Accepted Article
Article Type : Animal Experiment
Human Blood-Derived Endothelial Progenitor Cells Augment Vasculogenesis and Osteogenesis
Running title: Blood progenitors promote bone formation
Hadar Zigdon-Giladi, DMD, PhD1,2,3, Gal Michaeli-Geller, DMD2,3, Tova Bick, PhD2, Dina Lewinson, PhD2, Eli E. Machtei, DMD1,2,3 1
Department of Periodontology, School of Graduate Dentistry, Rambam Health Care
Campus, Haifa, Israel. 2
Research Institute for Bone Repair, Rambam Health Care Campus, Haifa, Israel.
3
The Rappaport Family Faculty of Medicine, Technion - Israel Institute of Technology,
Haifa, Israel.
Corresponding Author Dr. Hadar Zigdon-Giladi, Research Institute for Bone Repair Rambam Health Care Campus, P.O. Box 9602 Haifa, 31096, Israel This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jcpe.12325 This article is protected by copyright. All rights reserved.
Accepted Article
Tel. 972-4-8543606, Fax. 972-4-8542467 Email: [email protected]
KEYWORDS: cell therapy, extra-cortical bone augmentation, guided bone regeneration, endothelial progenitor cells, tissue engineering
Conflict of Interest and Source of Funding- the authors declare that they have no conflict of interest. This study was supported by Kamin grant 46293, Ministry of Industry Trade and Labor, Israel, and Ofakim grant, Rambam Health Care Campus, Haifa, Israel.
ABSTRACT Introduction: Endothelial progenitor cells (EPC) participate in angiogenesis and osteogenesis therefore have the potential to enhance extra-cortical bone formation.
Aim: To enhance extra-cortical
bone formation following local transplantation of human
peripheral blood derived EPC (hEPC) in a guided bone regeneration (GBR) nude rat calvaria model.
Materials and methods: hEPC were isolated from peripheral blood of healthy volunteers. Cells were cultured and characterized by flow cytometry for specific endothelial markers. Following exposure of nude rat calvaria, gold domes were filled with 106 hEPC mixed with βTCP (n=6). Domes filled with βTCP served as control (n=6). Rats were sacrificed after 3 months. New bone formation and blood vessel density were analyzed by histology and histomorphometry. Transplanted hEPC were located in the regenerated tissue using immunohistology.
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Results: Abundant vasculature was observed adjacent to the newly formed bone. According to histomorphometric analysis: blood vessel density was 7.5 folds higher in the hEPC compared with control group. Similarly, gained extra-cortical bone height (2.46±1.1 mm vs. 0.843±0.61 mm, p=0.01) and bone area fraction (19.42±7.48 % vs. 4.81±3.93%, p=0.001) were elevated following hEPC transplantation. Moreover, hEPC expressing human specific CD31 were integrated into blood vessel walls adjacent to newly formed bone.
Conclusion: In nude rat GBR calvaria model, transplantation of hEPC significantly enhanced vasculogenesis and osteogenesis.
Clinical Relevance Scientific rational for the study: Alveolar bone deficiency is a major problem in implant and reconstructive dentistry. The available surgical techniques to enhance extra-cortical bone augmentation are generally not satisfying mainly due to limited blood supply. Extra-cortical bone augmentation was tested in nude rat's calvaria using Guided Bone Regeneration (GBR) model in combination with human endothelial progenitor cells (hEPC).
Principal findings: Histological analysis revealed abundant vasculature adjacent to the newly formed bone. Cell transplantation significantly increased blood vessel density, extra-cortical bone height and bone area fraction.
INTRODUCTION Extra-cortical bone augmentation is a challenging endeavor with increasing clinical demand. In the field of implant and reconstructive dentistry, regeneration of the lost bone is essential for
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Fuchs, S., Dohle, E., Kolbe, M., Kirkpatrick, C.J. (2010) Outgrowth Endothelial Cells: Sources, Characteristics and Potential Applications in Tissue Engineering and Regenerative Medicine. Adv Biochem Eng Biotechnol 123:201-217. Fuchs, S., Jiang, X., Schmidt, H., Dohle, E., Ghanaati, S., Orth, C., Hofmann, A., Motta, A., Migliaresi, C., Kirkpatrick, C.J. (2009) Dynamic processes involved in the prevascularization of silk fibroin constructs for bone regeneration using outgrowth endothelial cells. Biomaterials 30:1329-1338.
Ghazali, N., Collyer, J.C., Tighe, J.V. (2013) Hemimandibulectomy and vascularized fibula flap in bisphosphonate-induced mandibular osteonecrosis with polycythaemia rubra vera. Int J Oral Maxillofac Surg 42:120-123.
Giannoudis, P.V., Einhorn, T.A., Marsh, D. (2007) Fracture healing: the diamond concept. Injury 38 Suppl 4:S3-6.
Gössl ,M., Mödder, U.I., Atkinson, E.J., Lerman, A., Khosla, S. (2008) Osteocalcin expression by circulating endothelial progenitor cells in patients with coronary atherosclerosis. J Am Coll Cardiol 52:1314-1325.
Irinakis, T. (2006) Rationale for socket preservation after extraction of a single-rooted tooth when planning for future implant placement. J Can Dent Assoc 72:917-922.
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peripheral blood and defined their ability to initiate neovascularization.
EPC express
endothelial markers and may differentiate into cells expressing osteogenic markers (Gössl et al. 2008). These EPC represent a small population with the capacity to proliferate, migrate and differentiate into cells that line the lumen of blood vessels (Luttun et al. 2002).
In our previous experiments we were able to demonstrate that implantation of expanded rat EPC (rEPC) under a rigid dome enhanced extra-cortical bone formation (Zigdon et al. 2013). The technology to use blood-derived progenitors provides a totally new approach to bone tissue engineering, and is clinically applicable. In order to pave the road for future clinical trials, the aim of this study was to evaluate whether human peripheral blood derived EPC (hEPC) stimulate extra-cortical
bone formation in GBR calvaria model. Secondary aim was to
investigate the role of hEPC in vasculogeneis.
MATERIALS AND METHODS The study protocol was initially approved by the committee for the supervision of animal experiments at the faculty of Medicine, Technion (I.I.T.) no. IL0530412, and by the Helsinki committee of Rambam medical center.
Isolation and expansion of hEPC: For isolation of hEPC, 50 ml blood was obtained from healthy volunteers who signed an informed consent. Pooled peripheral blood was collected into a sterile heparinaized tubes and hEPC were isolated as previously described for sheep EPC (Rozen et al. 2009) and rat EPC (Zigdon et al. 2013). Briefly: blood was diluted 1:1 with phosphate buffered saline (PBS). Mononuclear cells (MNCs) were isolated with density gradient centrifugation (LymphoprepTM, Axis-Shield, Oslo, Norway) and pelleted cells were resuspended in Endothelial Basal Medium (EBM-2) containing 20% heat inactivated fetal bovine serum (FBS),
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penicillin-streptomycin (Biological Industries Ltd., Beit Haemek, Israel) and supplemented with Endothelial Growth Medium (EGM-2MV SingleQuote; Clonetics, Cambrex Bio Science, MD, USA) that includes: vascular endothelial growth factor (VEGF), fibroblast growth factor-2, epidermal growth factor, insulin-like growth factor-1 and ascorbic acid. Cells were seeded on six-well plates coated with 5 µg/cm2 of fibronectin (Biological Industries Ltd., Beit Haemek, Israel) and grown at 37°C with humidified 95% air/5% CO2. After four days of culture, nonadherent cells were discarded by gentle washing with PBS, and fresh medium was applied. The attached cells were continuously cultured with complete EGM-2 medium. Cells were fed three times per week and were split when reached ~80% confluent by brief trypsinization using 0.5% trypsine in 0.2% EDTA (Biological Industries Ltd., Beit Haemek, Israel).
Characterization of hEPC: EPC were characterized by Flow cytometry (FACS) using FITC labeled antibodies specific for: CD14, CD34 (mouse anti human BD Biosciences, San Jose, CA, USA) and CD31 (LifeSpan BioSciences, Seattle, WA, USA).
5x105 cells in PBS were
incubated 30 minutes with antibodies according to the manufacturers’ recommendations. Negative controls were Mouse IgG1 FITC isotype (BD Biosciences, San Jose, CA, USA). Following washings x3, cells were resuspended in PBS and analyzed using FACScan and CellQuest software (Becton Dickinson & Co., Franklin Lakes, NJ).
Coating of β-tricalciumphosphate (βTCP) with fibronectin: Based on our previous results (Zigdon et al. 2014), synthetic β-tricalciumphosphate (βTCP) granules (0.6-1mm grain size, 40% porosity and 100-200µm pore size, Poresorb-TCP®, Lasak Ltd., Prague, Czech Republic) was chosen as scaffold for the present study. To enable attachment of cells, 0.2 g βTCP were coated with 50 µg fibronectin (Biological Industries Ltd., Beit Haemek, Israel) (Seebach et al. 2010).
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Cell transplantation: male nude (athymic) rats (Hsd: RH-FoxN1RNU, Harlan, IN, USA, 13 weeks, ~300 g) were used in order to allow xenogenic transplantation of human cells. Rats were anaesthetized by intramuscular injection of 100mg/kg bw Ketamin (Ketaset, Fort Dodge, Iowa, USA) and 5mg/kg bw Xylasin (Eurovet, Cuijk, Holland). 50mg/kg bw Cephalexin (Norbrook laboratories, Ireland) and 0.3 mg/kg bw Buprenorphine (Vetamarket, Israel) were injected s.c. pre-operatively and 3 days post operation. Surgical procedure was performed as previously described (Zigdon et al. 2013). Briefly, a U-shaped incision served to raise a full thickness skin flap and exposure of the parietal bone. Five perforations (1mm diameter) of the cortical bone were performed to allow passage of blood, cells and nutrients from the bone marrow into the space under the dome. Next, 106 hEPC (n=6) suspended in 50 µl medium or 50 µl medium without hEPC (control, n=6) were mixed with fibronectin coated βTCP and transplanted immediately under rigid gold domes (7mm radius, 5 mm height). The Domes were secured to the calvarium using fixation screws. Surgical flaps were repositioned and horizontal mattress sutures were performed (Fig. 1).. Each rat was kept in a separate cage and fed rat chow and water ad libitum for three months. Then, rats were sacrificed by CO2 asphyxiation and the domes were removed. The part of the calvarium surrounding the regenerated area was sawed out and specimens were fixed immediately in 4% paraformaldehyde for 2 days.
Histology: Fixed specimens were decalcified in 10% EDTA, (Sigma-Aldrich, MS, USA) for 4 weeks, cut into 2 halves in the midline, embedded in paraffin and sectioned (5 μm). For determination of bone morphology, sections were stained with Masson's trichrome.
Histomorphomentric analysis: Four Masson's trichrome stained sections (~20 μm apart) from each specimen were scanned by a panoramic digital slide scanner (3DHISTECH panoramic MIDI, Budapest, Hungary). For each specimen we calculated the means of 1) Extra-cortical
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bone height (ECBH): maximal gained bone height (in mm) measured from the top of the calvaria (Fig. 2) 2) Bone area fraction (BA): bone area in the newly regenerated tissue under the dome (Fig. 2). Above parameters were analyzed morphometrically using image-Pro software (Rockville, MD, USA).
Blood vessel density: Luminal structures perfused with red blood cells were identified as blood vessels (Bv). Ten sections were evaluated for each specimen. Bv density was defined as mean number of Bv in microscopic field (260X444 µm).
Immunohistology: sections were immuno-stained with human-specific mouse monoclonal antibody CD31 that do not react with rat CD31 (1:70, Thermo scientific, Fremont, CA, USA). H&E stain was used for general morphology.
Statistical analysis: A StatPlus®statistical package (AnalystSoft, Vancouver, BC, Canada) was used. Descriptive statistics that included means and medians, ranges and standard deviation (SD) were initially tabulated. Comparisons between hEPC and control groups were performed using t-tests. p≤0.05 was determined as significant.
RESULTS Isolation and expansion of hEPC: mononuclear cells were seeded on fibronectin coated plastic plates. 3-4 weeks after seeding, polyhedral cells appeared and rapidly replicated to form colonies (Fig. 3A). Self renewal was preserved for at least 5 passages.
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Characterization of hEPC: FACS analysis revealed that more than 99% of the cells were CD31 positive, an endothelial cell marker. Furthermore, 18% of the cells were CD34 positive, early hematopoietic and vascular-associated tissue cells marker. Importantly, cells were negative for CD14 (a hematopoietic cell marker) (Fig. 3B).
Follow-up of the healing process: Healing was un-eventful and all rats survived throughout the study. In all rats, new augmented hard tissue that is tightly attached to the original calvaria filled the space under the dome.
Descriptive histology: Histological sections revealed that the space under the dome was composed of bone, residual scaffold and connective tissue in different proportions. The newly formed bone was always continuous with the original calvaria (Fig. 4A and 4B). Islands of mature lamellar bone were observed surrounded by areas of residual scaffold and vascularized connective tissue (Fig. 4C). Blood vessels were observed adjacent to palisading of osteoblasts in the front of the newly formed bone demonstrating the coupling between osteogenesis and vasculogenesis (Fig. 4D).
Histomorphometric analysis revealed that hEPC significantly increased bone formation: Gained ECBH ranged from 0 to 1.8 mm (mean 0.84±0.61 mm) in the control group (βTCP alone). Addition of cells increased ECBH by three folds: hEPC mean ECBH 2.46±1.1 mm (range 1.1-4.1 mm; p=0.01 vs. control). In addition, overall bone area fraction (BA) was higher in the hEPC group compared with control (BA: 19.42±7.48 % vs.4.81±3.93 %, p=0.001; (Fig. 5A-B). The vasculogenic effect of hEPC was also demonstrated: blood vessel density in the regenerated tissue was 7.5 fold higher in the hEPC group compared with control (7.51±1 vs.
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1.08±0.2, ≤0.0001) .Moreover, immunohistological labeling of the transplanted (CD31+) cells revealed integration of hEPC in blood vessel walls (Fig. 6) adjacent to the newly formed bone.
DISCUSSION Efficient blood supply is necessary to deliver nutrients, oxygen and cells to regenerating sites. Several strategies aimed to increase angiogenesis of bone grafts were described: the use of vascularized bone graft (Ghazali et al. 2013), incorporation of angiogenic growth factors (VEGF) to osteoconductive scaffold (Sun X et al. 2013) or cell-based therapies using endothelial cells (Koob et al. 2011).
In the present study, the potential of hEPC to increase vasculogenesis and therefore to enhance extra- cortical bone regeneration was evaluated in an established rat calvarium model (Slotte and Lundgren 2002). In our previous studies, we demonstrated the concept of stimulating extra-cortical bone augmentation using rat EPC: rat EPC were isolated from inbred Lewis rats. In a similar rat calvaria GBR model, transplantation of rat EPC mixed with βTCP doubled extra-cortical bone height and significantly increased bone area and bone volume compared to βTCP control. Bone architecture and mineral density were not affected by cell transplantation (Zigdon et al. 2013). Therefore, as an additional step towards clinical trials, the aim of the present study was to investigate whether human cells are able to enhance extracortical bone regeneration similar to rat cells. Secondary aim was to investigate the role of hEPC in vasculogeneis. To this end, athymic nude rats were used in order to avoid immunogenic rejection of transplanted human cells. Indeed, the results of this study indicate that vasculogenesis and extra-cortical bone augmentation was significantly improved following transplantation of hEPC mixed with βTCP compared to control (βTCP alone). Moreover, the
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integration of the transplanted human cells in the newly formed blood vessels demonstrating their direct role in vasculogenesis.
hEPC were isolated from 50 ml peripheral blood and cultured in specific endothelial medium. Expanded cells presented unique cobblestone morphology and specific surface antigens that characterize EPC (Yoder et al. 2007, Hur et al. 2004, Hirschi et al. 2008). It was previously demonstrated that EPC participate in angiogenesis and vasculogenesis during embryonic and postnatal periods (Takahashi et al. 1999). EPC originate from bone marrow and been recruited to ischemic or injured sites by gradient of vasculogenic / angiogenic molecules (e.g. VEGF, erythropoietin) (Ferguson et al. 1999, Giannoudis et al. 2007). Their isolation from peripheral blood was first describe by Asahara (1997) and allowed the development of new therapeutic strategies in regenerative medicine. The current main clinical use of EPC is to treat ischemic tissue after acute myocardial infarct (Isner & Losordo 1999, Kalka et al. 2000). However, the therapeutic angiogenic effect of EPC was recently elaborated to treat unstable angina, stroke, diabetic microvasculopathies, pulmonary arterial hypertension, atherosclerosis, and ischemic retinopathies (Rafii et al. 2003, Ward et al. 2007, Sekiguchi et al. 2009, Tateishi-Yuyama et al. 2002, Jung & Roh 2008).
The results of this study indicate that hEPC transplantation significantly increase gained extracortical bone height. The gained ECBH in the hEPC transplanted group was higher than 2 mm in 66% of the samples; while in the control group, the highest value reached only 1.8 mm. These results are in agreement with our previous study using rat EPC (Zigdon et al. 2013) thus, indicating similar osteogenic potential of human EPC and rat EPC.
However since our
histomorphometric measurements in the previous study included the original rat calvaria, bone parameters were higher compared with the results of the current study. The results of this study
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can also be compared with the results of a recently published research that examined extracortical
bone augmentation in a rabbit tibia model. Gained ECBH 6 weeks following
transplantation of deproteinized bovine bone and rhPDGF covered by membrane ranged from 1.8 to 2 mm (Kämmerer et al. 2012). In another research, extra-cortical bone augmentation was tested in a rabbit calvaria model using titanium domes that were filled with deproteinized bovine bone w/wo 106 adipose derived mesenchymal stem cells. The results of this study were similar to our results despite the differences in the study design (e.g. animal model, scaffold, stem cell isolation and characterization): mean gained ECBH was 1.73 ±0.42 mm in scaffold plus GBR group and 2.8±0.6 mm in the stem-cell transplanted group, (Pieri et al. 2010). Additional encouraging results of the current study were the enhancement of bone area fraction following hEPC transplantation.
These results can be supported by numerous studies that used
rat EPC or sheep EPC to enhance bone regeneration and showed promising results: Rozen and colleagues (2009) created 3.2 cm segmental defect in sheep tibia. 2 weeks later, autologous sheep EPC were locally transplanted into the defect. Micro-CT and histological analyses demonstrated complete bridging of the gap in 7/8 of EPC treated group 12 weeks posttransplantation. In the control (non-transplanted) group connective tissue filled the defects. Atesok et al. 2010 transplanted rat EPC loaded on collagen sponge into 5 mm segmental bone defect in rat femur. At 10 weeks, all the animals in the EPC-treated group had complete union (7/7), but in the control group none achieved union (0/7). Recently, hEPC seeded on βTCP were used to treat segmental bone defect in rat femur (Seebach et al. 2010). Increased vascularization of the graft was observed one week following EPC transplantation (compared to βTCP or MSC transplantation), while enhancement of osteogenesis was observed 12 weeks post transplantation.
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The role of EPC in bone regeneration is still unclear. Understanding the mechanisms underlying extra-cortical bone formation by hEPC will further enlighten the interdependence of osteogenesis and vasculogenesis during bone regeneration. The cellular cross talk between endothelial cells and osteoprogenitors comprises paracrine mechanisms based on several growth factors (VEGF, BMP-2 and TGFβ) as well as direct cell to cell communication (Fuchs et al. 2009). Although this study didn't investigate hEPC mechanism of action, we gained insight into the role of hEPC in vasculogenesis by counting functional blood vessels in the regenerated site and by using specific human CD31 antibody in order to localize hEPC in the newly formed rat tissue.
According to our results, engraftment of hEPC into vessels walls indicate that these cells participate directly in vessel formation, therefore this cellular approach holds advantage over other bone tissue engineering techniques that use pro-angiogenic (VEGF) or pro-osteogenic (BMP) growth factors.
It seems that hEPC constitute powerful candidate cell type for bone regeneration. This cellbased approach seems feasible in clinical settings since EPC are easy to harvest, isolate, cultivate, characterize, and provide in a sufficient amount within 3-4 weeks.
Therefore, if this cell approach could be applied to patients suffering from severe alveolar bone atrophy, it would clearly improve the present clinical approaches, which still have several disadvantages, such as: two to three surgeries, high morbidity and unpredictable results. Despite the encouraging result, this preclinical study has several limitations due to the low animal number in each group, short follow up and the absence of mechanism of action of these cells. Therefore our future studies will be focused on these issues.
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Within the limits of this study, it can be concluded that local transplantation of hEPC mixed with βTCP stimulates blood vessel formation and extra-cortical bone augmentation in GBR rat calvaria model. Additional studies should be performed to further support these results and gain more information regarding EPC mechanism of action.
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Verhoeven ,J.W., Cune, M.S., Terlou, M., Zoon, M.A., de Putter, C. (1997) The combined use of endosteal implants and iliac crest onlay grafts in the severely atrophic mandible: a longitudinal study. Int J Oral Maxillofac Surg 26:351-7.
Ward, M.R., Stewart, D.J., Kutryk, M.J. (2007) Endothelial progenitor cell therapy for the treatment of coronary disease, acute MI, and pulmonary arterial hypertension: current perspectives. Catheter Cardiovasc Interv 70:983-998.
Yoder, M.C., Mead, L.E., Prater, D., Krier, T.R., Mroueh, K.N., Li, F., Krasich, R., Temm, C.J., Prchal, J.T., Ingram, D.A. (2007) Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals. Blood 109:1801–1809.
Zigdon-Giladi, H., Bick, T., Morgan, E.F., Lewinson, D., Machtei, E.E. (2013) Peripheral Blood Derived Endothelial Progenitor Cells Enhance Vertical Bone Formation. Clin Implant Dent Relat Res doi: 10.1111/cid.12078.
Zigdon, H., Lewinson, D., Bick, T., Machtei, E.E. (2014) Vertical bone augmentation using different osteoconductive scaffolds combined with barrier domes in the rat calvarium. Clin Implant Dent Relat Res 16:138-144.
Acknowledgments We would like to thank Dr. Margarita Filatov for assistance with cell characterization.
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LEGENDS TO FIGURES Fig. 1- Surgical procedure: A- U shape incision: full thickness flap elevation. B- Gold dome serving as GBR containing βTCP alone or βTCP plus hEPC. C- Gold dome fixed to the calvaria by fixation screws. D- Flap reposition and horizontal mattress sutures.
Fig. 2- Demonstration of histomorphometric measurements: A- Original rat calvaria (red) was separated from the newly formed tissue. Gained extracortical bone height (yellow arrows) and newly formed bone area (newly formed bone stained blue) were measured.
Fig. 3- Human EPC (hEPC): culture and characterization. A- hEPC were cultured for 30 days. Polygonal shaped cells are shown. B- Representative FACS analysis of hEPC: Top panel shows cells stained with isotype control. More than 99% of the cells were CD31+, 18% of the cells were CD34+ and all cell were negative for CD14. Fig. 4- Representative Masson's trichrome sections of tissue formed under a gold domes A and B- Newly formed tissue under the dome 3 months following transplantation of βTCP alone, control (A) or hEPC (B). An arbitrary dotted line separates the original carvaria (CL) from the augmented tissue (X2 magnification, scale bar- 1000µm). C- The insert seen in panel A was taken at X40 magnification (scale bar- 50µm). Augmented tissue composes of residual scaffold (RS), new bone (NB) with osteocytes (white arrows), osteoblasts (black arrows) and connective tissue (CT).
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D- The insert seen in panel B was taken at X40 magnification (scale bar- 50µm). Palisading of osteoblasts (yellow arrows) observed adjacent to blood vessels (white arrows).
Fig. 5- Histomorphometric analysis A- Gained extra-cortical
bone height (mm) following transplantation of hEPC or βTCP
(control), p≤0.01. B- Bone area fraction (%) following transplantation of hEPC or βTCP (control), * p≤0.001 Fig. 6- Blood vessel count and hEPC localization in the rat regenerated tissue A&B- Blood vessels (black arrows) adjacent to the newly formed bone in TCP control (A) and hEPC (B).
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Article Type : Animal Experiment
Human Blood-Derived Endothelial Progenitor Cells Augment Vasculogenesis and Osteogenesis
Running title: Blood progenitors promote bone formation
Hadar Zigdon-Giladi, DMD, PhD1,2,3, Gal Michaeli-Geller, DMD2,3, Tova Bick, PhD2, Dina Lewinson, PhD2, Eli E. Machtei, DMD1,2,3 1
Department of Periodontology, School of Graduate Dentistry, Rambam Health Care
Campus, Haifa, Israel. 2
Research Institute for Bone Repair, Rambam Health Care Campus, Haifa, Israel.
3
The Rappaport Family Faculty of Medicine, Technion - Israel Institute of Technology,
Haifa, Israel.
Corresponding Author Dr. Hadar Zigdon-Giladi, Research Institute for Bone Repair Rambam Health Care Campus, P.O. Box 9602 Haifa, 31096, Israel This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jcpe.12325 This article is protected by copyright. All rights reserved.
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Tel. 972-4-8543606, Fax. 972-4-8542467 Email: [email protected]
KEYWORDS: cell therapy, extra-cortical bone augmentation, guided bone regeneration, endothelial progenitor cells, tissue engineering
Conflict of Interest and Source of Funding- the authors declare that they have no conflict of interest. This study was supported by Kamin grant 46293, Ministry of Industry Trade and Labor, Israel, and Ofakim grant, Rambam Health Care Campus, Haifa, Israel.
ABSTRACT Introduction: Endothelial progenitor cells (EPC) participate in angiogenesis and osteogenesis therefore have the potential to enhance extra-cortical bone formation.
Aim: To enhance extra-cortical
bone formation following local transplantation of human
peripheral blood derived EPC (hEPC) in a guided bone regeneration (GBR) nude rat calvaria model.
Materials and methods: hEPC were isolated from peripheral blood of healthy volunteers. Cells were cultured and characterized by flow cytometry for specific endothelial markers. Following exposure of nude rat calvaria, gold domes were filled with 106 hEPC mixed with βTCP (n=6). Domes filled with βTCP served as control (n=6). Rats were sacrificed after 3 months. New bone formation and blood vessel density were analyzed by histology and histomorphometry. Transplanted hEPC were located in the regenerated tissue using immunohistology.
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Results: Abundant vasculature was observed adjacent to the newly formed bone. According to histomorphometric analysis: blood vessel density was 7.5 folds higher in the hEPC compared with control group. Similarly, gained extra-cortical bone height (2.46±1.1 mm vs. 0.843±0.61 mm, p=0.01) and bone area fraction (19.42±7.48 % vs. 4.81±3.93%, p=0.001) were elevated following hEPC transplantation. Moreover, hEPC expressing human specific CD31 were integrated into blood vessel walls adjacent to newly formed bone.
Conclusion: In nude rat GBR calvaria model, transplantation of hEPC significantly enhanced vasculogenesis and osteogenesis.
Clinical Relevance Scientific rational for the study: Alveolar bone deficiency is a major problem in implant and reconstructive dentistry. The available surgical techniques to enhance extra-cortical bone augmentation are generally not satisfying mainly due to limited blood supply. Extra-cortical bone augmentation was tested in nude rat's calvaria using Guided Bone Regeneration (GBR) model in combination with human endothelial progenitor cells (hEPC).
Principal findings: Histological analysis revealed abundant vasculature adjacent to the newly formed bone. Cell transplantation significantly increased blood vessel density, extra-cortical bone height and bone area fraction.
INTRODUCTION Extra-cortical bone augmentation is a challenging endeavor with increasing clinical demand. In the field of implant and reconstructive dentistry, regeneration of the lost bone is essential for
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Fuchs, S., Dohle, E., Kolbe, M., Kirkpatrick, C.J. (2010) Outgrowth Endothelial Cells: Sources, Characteristics and Potential Applications in Tissue Engineering and Regenerative Medicine. Adv Biochem Eng Biotechnol 123:201-217. Fuchs, S., Jiang, X., Schmidt, H., Dohle, E., Ghanaati, S., Orth, C., Hofmann, A., Motta, A., Migliaresi, C., Kirkpatrick, C.J. (2009) Dynamic processes involved in the prevascularization of silk fibroin constructs for bone regeneration using outgrowth endothelial cells. Biomaterials 30:1329-1338.
Ghazali, N., Collyer, J.C., Tighe, J.V. (2013) Hemimandibulectomy and vascularized fibula flap in bisphosphonate-induced mandibular osteonecrosis with polycythaemia rubra vera. Int J Oral Maxillofac Surg 42:120-123.
Giannoudis, P.V., Einhorn, T.A., Marsh, D. (2007) Fracture healing: the diamond concept. Injury 38 Suppl 4:S3-6.
Gössl ,M., Mödder, U.I., Atkinson, E.J., Lerman, A., Khosla, S. (2008) Osteocalcin expression by circulating endothelial progenitor cells in patients with coronary atherosclerosis. J Am Coll Cardiol 52:1314-1325.
Irinakis, T. (2006) Rationale for socket preservation after extraction of a single-rooted tooth when planning for future implant placement. J Can Dent Assoc 72:917-922.
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peripheral blood and defined their ability to initiate neovascularization.
EPC express
endothelial markers and may differentiate into cells expressing osteogenic markers (Gössl et al. 2008). These EPC represent a small population with the capacity to proliferate, migrate and differentiate into cells that line the lumen of blood vessels (Luttun et al. 2002).
In our previous experiments we were able to demonstrate that implantation of expanded rat EPC (rEPC) under a rigid dome enhanced extra-cortical bone formation (Zigdon et al. 2013). The technology to use blood-derived progenitors provides a totally new approach to bone tissue engineering, and is clinically applicable. In order to pave the road for future clinical trials, the aim of this study was to evaluate whether human peripheral blood derived EPC (hEPC) stimulate extra-cortical
bone formation in GBR calvaria model. Secondary aim was to
investigate the role of hEPC in vasculogeneis.
MATERIALS AND METHODS The study protocol was initially approved by the committee for the supervision of animal experiments at the faculty of Medicine, Technion (I.I.T.) no. IL0530412, and by the Helsinki committee of Rambam medical center.
Isolation and expansion of hEPC: For isolation of hEPC, 50 ml blood was obtained from healthy volunteers who signed an informed consent. Pooled peripheral blood was collected into a sterile heparinaized tubes and hEPC were isolated as previously described for sheep EPC (Rozen et al. 2009) and rat EPC (Zigdon et al. 2013). Briefly: blood was diluted 1:1 with phosphate buffered saline (PBS). Mononuclear cells (MNCs) were isolated with density gradient centrifugation (LymphoprepTM, Axis-Shield, Oslo, Norway) and pelleted cells were resuspended in Endothelial Basal Medium (EBM-2) containing 20% heat inactivated fetal bovine serum (FBS),
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penicillin-streptomycin (Biological Industries Ltd., Beit Haemek, Israel) and supplemented with Endothelial Growth Medium (EGM-2MV SingleQuote; Clonetics, Cambrex Bio Science, MD, USA) that includes: vascular endothelial growth factor (VEGF), fibroblast growth factor-2, epidermal growth factor, insulin-like growth factor-1 and ascorbic acid. Cells were seeded on six-well plates coated with 5 µg/cm2 of fibronectin (Biological Industries Ltd., Beit Haemek, Israel) and grown at 37°C with humidified 95% air/5% CO2. After four days of culture, nonadherent cells were discarded by gentle washing with PBS, and fresh medium was applied. The attached cells were continuously cultured with complete EGM-2 medium. Cells were fed three times per week and were split when reached ~80% confluent by brief trypsinization using 0.5% trypsine in 0.2% EDTA (Biological Industries Ltd., Beit Haemek, Israel).
Characterization of hEPC: EPC were characterized by Flow cytometry (FACS) using FITC labeled antibodies specific for: CD14, CD34 (mouse anti human BD Biosciences, San Jose, CA, USA) and CD31 (LifeSpan BioSciences, Seattle, WA, USA).
5x105 cells in PBS were
incubated 30 minutes with antibodies according to the manufacturers’ recommendations. Negative controls were Mouse IgG1 FITC isotype (BD Biosciences, San Jose, CA, USA). Following washings x3, cells were resuspended in PBS and analyzed using FACScan and CellQuest software (Becton Dickinson & Co., Franklin Lakes, NJ).
Coating of β-tricalciumphosphate (βTCP) with fibronectin: Based on our previous results (Zigdon et al. 2014), synthetic β-tricalciumphosphate (βTCP) granules (0.6-1mm grain size, 40% porosity and 100-200µm pore size, Poresorb-TCP®, Lasak Ltd., Prague, Czech Republic) was chosen as scaffold for the present study. To enable attachment of cells, 0.2 g βTCP were coated with 50 µg fibronectin (Biological Industries Ltd., Beit Haemek, Israel) (Seebach et al. 2010).
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Cell transplantation: male nude (athymic) rats (Hsd: RH-FoxN1RNU, Harlan, IN, USA, 13 weeks, ~300 g) were used in order to allow xenogenic transplantation of human cells. Rats were anaesthetized by intramuscular injection of 100mg/kg bw Ketamin (Ketaset, Fort Dodge, Iowa, USA) and 5mg/kg bw Xylasin (Eurovet, Cuijk, Holland). 50mg/kg bw Cephalexin (Norbrook laboratories, Ireland) and 0.3 mg/kg bw Buprenorphine (Vetamarket, Israel) were injected s.c. pre-operatively and 3 days post operation. Surgical procedure was performed as previously described (Zigdon et al. 2013). Briefly, a U-shaped incision served to raise a full thickness skin flap and exposure of the parietal bone. Five perforations (1mm diameter) of the cortical bone were performed to allow passage of blood, cells and nutrients from the bone marrow into the space under the dome. Next, 106 hEPC (n=6) suspended in 50 µl medium or 50 µl medium without hEPC (control, n=6) were mixed with fibronectin coated βTCP and transplanted immediately under rigid gold domes (7mm radius, 5 mm height). The Domes were secured to the calvarium using fixation screws. Surgical flaps were repositioned and horizontal mattress sutures were performed (Fig. 1).. Each rat was kept in a separate cage and fed rat chow and water ad libitum for three months. Then, rats were sacrificed by CO2 asphyxiation and the domes were removed. The part of the calvarium surrounding the regenerated area was sawed out and specimens were fixed immediately in 4% paraformaldehyde for 2 days.
Histology: Fixed specimens were decalcified in 10% EDTA, (Sigma-Aldrich, MS, USA) for 4 weeks, cut into 2 halves in the midline, embedded in paraffin and sectioned (5 μm). For determination of bone morphology, sections were stained with Masson's trichrome.
Histomorphomentric analysis: Four Masson's trichrome stained sections (~20 μm apart) from each specimen were scanned by a panoramic digital slide scanner (3DHISTECH panoramic MIDI, Budapest, Hungary). For each specimen we calculated the means of 1) Extra-cortical
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bone height (ECBH): maximal gained bone height (in mm) measured from the top of the calvaria (Fig. 2) 2) Bone area fraction (BA): bone area in the newly regenerated tissue under the dome (Fig. 2). Above parameters were analyzed morphometrically using image-Pro software (Rockville, MD, USA).
Blood vessel density: Luminal structures perfused with red blood cells were identified as blood vessels (Bv). Ten sections were evaluated for each specimen. Bv density was defined as mean number of Bv in microscopic field (260X444 µm).
Immunohistology: sections were immuno-stained with human-specific mouse monoclonal antibody CD31 that do not react with rat CD31 (1:70, Thermo scientific, Fremont, CA, USA). H&E stain was used for general morphology.
Statistical analysis: A StatPlus®statistical package (AnalystSoft, Vancouver, BC, Canada) was used. Descriptive statistics that included means and medians, ranges and standard deviation (SD) were initially tabulated. Comparisons between hEPC and control groups were performed using t-tests. p≤0.05 was determined as significant.
RESULTS Isolation and expansion of hEPC: mononuclear cells were seeded on fibronectin coated plastic plates. 3-4 weeks after seeding, polyhedral cells appeared and rapidly replicated to form colonies (Fig. 3A). Self renewal was preserved for at least 5 passages.
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Characterization of hEPC: FACS analysis revealed that more than 99% of the cells were CD31 positive, an endothelial cell marker. Furthermore, 18% of the cells were CD34 positive, early hematopoietic and vascular-associated tissue cells marker. Importantly, cells were negative for CD14 (a hematopoietic cell marker) (Fig. 3B).
Follow-up of the healing process: Healing was un-eventful and all rats survived throughout the study. In all rats, new augmented hard tissue that is tightly attached to the original calvaria filled the space under the dome.
Descriptive histology: Histological sections revealed that the space under the dome was composed of bone, residual scaffold and connective tissue in different proportions. The newly formed bone was always continuous with the original calvaria (Fig. 4A and 4B). Islands of mature lamellar bone were observed surrounded by areas of residual scaffold and vascularized connective tissue (Fig. 4C). Blood vessels were observed adjacent to palisading of osteoblasts in the front of the newly formed bone demonstrating the coupling between osteogenesis and vasculogenesis (Fig. 4D).
Histomorphometric analysis revealed that hEPC significantly increased bone formation: Gained ECBH ranged from 0 to 1.8 mm (mean 0.84±0.61 mm) in the control group (βTCP alone). Addition of cells increased ECBH by three folds: hEPC mean ECBH 2.46±1.1 mm (range 1.1-4.1 mm; p=0.01 vs. control). In addition, overall bone area fraction (BA) was higher in the hEPC group compared with control (BA: 19.42±7.48 % vs.4.81±3.93 %, p=0.001; (Fig. 5A-B). The vasculogenic effect of hEPC was also demonstrated: blood vessel density in the regenerated tissue was 7.5 fold higher in the hEPC group compared with control (7.51±1 vs.
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1.08±0.2, ≤0.0001) .Moreover, immunohistological labeling of the transplanted (CD31+) cells revealed integration of hEPC in blood vessel walls (Fig. 6) adjacent to the newly formed bone.
DISCUSSION Efficient blood supply is necessary to deliver nutrients, oxygen and cells to regenerating sites. Several strategies aimed to increase angiogenesis of bone grafts were described: the use of vascularized bone graft (Ghazali et al. 2013), incorporation of angiogenic growth factors (VEGF) to osteoconductive scaffold (Sun X et al. 2013) or cell-based therapies using endothelial cells (Koob et al. 2011).
In the present study, the potential of hEPC to increase vasculogenesis and therefore to enhance extra- cortical bone regeneration was evaluated in an established rat calvarium model (Slotte and Lundgren 2002). In our previous studies, we demonstrated the concept of stimulating extra-cortical bone augmentation using rat EPC: rat EPC were isolated from inbred Lewis rats. In a similar rat calvaria GBR model, transplantation of rat EPC mixed with βTCP doubled extra-cortical bone height and significantly increased bone area and bone volume compared to βTCP control. Bone architecture and mineral density were not affected by cell transplantation (Zigdon et al. 2013). Therefore, as an additional step towards clinical trials, the aim of the present study was to investigate whether human cells are able to enhance extracortical bone regeneration similar to rat cells. Secondary aim was to investigate the role of hEPC in vasculogeneis. To this end, athymic nude rats were used in order to avoid immunogenic rejection of transplanted human cells. Indeed, the results of this study indicate that vasculogenesis and extra-cortical bone augmentation was significantly improved following transplantation of hEPC mixed with βTCP compared to control (βTCP alone). Moreover, the
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integration of the transplanted human cells in the newly formed blood vessels demonstrating their direct role in vasculogenesis.
hEPC were isolated from 50 ml peripheral blood and cultured in specific endothelial medium. Expanded cells presented unique cobblestone morphology and specific surface antigens that characterize EPC (Yoder et al. 2007, Hur et al. 2004, Hirschi et al. 2008). It was previously demonstrated that EPC participate in angiogenesis and vasculogenesis during embryonic and postnatal periods (Takahashi et al. 1999). EPC originate from bone marrow and been recruited to ischemic or injured sites by gradient of vasculogenic / angiogenic molecules (e.g. VEGF, erythropoietin) (Ferguson et al. 1999, Giannoudis et al. 2007). Their isolation from peripheral blood was first describe by Asahara (1997) and allowed the development of new therapeutic strategies in regenerative medicine. The current main clinical use of EPC is to treat ischemic tissue after acute myocardial infarct (Isner & Losordo 1999, Kalka et al. 2000). However, the therapeutic angiogenic effect of EPC was recently elaborated to treat unstable angina, stroke, diabetic microvasculopathies, pulmonary arterial hypertension, atherosclerosis, and ischemic retinopathies (Rafii et al. 2003, Ward et al. 2007, Sekiguchi et al. 2009, Tateishi-Yuyama et al. 2002, Jung & Roh 2008).
The results of this study indicate that hEPC transplantation significantly increase gained extracortical bone height. The gained ECBH in the hEPC transplanted group was higher than 2 mm in 66% of the samples; while in the control group, the highest value reached only 1.8 mm. These results are in agreement with our previous study using rat EPC (Zigdon et al. 2013) thus, indicating similar osteogenic potential of human EPC and rat EPC.
However since our
histomorphometric measurements in the previous study included the original rat calvaria, bone parameters were higher compared with the results of the current study. The results of this study
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can also be compared with the results of a recently published research that examined extracortical
bone augmentation in a rabbit tibia model. Gained ECBH 6 weeks following
transplantation of deproteinized bovine bone and rhPDGF covered by membrane ranged from 1.8 to 2 mm (Kämmerer et al. 2012). In another research, extra-cortical bone augmentation was tested in a rabbit calvaria model using titanium domes that were filled with deproteinized bovine bone w/wo 106 adipose derived mesenchymal stem cells. The results of this study were similar to our results despite the differences in the study design (e.g. animal model, scaffold, stem cell isolation and characterization): mean gained ECBH was 1.73 ±0.42 mm in scaffold plus GBR group and 2.8±0.6 mm in the stem-cell transplanted group, (Pieri et al. 2010). Additional encouraging results of the current study were the enhancement of bone area fraction following hEPC transplantation.
These results can be supported by numerous studies that used
rat EPC or sheep EPC to enhance bone regeneration and showed promising results: Rozen and colleagues (2009) created 3.2 cm segmental defect in sheep tibia. 2 weeks later, autologous sheep EPC were locally transplanted into the defect. Micro-CT and histological analyses demonstrated complete bridging of the gap in 7/8 of EPC treated group 12 weeks posttransplantation. In the control (non-transplanted) group connective tissue filled the defects. Atesok et al. 2010 transplanted rat EPC loaded on collagen sponge into 5 mm segmental bone defect in rat femur. At 10 weeks, all the animals in the EPC-treated group had complete union (7/7), but in the control group none achieved union (0/7). Recently, hEPC seeded on βTCP were used to treat segmental bone defect in rat femur (Seebach et al. 2010). Increased vascularization of the graft was observed one week following EPC transplantation (compared to βTCP or MSC transplantation), while enhancement of osteogenesis was observed 12 weeks post transplantation.
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The role of EPC in bone regeneration is still unclear. Understanding the mechanisms underlying extra-cortical bone formation by hEPC will further enlighten the interdependence of osteogenesis and vasculogenesis during bone regeneration. The cellular cross talk between endothelial cells and osteoprogenitors comprises paracrine mechanisms based on several growth factors (VEGF, BMP-2 and TGFβ) as well as direct cell to cell communication (Fuchs et al. 2009). Although this study didn't investigate hEPC mechanism of action, we gained insight into the role of hEPC in vasculogenesis by counting functional blood vessels in the regenerated site and by using specific human CD31 antibody in order to localize hEPC in the newly formed rat tissue.
According to our results, engraftment of hEPC into vessels walls indicate that these cells participate directly in vessel formation, therefore this cellular approach holds advantage over other bone tissue engineering techniques that use pro-angiogenic (VEGF) or pro-osteogenic (BMP) growth factors.
It seems that hEPC constitute powerful candidate cell type for bone regeneration. This cellbased approach seems feasible in clinical settings since EPC are easy to harvest, isolate, cultivate, characterize, and provide in a sufficient amount within 3-4 weeks.
Therefore, if this cell approach could be applied to patients suffering from severe alveolar bone atrophy, it would clearly improve the present clinical approaches, which still have several disadvantages, such as: two to three surgeries, high morbidity and unpredictable results. Despite the encouraging result, this preclinical study has several limitations due to the low animal number in each group, short follow up and the absence of mechanism of action of these cells. Therefore our future studies will be focused on these issues.
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Within the limits of this study, it can be concluded that local transplantation of hEPC mixed with βTCP stimulates blood vessel formation and extra-cortical bone augmentation in GBR rat calvaria model. Additional studies should be performed to further support these results and gain more information regarding EPC mechanism of action.
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Acknowledgments We would like to thank Dr. Margarita Filatov for assistance with cell characterization.
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LEGENDS TO FIGURES Fig. 1- Surgical procedure: A- U shape incision: full thickness flap elevation. B- Gold dome serving as GBR containing βTCP alone or βTCP plus hEPC. C- Gold dome fixed to the calvaria by fixation screws. D- Flap reposition and horizontal mattress sutures.
Fig. 2- Demonstration of histomorphometric measurements: A- Original rat calvaria (red) was separated from the newly formed tissue. Gained extracortical bone height (yellow arrows) and newly formed bone area (newly formed bone stained blue) were measured.
Fig. 3- Human EPC (hEPC): culture and characterization. A- hEPC were cultured for 30 days. Polygonal shaped cells are shown. B- Representative FACS analysis of hEPC: Top panel shows cells stained with isotype control. More than 99% of the cells were CD31+, 18% of the cells were CD34+ and all cell were negative for CD14. Fig. 4- Representative Masson's trichrome sections of tissue formed under a gold domes A and B- Newly formed tissue under the dome 3 months following transplantation of βTCP alone, control (A) or hEPC (B). An arbitrary dotted line separates the original carvaria (CL) from the augmented tissue (X2 magnification, scale bar- 1000µm). C- The insert seen in panel A was taken at X40 magnification (scale bar- 50µm). Augmented tissue composes of residual scaffold (RS), new bone (NB) with osteocytes (white arrows), osteoblasts (black arrows) and connective tissue (CT).
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D- The insert seen in panel B was taken at X40 magnification (scale bar- 50µm). Palisading of osteoblasts (yellow arrows) observed adjacent to blood vessels (white arrows).
Fig. 5- Histomorphometric analysis A- Gained extra-cortical
bone height (mm) following transplantation of hEPC or βTCP
(control), p≤0.01. B- Bone area fraction (%) following transplantation of hEPC or βTCP (control), * p≤0.001 Fig. 6- Blood vessel count and hEPC localization in the rat regenerated tissue A&B- Blood vessels (black arrows) adjacent to the newly formed bone in TCP control (A) and hEPC (B).
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