The Role of Cellulose in the Formulation of Interconnected Macro by MMSE Journal

Mechanics, Materials Science & Engineering, April 2017 – ISSN 2412-5954

The Role of Cellulose in the Formulation of Interconnected Macro and Micoporous Biocompatible Hydroxyapatite Scaffolds 35 J. Anita Lett1, M. Sundareswari1, K. Ravichandran2, Amirdha Sher Gill1, J. Joyce Prabhkar3 1 – Department of Physics, Sathyabama University, Chennai, India 2 – Department of Analytical Chemistry, University of Madras, Chennai, India 3 – Department of General Surgery, Madras Medical College, Chennai, India DOI 10.2412/mmse.62.43.650 provided by Seo4U.link

Keywords: bone tissue engineering, pure hydroxyapatite scaffolds, cellulose, porosity.

ABSTRACT. In bone tissue engineering, ceramics are widely used as implant material to enhance bone growth formation or as drug release vehicle. In the existing work porous Hydroxyapatite scaffolds were prepared by polymeric replication method using Cellulose as a binding agent. The influence of binder on various sintering temperature were evaluated. The Hydroxyapatite scaffold sintered at 1150°C was characterized for phase purity, structural analysis and porosity measurements. Hence, it is possible to produce Hydroxyapatite scaffolds with highly inter connecting macro and micro pores with an apparent density of 0.944g/cm3 corresponding to 75% porosity.

Introduction. Tissue engineering is a field where cells, bone/ scaffolds and signals/factors are mutually joined with the endeavour to re-establish, preserve and improve tissue and organ utility. Various scaffold resources have been investigated worldwide with both positive and negative outcomes. Factors for the failure of scaffolds include undesired scaffold degradation commodities affecting cellular functions, unaffected responses elicited by the scaffold materials themselves, lack of cell adhesion appropriate to non-suitable surface properties, controversies in the degradation rate of the scaffold and the growth rate of the fresh tissue, or mechanical mismatches connecting scaffolds and the tissue at the implantation location. Calcium phosphates are amongst the most widely used resources for bone tissue regeneration. They can be man-made as gels, pastes and solid blocks or even as porous matrices, with orthopaedics and dentistry being their main areas of relevance. Hydroxyapatite (HAP) are the most frequently used calcium phosphates, owed to their Stoichiometric ratio (Ca/P) ratios close to that of natural bone and also for their stability when in contact with physiological environment. HAP is a major constituent of bone resource and is resorbed after a long time in the body, due to its biocompatibility [1-4]. The porous network or interconnected pores in HAP structure permit the tissue to penetrate, which further enhances the implant tissue attachment (Itoh et al). Several methods have been investigated to achieve the required porous scaffolds for instance, Sopyan et al has studied with pore-creating volatile particles, ceramic foaming methods and polymeric sponge process [5]. The polymeric sponge technique, which offers great flexibility, is particularly of interest due to its greater advantages such as opportunity to control the pore size, for several required complex shapes and straightforward process (Tian and Tian 2001).The polymeric sponge technique involves covering of open-cell polymeric foam with ceramic slurry followed by flaming out of polymeric foam in the course of sintering process which yields a duplication of the original polymer foam in the ceramic foam structure. However, the properties of the Hydroxyapatite scaffold prepared through the polymeric sponge technique are highly depend on the slurry properties together with homogeneity, rheology and dispersion (Zhang et al 2006). Monmaturapoj has reported 35

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the dispersant on the rheological behaviour of concentrated hydroxyapatite suspensions [6]. In this study, porous hydroxyapatite scaffolds were obtained using the polymer replication method, using cellulose as a binding agent and the morphology and its physio-chemical properties were studied. These scaffolds can be used as matrices for bone tissue engineering or as specific release vehicles [7]. Also, they may be functionalized with molecules such as collagen, chitosan, etc, in order to enhance their biological responses [8]. Material and Methodology. Sample Preparation. The Hydroxyapatite powder with an average crystallite size of approximately 40 nm, was prepared according to our previous work by sol-gel [9].The template used to prepare scaffolds were polyurethane sponges with an average pore size of 400 microns. The Cellulose, used as a binder was made into a homogenous mixture by mixing for 2 hr with water at 50 ºC. To fabricate scaffolds, a ceramic slurry containing 6 g of Hydroxyapatite powder, binder, tensioactive agent (A40 V Dispex provided from BASF) and water [6] were blended again for 2 hours to disperse thoroughly. The polyurethane sponges were dipped in the slurries and the excess slurry was removed and dried at room temperature for 2 days, followed by drying in the hot air furnace at 110 ºC until its completely dried. The polyurethane sponge used as template was removed by heating in a muffle furnace at 600oC for 2 hrs, followed by the densification of scaffold by sintering at 1150 oC for 4 hours. Now, these prepared scaffolds were cut into cubes for further characterization. Physio-Chemical Characterization. The microstructure of the hydroxyapatite scaffolds was characterized using scanning electron microscopy (FESEM: Supra VP35 Carl Zeiss, Germany) and the macro porous structure using optical stereo zoom microscope. The phase purity of the Hydroxyapatite scaffolds were determined by X-ray diffraction using a X’pertPro, Philips, The Netherlands with CuKα radiation over the 2θ range of 10°–80° with a step size of 0.05°. The functional group analysis of HAP scaffolds was carried out in the spectral range from 4000 to 650 cm−1 using a single beam Fourier transform infrared spectrometer (Agilent, Cary 630). The porosity and density measurements of the scaffolds were calculated by simple displacement techniques [8-9]. A scaffold of weight ‘W’ was immersed in a graduated cylinder containing a known volume (V1) of water until no air bubble emerged from the scaffold. The total volume of the water and scaffold was then recorded as V2. The volume difference (V2 – V1) was the volume of the skeleton of the scaffold. The scaffold was removed and the residual water was measured as V3. The apparent density of the scaffold (ρ), was evaluated using,



W V2  V3

(1)

The porosity of the open pores in the scaffold (ε), was evaluated using,



V1  V3 V2  V3

(2)

Results and Discussions. The nano-HAP powder used for fabricating scaffolds processed an elongated cylindrical shape with the crystallite size of 40 nm. The scaffolds were approximately 10 mm x 10 mm x 10 mm in size. The morphology of the fabricated Hydroxyapatite is shown in Fig.1, 2. It is found that the scaffolds replicated the pores in the sponges with a pore size of approximately 500 microns. Thus, highly porous Hydroxyapatite scaffolds were produced using the polymer MMSE Journal. Open Access www.mmse.xyz 144

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replication method as seen in Fig. 2.With SEM observations (Fig. 2), pore diameters ranging from 400 to 500 μm were observed. On the other hand, micropores (Fig. 3) of size lesser than micron were also visualised in the pore walls and wall struts. The microstructures of the scaffolds (Fig. 2) could be observed that the scaffold had a compact structure and that the pores were evenly distributed. The open as well as interconnected pore network was an essential factor for the scaffold to permit cell growth and the transportation of nutrients and metabolic waste. Gotz et al.[13] has reported that pore sizes around 300 µm were suggested for implants due to improved new bone and capillary formation. Hollister et al[14] conducted in vivo studies on HAP scaffolds with pore diameters ranging between 400 µm and 1200 µm and inferred no significant difference in bone growth for scaffolds of all pore sizes.

Fig. 1. Macro pores of Scaffold.

2µm Fig. 2 Micro pores of Scaffold.

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Fig. 3. XRD pattern of Sintered Scaffold.

40 20

3500

3000

2500

2000

1500

1000

635.08 603.13 569.83 479.08 464.80 448.99 440.38

1093.41 1041.49

1639.41

2078.54 2003.14 1934.21

3448.35

-20

-0

Transmittance [%]

100

The XRD patterns of the scaffolds prepared is shown in Fig. 3. All of the peaks matched with the JCPDS pattern 09-0432 for HAP, which suggested that no other phases were present, as shown in Fig. 3. All of the peaks were attributed to the HAP phase and no additional peaks were observed. The results indicated that the HAP did not decompose after sintering. The characteristic peaks at two theta 31.7° corresponding to 211 diffraction became narrower and sharper for sintering temperature 1150°C. These data confirms that the major phase as Hydroxyapatite and absence of impurity such as calcium phosphates and calcium oxide are clearly identified [22] .

500

Wavenumber cm-1

Fig. 4. FTIR pattern of Sintered Scaffold. The FT-IR spectra of the synthesized HAP scaffold prepared using cellulose as a binding agent is shown in Fig.4. In the FTIR spectra, the bands at 3570 and 630 cm-1[15] were recognized to the hydroxyl stretching bands and bending bands of HAP, respectively. The broad absorption band from 3600 to 3300 cm-1[15] indicated the existence of the bending mode of absorbed water[16]. The bands at 1093 and 1041 cm-1 were assigned antisymmetric ʋ3 [PO43− ] P-O stretching mode and the ʋ1 P-O symmetric stretching mode was detected at 962 cm-1 [15, 17-18]. The bands at 603 and 569 cm-1 were attributed to components of the triply degenerate ʋ4 O-P-O bending modes. The Scaffold prepared using cellulose showed high porosity of 75% with apparent density of 0.944 g/cm3. The mechanical properties are strongly subjective by apparent density [18]. In trabecular bone,

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the apparent density ranges from 0.14g/cm3 to 1.10g/cm3 [18]. Porosity is based on the presence of open pores. It was found that mechanical properties varied with binders used. Moreover, a few studies [19, 20] have reported that the decomposition of HAP would reduce its mechanical properties. Hence, a symmetry is to be maintained between the porosity and apparent density for precise purposes, since higher mechanical strength corresponds to higher density, while a high porosity provides a surrounding favourable for living organism[21]. Summary. In this work, porous scaffolds were prepared using a polymeric sponge template method using cellulose as a binding agent. A well defined elongated cylindrical HAP crystals with negligible agglomeration was used to fabricate these scaffolds. The FESEM results exposed that the porous Hydroxyapatite scaffolds acquired macro pores and micro pores that emerges to be interconnected with a homogenous porous network (Fig. 3).The scaffolds comprises pure crystalline Hydroxyapatite phase and no additional phase were produced through the spongy technique as confirmed by XRD and FTIR. The scaffold prepared with cellulose in spite of high porosity (75%) appeared to report an apparent density of 0.944 g/cm3 comparable with trabecular bone (0.14 g/cm3 to 1.10 g/cm3). Thus, it is possible to produce porous scaffolds with varied porosity and density. Such scaffolds can find its application for tissue engineering in non load bearing applications or even as a vehicle for the delivery of biological molecules. Currently, studies are being performed in order to incorporate collagen type I in these porous constructs, to improve their potential applications. References [1] J.R. Woodard, A.J. Hilldore, S.K. Lan, C.J. Park, A.W. Morgan, J.A. Eurell, et al., ” The mechanical properties and osteoconductivity of hydroxyapatite bone scaffolds with multi-scale porosity“, Biomaterials 28 (1) (Jan 2007) 45. [2] L.D. Harris, B.S. Kim, D.J. Mooney, ” Open pore biodegradable matrices formed with gas foaming”, Journal of Biomedical Materials Research 42 (3) (Dec 5 1998) 396. [3] L.A. Cyster, D.M. Grant, S.M. Howdle, F.R. Rose, D.J. Irvine, D. Freeman, et al., ” he influence of dispersant concentration on the pore morphology of hydroxyapatite ceramics for bone tissue engineering”, Biomaterials 26 (7) (Mar 2005) 697. [4] Q. Fu, M.N. Rahaman, B.S. Bal, W. Huang, D.E. Day, ” Freeze Extrusion Fabrication of 13-93 Bioactive Glass Scaffolds for Bone Repair“, Journal of Biomedical Materials Research A 82 (1) (Jul 2007) 222. [5] Sopyan, Porous hydroxyapatite for artificial bone applications, Science and Technology of Advanced Materials 8 (2007) 116–123, [6] N. Monmaturapoj, Influence of preparation method on hydroxyapatite porous scaffolds, Bull. Mater. Sci., Vol. 34, No. 7, December 2011, pp. 1733–1737. I [7] T.M. Chu, D.G. Orton, S.J. Hollister, S.E. Feinberg, J.W. Halloran, ”MecCalcium biomineralization in the radular teeth of the chiton, Acanthopleura hirtosahanical and in vivo performance of hydroxyapatite implants with controlled architectures“, Biomaterials 23 (5) (Mar2002) 1283. [8] A. Tampieri, G. Celotti, S. Sprio, A. Delcogliano, S. Franzese, Biomaterials 22 (2001) 1365. [9] J. Anita Lett, M. Sundareswari, K. Ravichandran, Porous hydroxyapatite scaffolds for orthopedic and dentalapplications - the role of binders, Materials Today: Proceedings 3 (2016) 1672–1677 [10] S.M. Zhang, F.Z. Cui, S.S. Liao, Y. Zhu, L. Han, ” Synthesis and biocompatibility of porous nano-hydroxyapatite/collagen/alginate composite”, Journal of Materials Science 14 (7) (Jul 2003) 641. [11] S. Yunoki, T. Ikoma, A. Monkawa, E. Marukawa, S. Sotome, K. Shinomiya, et al., Journal of Biomaterials Science 18 (4) (2007) 393. MMSE Journal. Open Access www.mmse.xyz 147

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[12] P. Sepulveda, F.S. Ortega, M.D.M. Innocentini, V.C. Pandolfelli, Journal of the American Ceramic Society, ” Properties of Highly Porous Hydroxyapatite Obtained by the Gelcasting of Foams”, 83 (12) (Dec 2000) 3021. [13] Gotz, H. E. et al. Effect of surface finish on the osseointegration of laser-treated titanium alloy implants. Biomaterials 25, 4057–4064 (2004). [14] Hollister, S. J. et al. Engineering craniofacial scaffolds. Orthod. Craniofac. Res. 5, 162–173 (2005). [15] Mehdi Kazemzadeh Narbat, Fariba Orang, Mehran Solati Hashtjin and Azadeh Goudarzi, “Fabrication of Porous Hydroxyapatite-Gelatin Composite Scaffolds for Bone Tissue Engineering”, Iranian Biomedical Journal 10 (4): 215-223 (October 2006) [16]. C. Guzmán Vázquez, C. Piña Barba and N. Mungu´ıa, ; Revista Mexicana De Fi´Sica, ” Stoichiometric hydroxyapatite obtained by precipitation and sol gel processes”, Vol.51, No.3, pp. 284–293, 2005. [17] T. Anee Kuriakose, S. Narayana Kalkuraa, M. Palanichamy, D. Arivuoli, Karsten Dierks, G. Bocelli, C. Betzel,, ” A novel low temperature sol–gel synthesis process for thermally stable nano crystalline hydroxyapatite, “Journal of Crystal Growth Vol.263, pp.517–523, 2004. [18] Evans, L.A., Macey, D.J. and Webb, ” Calcium biomineralization in the radular teeth of the chiton, Acanthopleura hirtosa“, (1992), Calcif Tissue Int. 51: 78-82. [19] Li, S., Izui, H., Okano, M. &Watanabe, T. The effects of sintering temperature andpressure on the sintering behavior of hydroxyapatite powder prepared by spark plasma sintering. J. Biomech. Eng. 3, 1–12 (2008). [20] Khalil, K. A., Won Kim, S. & Kim, H. Y. Consolidation andmechanical properties of nanostructured hydroxyapatite- (ZrO2 1 3 mol% Y2O3) bioceramics by highfrequency induction heat sintering. Mat. Sci. Eng. A-Struct. 456, 368–372 (2007). [21] Gu, Y.W., Loh, N. H., Khor, K. A., Tor, S. B. & Cheang, P. Spark plasma sintering of hydroxyapatite powders. Biomaterials 23, 37–43 (2002).

Cite the paper J. Anita Lett, M. Sundareswari, K. Ravichandran, Amirdha Sher Gill, J. Joyce Prabhkar (2017). The Role of Cellulose in the Formulation of Interconnected Macro and Micoporous Biocompatible Hydroxyapatite Scaffolds. Mechanics, Materials Science & Engineering, Vol 9. doi:10.2412/mmse.62.43.650

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