744 BG753663 R83178 AW297717 BE777770 H62319 H29682 BQ021774 R81620 H39844 H49329 H58317 H50781 R46436 R68004 R48131 H74123 H48337 H72135 BM980948 H45907 H03415 H17697 BF793857 H56543 BE277159 R18627 R28340 T86617 R65621

Symbol TUBB3 PKM2 MPST RAG1AP1 ACADVL HLA-B ABHD12 TUBB4 HSD17B7 CPD ATF4 SLFN5 KIAA0701 ACTA2 TXK SNAPC3 BANF1 STARD4 ZNF131 FAM118A PCBP2 SH3BP2 RCHY1 PLCG2 MTMR3 HDGF KIAA0319L YPEL3 COG7 HMGN1 MCM3APAS NDUFV1 APPBP2 MAZ MYBL2 CSF2RA

Cytoband 16q24.3 15q22 22q13.1 1q22 17p13-p11 6p21.3 20p11.21 19p13.3 1q23 17q11.2 22q13.1 17q12 12q23.1 10q23.3 4p12 9p22.3 11q13.1 5q22.1 5p12-p11 22q13 12q13.12-q13.13 4p16.3 4q21.1 16q24.1 22q12.2 1q21-q23 1p34.2 16p11.2 16p12.1 21q22.3|21q22.2 21q22.3 11q13 17q21-q23 16p11.2 20q13.1 Xp22.32 and Yp11.3

Score(d) 2,74 2,40 2,35 2,29 2,29 2,24 2,21 2,18 2,14 2,12 2,10 2,08 2,07 2,05 2,03 2,02 2,01 2,01 2,00 1,97 1,97 1,97 1,95 1,94 1,93 1,93 1,92 1,92 1,90 1,89 1,89 1,88 1,85 1,85 1,84 1,84

GenBank R66021 H77767 R89805 BQ073363 R97618 R28062 BG764810 R89791 H63198 BG567139 H20905 R99225 H43825 R83637 R73398 H00542 R89913 H57195 BQ070537 H84133 R47761 R99693 R71957 H30017 R60608 BG477018 BM928522 H12136 H52445 BQ050102 H73844 H47468 AI820750 H51848 BI761161 R97287

Symbol DDX21 RNF19 ELOVL7 SF3B4 TBC1D22A LRP8 IMPDH2 WDR26 RABIF HIGD1A PON2 KRTAP4-7 BAT2 CDGAP FBXO9 CASD1 CD58 CSF1R DDOST ZNF610 HFE ZNF43 SLC27A1 PIK3R2 NETO2 PRKCD RPN2 DOCK1 LRRC31 PSMB2 WDR23 LTV1 AHSA2 PAPPA2 CD63 FAM114A1

Cytoband 10q21 8q22 5q12.1 1q12-q21 22q13.3 1p34 3p21.2 1q42.11 1q32-q41 3p22.1 7q21.3 17q12-q21 6p21.3 3q13.32-q13.33 6p12.3-p11.2 7q21.3 1p13 5q33-q35 1p36.1 19q13.33 6p21.3 19p13.1-p12 19p13.11 19q13.2-q13.4 16q11 3p21.31 20q12-q13.1 10q26.13-q26.3 3q26.2 1p34.2 14q12 6q24.2 2p15 1q23-q25 12q12-q13 4p14

Score(d) 1,83 1,83 1,81 1,78 1,76 1,76 1,75 1,74 1,73 1,72 1,72 1,71 1,71 1,71 1,71 1,71 1,71 1,70 1,69 1,69 1,68 1,67 1,67 1,66 1,64 1,63 1,62 1,62 1,62 1,61 1,61 1,60 1,59 1,58 1,58 1,58

GenBank H84572 H77780 H85307 H66641 BF312472 H60337 H58260 W02020 H12668 H27728 H59530 H10626 R56388 R50904 H86677 H13805 H74119 H12182 R71153 R19064 BI520063 R90994 H50871 N78217 R50637 AW027708 H03723 H59348 R66182 H83982 H60117 BM913573 H45243 H83233 R96415

Symbol LRCH3 TBCA KRAS PAXIP1 MDH2 EMP2 NARG1L FTL RASL11B C9orf37 CHEK1 BAHD1 YWHAZ RHOF FZR1 C1orf96 SEC61B RAB11B TRAPPC1 C12orf47 DULLARD BIRC6 TRIM34 MICA PVRL1 STARD3 YRDC RSC1A1 RCN2 HMGN1 HEL308 HNRPA1 GZF1 MDH1 RHAG

Cytoband 3q29 5q14.1 12p12.1 7q36 7cen-q22 16p13.2 13q14.11 19q13.3-q13.4 4q12 9q34.3 11q24-q24 15q15.1 8q23.1 12q24.31 19p13.3 1q42.13 9q22.32-q31.3 19p13.2 17p13.1 12q24.12 17p13 2p22-p21 11p15 6p21.3 11q23.3 17q11-q12 1p34.3 1p36.1 15q23 21q22.3|21q22.2 4q21.23 12q13.1 20p12.3-p11.21 2p13.3 6p21.1-p11

Score(d) 1,57 1,57 1,56 1,55 1,55 1,54 1,53 1,52 1,52 1,52 1,51 1,50 1,50 1,49 1,49 1,49 1,49 1,48 1,47 1,47 1,46 1,46 1,45 1,45 1,44 1,44 1,44 1,44 1,43 1,42 1,42 1,42 1,41 1,41 1,40

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Table II. Down-regulated genes GenBank AA037640 H10763 BM925458 R68707 R28233 R62649 BG618635 R52847 R74296 T83077 BI553961 H15190 T78062 H29912 H14912 R55417 H21862 AA293401 R67303 R54534 R88034 H00239 H15096 T97034 BE250543 H06392 H22747 BM808425 R10213 T73993 AI672432 R92843 R79544 BG118607 R39664 R26013 BE884107 R92854 H10435 T95161 H30858 T84620 R25792 H10424 H23029 T72691 H23379 R59260 H59985 R53387 R63640 H53175 R19686 H86851 H61030 R09139 R61761 R14324 R10548 T80236 R31395 H14394 R32937 BM010025 R99052 BM910117 BG337015 R02755 R14442 R93690 R82389 T74081 T84134 H25007 R51133 BM564865 T85956 R14519 R93664 T96195 T78359 H00220 H49424 R71138

Symbol C1orf213 GRIA3 C16orf5 SPOCK1 FCHO2 ARHGEF15 FGL1 RAB18 METT11D1 OCIAD1 CDC42SE2 SLC26A11 SOBP KCNJ4 PCSK9 BDP1 CACNG4 LONRF2 NEO1 DICER1 DBNDD1 ABCF2 PCDH7 DPY19L1 ARMC7 STOML1 AGT CDC27 PPP2R1B ZADH1 DUSP28 GLIS3 WDR68 C12orf4 CYP19A1 SPIN1 ERGIC2 PSG9 BRAP C1orf21 PPFIA4 HMGCS1 ME2 MAP6 PIAS2 FLJ32255 SCG3 MEF2C PARP12 TLE2 ZNF605 PISD LRRC3B THOC2 REXO2 MYH14 ENTPD6 STK38L RHD ADCY2 OSBPL10 DFNA5 RCBTB1 STAT3 BLVRB RAB25 DUSP5 SENP8 PCSK5 HSD17B4 PDHX SC5DL HMBS KCTD20 SPG3A PDPK1 PDZK1 ALMS1 SAE1 ELF4 RASSF2 SH3GLB2 GALNT9 PGAP1

Cytoband 1p36.12 Xq25-q26 16p13.3 5q31 5q13.2 17p13.1 8p22-p21.3 10p12.1 14q11.2 4p11 5q31.1 17q25.3 6q21 22q13.1 1p32.3 5q13 17q24 2q11.2 15q22.3-q23 14q32.13 16q24.3 7q36 4p15 7p14.3 17q25.1 15q24-q25 1q42-q43 17q12-q23.2 11q23.2 14q24.3 2q37.3 9p24.2 17q23.3 12p13.3 15q21.1 9q22.1-q22.3 12p11.22 19q13.2 12q24 1q25 1q32.1 5p14-p13 6p25-p24|18q21 11q13.3 18q21.1 5p12 15q21 5q14 7q34 19p13.3 12q24.33 22q12.2 3p24 Xq25-q26.3 11q23.1-q23.2 19q13.33 20p11.2-p11.22 12p11.23 1p36.11 5p15.3 3p22.3 7p15 13q14 17q21.31 19q13.1-q13.2 1q22 10q25 15q23 9q21.3 5q21 11p13 11q23.3 11q23.3 6p21.31 14q22.1 16p13.3 1q21 2p13 19q13.32 Xq26 20pter-p12.1 9q34 12q24.33 2q33.1

Score(d) -4,94 -3,15 -3,13 -3,12 -3,11 -3,11 -3,09 -3,05 -3,02 -2,98 -2,98 -2,95 -2,93 -2,93 -2,90 -2,89 -2,89 -2,89 -2,88 -2,87 -2,87 -2,85 -2,83 -2,83 -2,82 -2,82 -2,79 -2,78 -2,76 -2,75 -2,74 -2,74 -2,73 -2,72 -2,72 -2,71 -2,70 -2,68 -2,66 -2,66 -2,66 -2,65 -2,65 -2,65 -2,64 -2,64 -2,64 -2,64 -2,64 -2,64 -2,63 -2,63 -2,62 -2,61 -2,61 -2,61 -2,61 -2,61 -2,60 -2,60 -2,60 -2,59 -2,58 -2,58 -2,58 -2,57 -2,57 -2,56 -2,54 -2,54 -2,53 -2,53 -2,52 -2,52 -2,52 -2,52 -2,52 -2,50 -2,50 -2,49 -2,49 -2,49 -2,49 -2,48

Journal of Osteology and Biomaterials

GenBank H22769 R59885 BI905854 R87379 AA037312 R39430 BI855956 H40732 R80287 H20904 R71951 H15995 R12880 R13988 H12138 R25804 R27505 R98179 T78984 R35160 R87370 H01632 R32534 R02835 T71406 H45110 BF112255 T80488 R48876 BE872084 H27657 R93684 H17600 H06235 H04585 BE467876 H23356 T74351 H83379 BG571413 R51450 H12175 H68047 H03783 H27730 BE272987 H01124 R00992 R13561 R14146 H23304 T80557 H38026 T74240 R02693 R25303 R67226 T78280 BQ009024 H23019 R09658 R10567 R67698 H23380 R95735 R94802 R77518 R36054 AI733547 T97532 R14363 H26658 R25307 H59180 BE899360 BM479478 H85310 BI253920 H25173 T74284 R25121 AI820826 R77522 H42171

Symbol MPP1 KCNT2 GDPD3 FXYD6 ATPAF1 TRIM24 LIPE PPARBP RNASE6 CCDC6 ZNF76 WDR85 TRUB1 EGR3 AP2B1 CAND2 TRIM52 HLA-DPA1 RGPD5 IGF2R SEMA6B GALC N4BP1 RAB12 TNFRSF10C IER5 TLK2 SUPT6H NAP1L4 VAV2 GIT1 SNX27 MAP1B FAM102B CYP19A1 SOX1 CADM1 JAKMIP2 MTSS1 PSG9 USP46 NRARP EPSTI1 CDH5 PPP2R1B NEK6 ADAMTS3 GLYAT MAPKAPK2 GSTM5 SYNGR1 HMG2L1 ARR3 HSPA1A UPF3A HSDL2 CNOT6L HAT1 SLC9A6 TGFBR1 MKI67IP CRBN HYAL4 VPS41 CDCA2 ZC3H13 LSM14A C6orf85 LONP2 SPTA1 HDAC5 MTA1 CACNA1D CYP39A1 PPT2 UNC5A SLC44A5 MVK NRG1 RGS4 ECHDC1 C10orf61 TMTC3 RAB6A

Cytoband Xq28 1q31.3 16p11.2 11q23.3 1p33-p32.3 7q32-q34 19q13.2 17q12-q21.1 14q11.2 10q21 6p21.3-p21.2 9q34.3 10q25.3 8p23-p21 17q11.2-q12 3p25.1 5q35.3 6p21.3 2q13 6q26 19p13.3 14q31 16q12.1 18p11.22 8p22-p21 1q25.3 17q23 17q11.2 11p15.5 9q34.1 17p11.2 1q21.3 5q13 1p13.3 15q21.1 13q34 11q23.2 5q32 8p22 19q13.2 4q12 9q34.3 13q13.3 16q22.1 11q23.2 9q33.3-q34.11 4q13.3 11q12.1 1q32 1p13.3 22q13.1 22q13.1 Xcen-q21 6p21.3 13q34 9q32 4q13.3 2q31.2-q33.1 Xq26.3 9q22 2q14.3 3p26.3 7q31.3 7p14-p13 8p21.2 13q14.12 19q13.11 6p25.2 16q12.1 1q21 17q21 14q32.3 3p14.3 6p21.1-p11.2 6p21.3 5q35.2 1p31.1 12q24 8p12 1q23.3 6q22.33 10q23.33 12q21.32 11q13.3

Score(d) -2,47 -2,47 -2,46 -2,46 -2,46 -2,46 -2,45 -2,44 -2,44 -2,44 -2,44 -2,44 -2,43 -2,42 -2,42 -2,42 -2,41 -2,41 -2,41 -2,41 -2,39 -2,39 -2,39 -2,39 -2,38 -2,38 -2,38 -2,36 -2,36 -2,36 -2,36 -2,35 -2,35 -2,35 -2,34 -2,33 -2,33 -2,33 -2,32 -2,32 -2,32 -2,32 -2,32 -2,31 -2,31 -2,30 -2,30 -2,30 -2,29 -2,29 -2,29 -2,29 -2,28 -2,26 -2,24 -2,23 -2,22 -2,22 -2,21 -2,20 -2,20 -2,20 -2,20 -2,18 -2,18 -2,17 -2,16 -2,16 -2,15 -2,14 -2,14 -2,13 -2,13 -2,12 -2,12 -2,12 -2,11 -2,11 -2,11 -2,10 -2,10 -2,10 -2,10 -2,10

GenBank R09977 R13941 H12683 BI092679 R55445 R24595 T95199 R59506 H16921 T65667 H63698 R22258 T83263 H48691 T71619 R28325 H51444 R19557 R36586 R24814 BE258194 H62270 BE880112 H62028 R35752 R17265 R09746 R17249 R88035 R51152 H08877 R52038 BI667959 R84665 R35150 R13758 H04030 R82740 BG680435 R60329 H54375 R63888 T83154 BQ054389 BM924060 H14396 R33743 R95822 H19860 H70133 H04421 R36515 BM476209 T99398 H16011 H84539 R35530 R97904 T78739 BM452073 BI257812 R94806 R32214 R31302 R96409 BM549305 R23489 R52735 R65925 R17860 H08441 T71508 BF001524 R88458 T96831 H60356 H08597 BQ073014 R71971 T75092 R67169 R26344 R24816 R20382

Symbol Cytoband C6orf85 6p25.2 PLEKHA5 12p12 PSG8 19q13.31 H19 11p15.5 SLC29A1 6p21.2-p21.1 SCRG1 4q31-q32 MT1JP 16q13 HERC5 4q22.1 PPEF1 Xp22.2-p22.1 RYR2 1q42.1-q43 NAPE-PLD 7q22.1 PIK3CA 3q26.3 NIF3L1 2q33 AASS 7q31.3 KNTC1 12q24.31 YPEL3 16p11.2 C1orf128 1p36.11 TMCC3 12q22 INPP5E 9q34.3 NTRK3 15q25 PWWP2 10q26.3 GHDC 17q21.2 SP2 17q21.32 DYRK3 1q32.1 P2RY14 3q24-q25.1 HGSNAT 8p11.1 GULP1 2q32.3-q33 ZSCAN16 6p22.1 CEP250 20q11.22-q12 F8 Xq28 MTP18 22q COL5A3 19p13.2 RTN1 14q23.1 ABCC8 11p15.1 ARHGAP19 10q24.1 SKIP 2q36 CLDN1 3q28-q29 STS Xp22.32 CUEDC1 17q23.2 BAG4 8p12 C4orf18 4q32.1 ZNF148 3q21 ORC6L 16q12 ATP5G1 17q21.32 SGCB 4q12 GDA 9q21.13 MTAC2D1 14q32.12 EPB41L2 6q23 CCDC136 7q33 RGS6 14q24.3 DUSP1 5q34 GJA5 1q21.1 DACH1 13q22 DYNC1H1 14q32.3-qter|14q32 ZCCHC11 1p32.3 YTHDF2 1p35 RAD23B 9q31.2 SPP1 4q21-q25 EPHB2 1p36.1-p35 DYRK4 12p13.32 BACE1 11q23.2-q23.3 NSMCE4A 10q26.13 DLX4 17q21.33 PCYOX1 2p13.3 SGCB 4q12 DDX17 22q13.1 ZNF354A 5q35.3 THAP8 19q13.12 TPCN1 12q24.13 TCEAL8 Xq22.1 PTPRA 20p13 KCNU1 8p11.22 PKP3 11p15 RANGAP1 22q13 IFNAR1 21q22.1|21q22.11 SH3PXD2A 10q24.33 PLCXD3 5p13.1 DBNDD2 20q13.12 TRIM41 5q35.3 PCDH17 13q21.1 CHAF1B 21q22.13 FAM46A 6q14 NCAPD3 11q25 PAP2D 1p21.3

Score(d) -2,09 -2,09 -2,08 -2,08 -2,07 -2,07 -2,07 -2,06 -2,06 -2,05 -2,05 -2,04 -2,04 -2,04 -2,03 -2,03 -2,03 -2,02 -2,02 -2,02 -2,02 -2,02 -2,00 -2,00 -2,00 -1,99 -1,99 -1,98 -1,98 -1,97 -1,97 -1,96 -1,96 -1,96 -1,96 -1,95 -1,95 -1,95 -1,94 -1,94 -1,94 -1,94 -1,94 -1,94 -1,94 -1,93 -1,93 -1,93 -1,93 -1,92 -1,92 -1,92 -1,91 -1,90 -1,90 -1,90 -1,89 -1,89 -1,88 -1,88 -1,87 -1,87 -1,85 -1,85 -1,85 -1,84 -1,84 -1,84 -1,84 -1,83 -1,83 -1,82 -1,82 -1,81 -1,81 -1,80 -1,80 -1,80 -1,80 -1,80 -1,80 -1,80 -1,79 -1,79

Sollazzo V. et al.

GenBank R82716 R10978 R12356 T74007 H50008 R10566 R15156 AI820766 R65643 R88547 R11718 R97935 R69649 R05983 H18866 H08348 R32695 R59315 R25125 H19859 H03156 T75516 BG698179 R71263 R97943 H47561 R92123 H06404 R72674 R23434 R69681 R17859 T70417 R27521 H16162 R55696 H03163 R11158 H10921 T64794 T75436 H28548 H30857 H42534 H10450 H20645 H43956 T75514

Symbol ISG15 ST7L MAOB NCSTN RNASET2 MSL3L1 KCNA6 ATP6V1B1 ATP8A1 HEPACAM TCF4 CDC34 PSG5 HAL CHRM3 SNRPB2 NNAT POLD3 NDUFC2 RABAC1 GFPT1 RREB1 ITPR2 TAF4 ZNF26 MRPL11 MRE11A PCTK1 C9orf6 MBTPS2 SLC16A5 ULK3 REC8 TRDN N4BP1 KIAA1107 SLC37A1 MRPL40 SCN3B SYMPK MAPK10 ADIPOQ MLH1 PRICKLE2 RAPH1 HS6ST1 C10orf116 KCTD6

Cytoband 1p36.33 1p13.2 Xp11.23 1q22-q23 6q27 Xp22.3 12p13 2p13.1 4p14-p12 11q24.2 18q21.1 19p13.3 19q13.2 12q22-q24.1 1q43 20p12.2-p11.22 20q11.2-q12 11q14 11q14.1 19q13.2 2p13 6p25 12p11 20q13.33 12q24.33 11q13.3 11q21 Xp11.3-p11.23 9q31.3 Xp22.1-p22.2 17q25.1 15q24.1 14q11.2-q12 6q22-q23 16q12.1 1p22.1 21q22.3 22q11.21 11q23.3 19q13.3 4q22.1-q23 3q27 3p21.3 3p14.1 2q33 2q21 10q23.2 3p14.3

Score(d) -1,78 -1,78 -1,78 -1,78 -1,77 -1,76 -1,76 -1,75 -1,75 -1,74 -1,73 -1,72 -1,72 -1,72 -1,71 -1,71 -1,70 -1,70 -1,70 -1,70 -1,69 -1,68 -1,68 -1,68 -1,68 -1,68 -1,68 -1,67 -1,67 -1,67 -1,67 -1,66 -1,66 -1,66 -1,66 -1,66 -1,66 -1,65 -1,65 -1,65 -1,65 -1,65 -1,65 -1,65 -1,64 -1,64 -1,64 -1,64

ies present in the existing literature have so far focused only on a few aspects of cell activities or they have been performed using different types of signals in different experimental conditions. The relevance of this study is that an overall analysis of the effects of PEMF on human osteoblast-like cells in vitro was made throughout microarray technology. Specifically the effect of PEMF exposure on human MG-63 osteosarcoma cell line was investigated. Hybridization of mRNA-derived probes to c-DNA microarrays allowed us to perform systemic analysis of expression profiles for thousand of genes simultaneously

GenBank H17693 R13127 R19718 H70413 H69624 R19760 R25565 BM995424 R18433 BG572617 H20342 R01835 R10051 H18570 R12018 H10630 R06495 R53686 H23402 R74000 R19537 T72724 T80698 T71409 T84526 R13054 T75247 H08892 AI792235 T78686 H46054 R19519 R11717 R55494 H01149 H77659 H21130 T74131 H03789 H58311 R12657 R78066 AI653890 BM127457 R50467 R13564 R22203 H59179

Symbol PIK3R1 CLPB STXBP5 NAG SFRS3 PPP1R16B ZNF616 ADD3 OPCML MTCH2 ZNF507 C10orf10 HKDC1 SLC1A2 MARK2 TMEM131 TP53AP1 MAST2 STIM2 GOLPH3L MBD5 KIAA1648 GLYATL1 XRN1 ZNF533 MMP16 PLEKHA7 SEMA4F PGPEP1 DOCK3 RHBDF1 BCR DUSP11 PLVAP INPP5D TUBG1 RANGAP1 MGC5566 IMPA1 F5 PAFAH1B3 MFSD2 DDX19A PAPPA2 PLA2G2A CXXC4 PRPF38B SPP2

Cytoband 5q13.1 11q13.4 6q24.3 2p24 6p21 20q11.23 19q13.33 10q24.2-q24.3 11q25 11p11.2 19q13.11 10q11.21 10q21.3 11p13-p12 11q12-q13 2q11.2 7q21.1 1p34.1 4p15.2 1q21.2 2q23.1 22q12.1 11q12.1 3q23 2q31.2-q31.3 8q21 11p15.1 2p13.1 19p13.11 3p21.31 16p13.3 22q11|22q11.23 2p13.2 19p13.2 2q37.1 17q21 22q13 20q13.12 8q21.13-q21.3 1q23 19q13.1 1p34.2 16q22.1 1q23-q25 1p35 4q22-q24 1p13.3 2q37-qter

Score(d) -1,64 -1,63 -1,63 -1,63 -1,62 -1,61 -1,61 -1,61 -1,61 -1,61 -1,61 -1,60 -1,60 -1,59 -1,59 -1,59 -1,58 -1,58 -1,58 -1,58 -1,57 -1,57 -1,57 -1,57 -1,56 -1,56 -1,56 -1,55 -1,55 -1,55 -1,55 -1,55 -1,55 -1,55 -1,55 -1,55 -1,55 -1,55 -1,55 -1,55 -1,54 -1,54 -1,54 -1,53 -1,53 -1,53 -1,53 -1,52

and to provide primary information on transcriptional changes related to PEMF action. Since DNA microarray is a molecular technology that enables the analysis of gene expression in parallel on a very large number of genes, spanning a significant fraction of the human genome, it gives a global view on the genetic effect of a factor on a cell system. In fact DNA microarray not only is a quantitative analysis but also is a qualitative one, differentiating a single gene among thousand sequences. We can have do the same observations for the TE-85 cells, via others genes. In response to PEMF, TE-85 cell line up-

GenBank T81514 R06513 BM014426 BF058993 T95376 R07975 BG563170 R21172 T97408 H61757 T77427 H84549 R60412 T97543 H58086 H46384 H10341 R22997 R73462 R19755 BI871497 H03146 H13007 T95213 N28016 R17328 H45525 T77476 H58361 BE535315 T79975 T77428 T82030 BI260503 R02753 R70843 R73278 T97909 H03906 T77421 H62959 T80020 H23315 R70894 T67154 H08432

Symbol TGOLN2 CD5L DAB2IP COL6A3 SBNO2 C1orf50 CHN2 CREB5 BAG1 ELK4 ZNF783 C22orf32 SAFB TMEM44 ADCY7 GSDML SULT4A1 SLC30A2 ATHL1 SCN8A SF3A1 OSBPL10 PCDH19 PRPF38B GALT LRRC29 RHOG GNA13 SLC35A4 GLB1 AGGF1 ELOVL5 MED10 SEC22C COBL HTRA1 ICAM3 KIAA0232 FN1 DUSP26 ENO3 MAP4 PTPRR TGIF1 IMMP2L SNUPN

Cytoband 2p11.2 1q21-q23 9q33.1-q33.3 2q37 19p13.3 1p34.2 7p15.3 7p15.1 9p12 1q32 7q36.1 22q13.2 19p13.3-p13.2 3q29 16q12-q13 17q12 22q13.2-q13.31 1p35.3 11p15.5 12q13 22q12.2 3p22.3 Xq13.3 1p13.3 9p13 16q22.1 11p15.5-p15.4 17q24.3 5q31.3 3p21.33 5q13.3 6p21.1-p12.1 5p15.31 3p22.1 7p12.1 10q26.3 19p13.3-p13.2 4p16.1 2q34 8p12 17pter-p11 3p21 12q15 18p11.3 7q31 15q24.2

123

Score(d) -1,52 -1,52 -1,51 -1,51 -1,51 -1,50 -1,50 -1,49 -1,49 -1,49 -1,49 -1,49 -1,49 -1,48 -1,48 -1,48 -1,48 -1,47 -1,47 -1,47 -1,47 -1,47 -1,46 -1,46 -1,46 -1,46 -1,45 -1,45 -1,45 -1,44 -1,44 -1,44 -1,44 -1,44 -1,44 -1,44 -1,44 -1,43 -1,43 -1,43 -1,42 -1,42 -1,41 -1,41 -1,41 -1,41

regulate and down-regulate several genes related to osteogenesis. Among the up-regulated genes, ATF4 (activating transcription factor 4) was interesting in that it encodes a transcription factor also isolated and characterized as the cAMP-response element binding protein 2 (CREB-2) that function together with Runx2 to control the transcriptional activity of mature osteoblasts. ATF4 interacts with Runx2 to regulate the transcriptional activity of osteocalcin23 and may mediate some of the responses of osteoblasts to PTH.24 ATF4, finally, phosphorylated by the kinase Rsk2, controls amino acid transportation in osteoblasts, an im-

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124 Sollazzo V. et al.

Journal of Osteology and Biomaterials

and modulating the gene expression of osteoblasts and / or through the degradation of specific matrix proteins29. SSP1 encodes for osteopontin (OPN), one of the critical mediators required for unloading-induced bone loss30. ADAMTS 3 is responsible of pro-collagen II degradation and MMP16 actives MMP-2, a matrix metallopeptidase involved in arthritis31. PEMF also modulate the expression of CACNG4 that decreases calmodulin32. In conclusion, our data provides a detailed description of the effects of PEMFs on human osteoblast-like cells in vitro. PEMFs seem to exert an anabolic effect on cells. In particular, they are consistent with the abundant preclinical and clinical findings demonstrating a positive effect of PEMFs on osteogen-

esis. PEMFs stimulation induces bone healing in patients, shortens the time of the healing processes, and stimulates the healing of nonunions. Exposure to PEMFs acts on cell behavior in different ways. More specifically, PEMFs stimulate cell proliferation and induce both osteoblastogenesis and differentiation of osteoblasts. Moreover, PEMFs promote ECM apposition and mineralization, while at the same time decrease the degradation and absorption processes of ECM. These data suggest a more comprehensive explanation of the observed clinical effect of PEMFs on the induction of osteogenesis. Given their broad effects, PEMFs might be useful in other fields such as regenerative medicine.

SAM Plotsheet

Observed Score

portant step in bone formation. ATF4 invalidation leads to a severe bone phenotype characterized by decreased bone formation: inactivation of Rsk2 leads to the Coffin â&#x20AC;&#x201C;Lowry syndrome whereas increased Rsk2 activity leads to neurofibromatosis type25. The down-regulated genes induced by PEMF, interestingly was HDAC5 that deacetylate Runx2, allows the protein to undergo Smurf-mediated degradation. It has been observed that pharmacological inhibition of HDACdependent deacetylation enhances the osteogenic activity of BMP-2, by increasing Runx2 activity and stability. The consequent osteoblast differentiation and increase bone formation may provide a new theoretical basis for developing therapeutic agents against osteoporosis 26. PEMF also down-regulate DUSP26 and DUSP5 that have a phosphatase action inhibiting the MAP kinase27. The down regulation of genes like TNFRSF10C, HTRA1, SPP1, ADAMST3 and MMP16 causes bone formation. TNFRSF10C is responsable of the osteoclasts activation and HTRA1 inhibit osteoblasts mineral deposition and the matrix calcification by the abolition of BMP-2 signal28. HTRA1 encodes a member of the family of serine proteases and is considered a regulator of cell growth. HtrA1 promotes cartilage degradation by inhibiting the TGF-beta signal and digesting components of cartilage matrix as aggrecano, decorin, fibromodulina and collagen type II soluble. HTRA1 inhibits mineral deposition by osteoblasts. This suggests that HTRA1 may regulate matrix calcification trough the inhibition of BMP-2 signal

Expected Score

Figure 2. A statistical analysis (see materials and methods chapter) of microarray (SAM) plot of TE85 exposed to PEMFs versus control is shown. Expected differentially expressed genes are reported on the x axis, whereas observed differentially expressed genes are reported on the y axis. Downregulated genes (green) are located in the lower left side of the graph; upregulated genes (red) are in the upper right side; genes with different expression but statistically insignificant are shown in black. Parallel lines drawn from the lower left to upper right squares are the cutoff limits. The solid line indicates the equal value of observed and expected differentially expressed genes. Significant: 1327; Median number of false positives: 0; False Discovery Rate (%): 0. Tail strength (%): 53.7; se (%): 76.5.

Sollazzo V. et al.

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6. Mammi GI, Rocchi R, Cadossi R, Massari L, Traina GC. The electrical stimulation of tibial osteotomies. Double-blind study. Clin Orthop Relat Res 1993;246-53.

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P-15 cell-binding domain derived from collagen: analysis of MG63 osteoblastic-cell response by means of a microarray technology. J Periodontol 2004;75:66-83. 21. Carinci F, Piattelli A, Stabellini G, Palmieri A, Scapoli L, Laino G, Caputi S, Pezzetti F. Calcium sulfate: analysis of MG63 osteoblast-like cell response by means of a microarray technology. J Biomed Mater Res B Appl Biomater 2004;71:260-7. 22. Carinci F, Volinia S, Pezzetti F, Francioso F, Tosi L, Piattelli A. Titanium-cell interaction: analysis of gene expression profiling. J Biomed Mater Res B Appl Biomater 2003;66:341-6. 23. Xiao G, Jiang D, Ge C, Zhao Z, Lai Y, Boules H, Phimphilai M, Yang X, Karsenty G, Franceschi RT. Cooperative interactions between activating transcription factor 4 and Runx2/Cbfa1 stimulate osteoblast-specific osteocalcin gene expression. J Biol Chem 2005;280:30689-96. 24. Jiang D, Franceschi R T, Boules H, Xiao G. Parathyroid hormone induction of the osteocalcin gene. Requirement for an osteoblast-specific element 1 sequence in the promoter and involvement of multiple-signaling pathways. J Biol Chem 2004;279:532937. 25. Yang X, Matsuda K, Bialek P, Jacquot S, Masuoka HC, Schinke T, Li L, Brancorsini S, Sassone-Corsi P, Townes TM, Hanauer A, Karsenty G. ATF4 is a substrate of RSK2 and an essential regulator of osteoblast biology; implication for Coffin-Lowry Syndrome. Cell 2004;117:387-98. 26. Jeon EJ, Lee KY, Choi NS, Lee MH, Kim HN, Jin YH, Ryoo HM, Choi JY, Yoshida M, Nishino N, Oh BC, Lee KS, Lee YH, Bae SC. Bone morphogenetic protein-2 stimulates Runx2 acetylation. J Biol Chem 2006;281:16502-11. 27. Vasudevan SA, Skoko J, Wang K, Burlingame SM, Patel PN, Lazo JS, Nuchtern JG, Yang J. MKP-8, a novel MAPK phosphatase that inhibits p38 kinase. Biochem Biophys Res Commun 2005;330:511-8.

Journal of Osteology and Biomaterials

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LEADING LEADINGREGENERATION REGENERATION