Intraoperative Adjunctive Stem Cell Treatment in Patients with Critical Limb Ischemia

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Intraoperative Adjunctive Stem Cell Treatment in Patients with Critical Limb Ischemia Using a Novel Point-of-Care Device R. Kolvenbach,1 Carla Kreissig,1 Catherine Cagiannos,1 Rana Afifi,2,3 and Eva Schmaltz,1 Du¨sseldorf, Germany

Introduction: In a prospective trial we tested whether adjunctive intraoperative stem cell treatment in patients with critical limb ischemia (CLI) can be performed safely in combination with bypass surgery and/or interventional treatment. The end point of our study was the safety and integrity of a novel point-of-care system used in patients with CLI. Methods: We included only patients with CLI and tissue loss according to Rutherford categories 4-6. The Harvest Bone Marrow Aspirate Concentrate System consists of an automated, microprocessor-controlled dedicated centrifuge with decanting capability and the accessory BMAC Pack for processing a patient’s bone marrow aspirate (BMA). The centrifuge is portable and enables BMA to be rapidly processed in the operating room to provide an autologous concentrate of nucleated cells for immediate injection. The surgeon aspirated 120 ml BMA from the iliac crest.

Results: Eight consecutive patients were treated according to the study protocol. The mean follow-up period was 9.2 months (range 2-18). Stem cells were always injected during the final revascularization attempt. One minor amputation and two major amputations were required. In five of eight patients there was a discrete increase in the ankle-brachial index postestem cell treatment. The dose of stem cells after centrifugation was 17.2 (range 13.8-54.2) 10E6 CD34-positive cells and 7.8 (range 1.8-35.9) 10E6 CD133-positive cells. The injected dose of VEGFR-2-coexpressing stem cells was 0.5-5.7 10E4.

Conclusion: We were able to show that the buffy coat preparation using a point-of-care system is a simple and fast method to enrich stem cells from BMAs. This automated system gives high recovery rates and good reproducibility.

INTRODUCTION Bone marrowederived stem cells not only give rise to all cell lines of the hematopoietic system but also seem to have the potential to transdifferentiate into somatic cell lines. Observations in animal models and experimental studies in humans suggest 1 Department of Vascular Surgery and Endovascular Therapy, Augusta Hospital and Catholic Clinics, Du¨sseldorf, Germany.

2 Stem Cell Department of Red Cross Blood Service West, Du¨sseldorf, Germany.

3 Department of Vascular Surgery, Carmel Medical Center, Haifa, Israel.

Correspondence to: Ralf Kolvenbach, MD, PhD, Department of Vascular Surgery and Endovascular Therapy, Augusta Hospital and Catholic Clinics, Du¨sseldorf, Germany, E-mail: Kolvenbach@VKKD-Kliniken.de Ann Vasc Surg 2009; -: 1-5 DOI: 10.1016/j.avsg.2009.07.018 Ó Annals of Vascular Surgery Inc. Published online: --,-

that these cells can initiate blood vessel growth in ischemic regions.1,2 Peichev et al.3 defined CD34positive stem cells which coexpress CD133 and VEGFR-2 as endothelial progenitor cells (EPCs).

In a prospective trial we tested whether adjunctive intraoperative stem cell treatment in patients with critical limb ischemia (CLI) can be performed safely in combination with bypass surgery and/or interventional treatment.

The end points of our study were limb salvage and integrity of the novel point-of-care system used. Concerning cell yield, we wanted to answer three questions: How many stem cells can be harvested in 120 mL bone marrow aspirate (BMA)?

How is the stem cell recovery after preparation of the mononuclear cell fraction from BMAs? Is there a correlation between stem cell count administered to the patient and clinical outcome?4 We report for the first time the intraoperative use of a point1

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Annals of Vascular Surgery

of-care device which permits stem cell treatment as an adjunct to bypass surgery or interventional therapy.

Cell viability was determined by flow cytometry with 7AAD staining. Examinations were done on bone marrow samples before and after preparation.

MATERIALS AND METHODS

Device Description and Sample Preparation

The clinical study was performed in cooperation with the Stem Cell Department of Red Cross Blood Service West. Eight consecutive patients were prospectively included in the study. Informed consent was obtained in all cases, explaining to the patient the experimental character of cell treatment.

The study was initiated after institutional review board approval.

We included only patients with CLI and tissue loss according to Rutherford categories 4-6. In order to have a uniform group of patients, cases with thromboangitis obliterans (Buerger disease) were excluded. Exclusion criteria were as follows: 1. Life expectancy <6 months due to concomitant illnesses.

2. Known active malignancy or results outside of normal limits from the following tests: peroxidaseeantiperoxidase, chest X-ray, prostate-specific antigen, mammogram, and Hemoccult unless follow-up studies reveal patient to be cancer-free.

3. Medical risk that precludes anesthesia (conscious sedation) or American Society of Anesthesiologists class 5.

4. Life-threatening complications of ischemia necessitating immediate amputation.

5. Uncorrected iliac artery occlusion on index side.

Patients were followed at monthly intervals. The major end point of this study was limb salvage. Additional parameters studied were pain, ankle-brachial index (ABI), ulcer healing, and amputation. We performed pre- and postinterventional magnetic resonance angiography studies (1.5 T; Siemens Magnetom, Erlangen, Germany). The study was combined with contrast injection to provide dynamic clinical information, including the evaluation of abnormal vascular anatomy as well as vascular hemodynamic and perfusion measurements. Pedal arch anatomy could not in all cases be studied due to software problems. Calculation of runoff scores therefore concentrated on tibial and peroneal arteries.

To determine the cell count of EPCs, we used fluorescence-activated cell sorting analysis with EPICS-XL, a four-color flow cytometer (Beckman Coulter, Fullerton, CA). Antibodies were provided from R&D Systems (Minneapolis, MN; VEGFR-2) and Beckman Coulter (CD34, CD133, and CD45).

The Smart Prep BMAC system is designed to concentrate a buffy coat of 20 mL from whole bone marrow aspirate of 120 mL. The SmartPReP2Ă&#x2019; Bone Marrow Aspirate Concentrate System (Harvest, Plymouth, MA) consists of an automated, microprocessorcontrolled dedicated centrifuge with decanting capability and the accessory BMAC IDE PAD Pack for processing a patientâ&#x20AC;&#x2122;s BMA. The centrifuge is portable and enables BMA to be rapidly processed in the operating room to provide an autologous concentrate of nucleated cells for immediate injection. The accessory pack contains processing kits including a functionally closed dual-chamber sterile processing disposable that allows the preparation of an autologous concentrated cellular product from a small volume (120 mL) of BMA (Fig. 1). There are two containers in the kit, and hence in the centrifuge, each with a volume of 60 mL. After one run, 10 mL can be gained from each container. A maximum volume of 20 mL can be injected.

The surgeon aspirated 120 mL BMA from the iliac crest. During processing, a floating disk in the marrow chamber automatically rises to the top of the packed red cell layer as it builds during centrifugation, separating the red cells from plasma (containing all other cells). Plasma is then automatically decanted into the BMAC chamber of the processing disposable while the floating separation disk minimizes the transfer of red cells. The sample is then centrifuged further, producing a cell concentrate button and a layer of cell-poor plasma. At the completion of the processing cycle, each processing disposable is removed from the centrifuge and a specified volume of plasma is aspirated out of the chamber using a sterile syringe and spacer that automatically leaves a sufficient volume of plasma for resuspending the cell concentrate button. The ready-to-inject stem cell fraction has a volume of 20 mL (Fig. 2).

The pattern of injection sites was linear, overlying the areas of critical limitation to arterial flow (tibial or pedal arteries) and as distal as possible. In contrast to other protocols, we did not inject into the proximal gastrocnemius muscle, to have an optimal concentration at the site where it was needed. Injections were spaced 1-2 cm apart; therefore, a total length of 20-40 cm could be treated. If an ulcer or tissue loss was present, injections were placed

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Fig. 1. Schematic drawing of stem cell processing. In each case 120 ml of bone marrow are harvested. This requires two container sets. A total amount of 20 ml of mononuclear cell fraction can be obtained.

around the perimeter of the ulcer/wound spaced at intervals of 1-2 cm and extended under the ulcer bed/wound as appropriate. All injections were performed immediately after harvesting and cell processing, before the surgical procedure.

RESULTS Eight consecutive patients were treated according to the study protocol. The mean follow-up period was 9.2 months (range 2-18). All patients suffered from CLI, rest pain, tissue necrosis, or gangrene. The mean age of the patients was 62.3 (range 39-81) years. All patients had at least one bypass operation, and 4/8 had two bypass attempts; in four cases additionally an endovascular procedure had been performed prior to stem cell treatment (Table I). Stem cells were always injected during the last revascularization attempt. One minor amputation and two major amputations were required (Table II).

Four of eight cases had bypass occlusion due to poor or nonexistent runoff. In two of these an attempt was made to perform bypass thrombectomy but without success. One of these cases developed gangrene and septic symptoms requiring urgent major limb amputation. There was no correlation between stem cell treatment and bypass patency considering the large number of patients where a saphenous vein bypass was no longer an option.

Cell counts and hematocrit did not change after bone marrow aspiration and stem cell injection.

In five of eight patients there was an increase in the ABI postestem cell treatment. The number of patients in this safety and feasibility trial was too small to show a correlation between stem cell treatment and an increase in ABI. There was no correlation between cell counts and clinical outcome in this small cohort of patients. Therefore, bypass patency could not be analyzed in relation to cell numbers.

There were no side effects related to bone marrow aspiration or to intramuscular stem cell injection.

Cell Analysis Within 20 min bone marrow aspiration and buffy coat preparation were completed. After sample withdrawal for quality control, the stem cell product was ready for injection. The dose of stem cells after centrifugation was 17.2 (range 13.8-54.2) 10E6 CD34-positive cells and 7.8 (range 1.8-35.9) 10E6 CD133-positive cells. The injected dose of VEGFR2-coexpressing stem cells was 0.5 to 5.7 10E4.

In our experience there was a learning curve of two cases to obtain perfect recovery rates for CD34-positive cells (41-99%), CD133-positive cells (37-99%), and EPCs (13-67%). Up to 99% of cells were viable after centrifugation (Table III).

DISCUSSION Stem cells can be administered in different ways and from different sources: Among these are stem cells from peripheral blood, bone marrow, or umbilical

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Fig. 2. Centrifuge container with three layers in large separation chamber (arrow) from bottom to top red cells and granulocytes. In small separation chamber mononuclear cells at the bottom and platelet rich plasma on top.

cord blood.5 Numerous studies have concentrated on progenitor cells or on highly selective CD133 cells to treat patients with end-stage heart disease. Intensive research has been conducted to discover the cellular pathways responsible for the various effects of stem cell therapy.5-10 Additional release of paracrine mediators by incorporated stem or progenitor cells may amplify the response to cellular treatment by attracting circulating progenitor cells and/or tissue-resident stem cells. Several trials involving patients who present a worst-case scenario have shown the efficacy and safety of stem cell therapy in CLI.11,12 Tateishi-Yuyama et al.12 and the Therapeutic Angiogenesis by Cell Transplantation (TACT) study investigators performed a prospective randomized controlled trial in patients with peripheral arterial disease. They reported a significant increase in transcutaneous oxygen pressure and pain-free walking time and decrease in rest pain in 22 patients with leg ischemia after intramuscular injection of bone marrowederived mononuclear cells.

Annals of Vascular Surgery

In another recently published study the efficacy and safety of autologous bone marrow-mononuclear cell implantation in patients with CLI due to thromboangiitis obliterans (Buerger disease) was evaluated.13 The study comprised 28 patients who suffered from thromboangiitis obliterans and unilateral CLI. Only one patient required toe amputation during follow-up. A change in the ankle-brachial pressure index >0.15 was achieved in eight patients at 3 months and in 14 patients at 6 months compared with baseline values.

What technical variables might influence outcome? Surprisingly, none of the studies reported so far showed an association between cell numbers infused and extent of functional improvement over the ranges tested.14,15 Although the volumes of bone marrow harvested, the cell isolation procedures, and the number of cells infused varied considerably among trials, the reported improvements were nearly identical.

One of the major advantages of the device described here is that it can be used in an operating room setting to treat vascular as well as cardiac surgical patients. In less than 30 min the cells are ready for injection. There is only one other system available which is designated as a point-of-care device that permits intraoperative stem cell treatment as an adjunct to bypass surgery or interventional therapy.16 Comparing the SmartPrep (Harvest) and the Sepax system (Biosafe, Eysins, Luzern, Switzerland), the stem cell counts in the injected mononuclear cell fraction and recovery rates are better using the first one. The CD34-positive stem cell counts reported by Aktas et al.17 are 2.25 to 11.04 10E6 cells. Our CD34-positive cell yield was 13.8 to 54.2 10E6. The CD34 recovery rate was 17.9-65.1% for Sepax and 41-99% for the SmartPrep system. We were able to show that the buffy coat preparation using the SmartPrep system is a simple and fast method to enrich stem cells from BMAs. This automated system gives high recovery rates and good reproducibility.

There are cases where stem cell treatment takes too long and is initiated too late to have any beneficial effect. We also have to question the one-time treatment regimen used in these cases. Especially in cases with critical ischemia, repetitive treatment protocols may prove to be more beneficial. A prospective study must show whether adjunctive cellular treatment in patients with critical ischemia, a prosthetic bypass, and no vein for a crural bypass available can improve outcome.

Large-scale randomized, controlled, multicenter trials will be essential to evaluate the long-term safety and efficacy of stem cell and EPC therapy for

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Table I. Previous operations and interventional procedures Patient

Previous operations

Endovascular interventions

1 2 3 4 5 6 7 8

Fem.crural PTFE, Miller cuff Fem.popI, Fem.PIII, PTFE, Linton patch Fem.crural PTFE, Miller cuff Fem.PIII, vein, Fem.crural PTFE, Miller cuff Fem.PI PTFE, Fem.truncal lesser saphenous vein Sympathectomy, Fem.PIII vein, Fem.crural PTFE Fem.PIII, lesser saphenous Fem.PIII, PTFE, Miller cuff

Subintimal PTA PTA

PTA Subintimal PTA

Fem.crural, femorocrural polytetrafluoroethylene (PTFE) bypass; Fem.popI, femoropopliteal segment I PTFE bypass; Fem.PIII vein, femoral below-knee vein bypass; Fem.truncal, femorotruncal bypass; PTA, percutaneous transluminal angioplasty.

Table II. Clinical categories and outcome Patient

Rutherford categories

Runoff score crural arteries

1 2 3 4 5 6 7 8

4 4 5 4 4 6 6 4

3 9 6 7 5 9 9 5

Bypass occluded

x x

x x

ABI preop

ABI postop

0.5 0.0 0.2 0.4 0.0 0.0 0.2 0.5

0.8 0.2 0.4 0.4 0.4 0.0 0.2 0.8

Minor amputation

Major amputation

Yes

Yes Yes

Table III. Cell characteristics and viability Patient

CD34 in product

CD34 recovery

CD133 in product

CD133 recovery

EPC in product

EPC recovery

Viability after preparation

1 2 3 4 5 6 7 8

17.2 10E6 14.8 10E6 15.7 10E6 29.6 10E6 32.9 10E6 54.2 10E6 13.8 10E6 22.2 10E6

56% 71% 81% 99% 99% 98% 41% 99%

7.8 10E6 1.8 10E6 4.65 10E6 18.4 10E6 11.6 10E6 35.9 10E6 7.6 10E6 10.7 10E6

60% 43% 79% 99% 80% 99% 37% 78%

1.2 10E4 5.7 10E4 0.5 10E4 1.6 10E4 1.6 10E4 1.0 10E4 n.t.

4.8 10E4

33% 21% 13% 67% 67% 41% n.t.

62%

91.1% 96.6% 73.9% 94.8% 95.1% 99.1% 81.3% 83%

CD34 recovery, 80 (41-99)%; CD133 recovery, 72 (37-99)%; EPC recovery, 43 (13-67)%.

treatment of tissue ischemia and vessel repair. Since only short- and intermediate-term follow-up data are available, there are still concerns of potential side effects such as neovascularization of occult neoplasias and the development of age- and diabetes-related vasculopathies. The consensus document of the European Society of Cardiology stresses the importance of robust end points when evaluating the results of stem cell therapy. Also in vascular patients the end points should focus on robust clinical outcomes as well as major adverse advents, subjective benefit, and economic gain. Outcome measures for future trials should be standardized to

permit comparisons between different groups.18,19 We conclude that stem cell therapy can be used as an adjunct to bypass procedures in highly selected cases. Long-term results have to prove whether this approach results in improved limb salvage.

REFERENCES 1. Cannon RO. Cardiovascular potential of bone marrow derived stem and progenitor cells. Cytotherapy 2004;6: 602-607.

2. Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275:964-967.

ARTICLE IN PRESS 6 Kolvenbach et al.

3. Peichev M, Naiyer AJ, Pereira D, et al. Expression of VGFER2 and CD133 by circulating CD34-positive cells identifies a population of functional endothelial precursors. Blood 2000;95:952-958.

4. Higashi Y, Kimura M, Hara K, et al. Autologous bone marrow nuclear cell implantation improves endotheliumdependent vasodilatation in patients with limb ischemia.

Circulation 2004;109:1215-1218.

5. Lin Y, Weisdorf DJ, Solovey Hebbel RP. Origins of circulating endothelial cells and endothelial outgrowth from blood.

J Clin Invest 2000;105:1-7.

6. Assmus B, Schachinger V, Teupe C, et al. Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI). Circulation 2002; 106:3009-3017.

7. Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomized controlled clinical trial. Lancet 2004;364:141-148.

8. Dimmeler S, Zeiher AM, Schneider MD. Unchain my heart: the scientific foundations of cardiac repair. J Clin Invest 2005;115:572-583.

9. Scha¨chinger V, Erbs S, Elsa¨sser A, et al. Intracoronary bone marrow-derived progenitor cells in acute myocardial infarction. N Engl J Med 2006;355:1210-1221.

10. Lunde K, Solheim S, Aakhus S, et al. Intracoronary injection of mononuclear bone marrow cells in acute myocardial infarction. N Engl J Med 2006;355:1199-1209.

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11. Esato K, Hamano K, Li TS, et al. Neovascularization induced by autologous bone marrow cell implantation in peripheral arterial disease. Cell Transplant 2002;11:747-752.

12. Tateishi-Yuyama E, Matsubara H, Murohara T, et al. Therapeutic angiogenesis for patients with limb ischemia by autologous transplantation of bone-marrow cells: a pilot study and a randomized controlled trial. Lancet 2002;360:427-435.

13. Durdu S, Akar AR, Arat M, et al. Autologous bone marrow mononuclear cell implantation for patients with Rutherford grade II-III thromboangiitis obliterans. J Vasc Surg 2006;44: 732-739.

14. Ishida A, Ohya Y, Sakuda H, et al. Autologous peripheral blood mononuclear cell implantation for patients with peripheral arterial disease improves limb ischemia. Circ J 2005;69:1260-1265.

15. Cannon RO. Cardiovascular potential of bone marrow derived stemand progenitor cells. Cytotherapy 2004;6:602-607.

16. Kolvenbach R, Cagiannos C, Kreissig C, Ludwig E. Stem cell use in critical limb ischemia. J Cardiovasc Surg (Torino) 2007;48:39-44.

17. Aktas M, Radke TF, Strauer BE, et al. Separation of adult bone marrow nuclear cells using the automated closed separation system Sepax. Cytotherapy 2008;10:203-213.

18. Rosenzweig A. Cardiac cell therapydmixed results from mixed cells. N Engl J Med 2006;355:1274-1277.

19. Bartunek J, Dimmler S, Drexler H, et al. The consensus of the task force of the European Society of Cardiology concerning the clinical investigation of the use of autologous adult stem cells for repair of the heart. Eur Heart J 2006;27:1338-1340.