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Coculture of peripheral blood CD34+ cell and mesenchymal stem cell sheets increase the formation of bone in calvarial critical-size defects in rabbits
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British Journal of Oral and Maxillofacial Surgery 52 (2014) 134–139
Coculture of peripheral blood CD34+ cell and mesenchymal stem cell sheets increase the formation of bone in calvarial critical-size defects in rabbits Guanghui Li a,b,1 , Xi Wang c,1 , Jian Cao d , Zhaoyu Ju a , Dongyang Ma d , Yanpu Liu a,∗∗ , Junrui Zhang a,∗ a
Department of Oral and Maxillofacial Surgery, School of Stomatology, Fourth Military Medical University, Xi’an 710032, China Department of Stomatology, PLA 252 Hospital, Baoding 071000, China c Department of Orthodontics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China d Department of Oral and Maxillofacial Surgery, Lanzhou General Hospital, Lanzhou Command of PLA, Lanzhou 730000, China b
Accepted 11 October 2013 Available online 6 November 2013
Abstract The reconstruction of large bony defects remains a clinical challenge, and angiogenesis and neovascularisation are being given more attention in bone tissue engineering. In this study we cocultured peripheral blood CD34+ cells (PB-CD34+ cells), an endothelial progenitor cell/haematopoietic stem cell-enriched population, with bone marrow-derived mesenchymal stem cells (MSC) to investigate their potential for bony regeneration. Cocultured cells showed better osteogenic differentiation than MSC alone in vitro. The cocultured cells and MSC sheets were also composited with hydroxyapatite and implanted in calvarial critical-size defects in rabbits. The rabbits were killed before microcomputed tomographic (MicroCT) and histological analysis. The results showed that cocultured cell composites had promoted bony regeneration more efficiently by 8 weeks after implantation. Our results indicate that the coculture of PB-CD34+ cells and MSC increases bony regeneration in calvarial critical-size defects in rabbits, and provide a new promising therapeutic strategy to aid skeletal healing. © 2013 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Keywords: Peripheral blood CD34+ cells; Mesenchymal stem cells; Co-culture; Bone regeneration; Rabbit calvarial critical-size defect; Cell sheet transplantation
Introduction ∗
Corresponding author at: Department of Oral and Maxillofacial Surgery, School of Stomatology, Fourth Military Medical University, #145 Changlexi Road, Xi’an, Shannxi 710032, China. Tel.: +86 29 84772534; fax: +86 29 83224470. ∗∗ Corresponding author at: Department of Oral and Maxillofacial Surgery, School of Stomatology, Fourth Military Medical University, #145 Changlexi Road, Xi’an, Shannxi 710032, China. Tel.: +86 29 84772532; fax: +86 29 83224470. E-mail addresses: [email protected] (Y. Liu), [email protected] (J. Zhang). 1 Guanghui Li and Xi Wang contributed equally to the research, and should be regarded as joint first author.
The reconstruction of large bony defects caused by tumours, trauma, or inflammation remains a major clinical challenge. Tissue engineering is a promising technique for bony regeneration, in which osteogenic cells are combined with scaffolds to construct tissue-engineered bone. Mesenchymal stem cells (MSC) have gained much interest because of their therapeutic potential.1 However, the size of the regenerated bone is often limited by lack of vessels, and this prevents sufficient nutritional support getting to the entire bone graft. For efficient bony regeneration by tissue engineering, rapid neovascularisation of the implanted graft is essential.2
0266-4356/$ – see front matter © 2013 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bjoms.2013.10.004
G. Li et al. / British Journal of Oral and Maxillofacial Surgery 52 (2014) 134–139
Some research workers have recently studied the coimplantation or potential coculture of endothelial progenitor cells with MSC for bone tissue engineering,3,4 and shown that they can form a prevascular network during coculture and increase osteoblastic activity when the 2 types of cell are in direct contact. CD34+ cells that contain intensive endothelial progenitor and haematopoietic stem cells5 are capable of inducing neovascularisation in vivo.6,7 CD34+ cells have also been reported to differentiate into osteoblasts in vitro.8–10 These findings strongly suggest that CD34+ cells have therapeutic potential for both vasculogenesis and osteogenesis. Cell sheet engineering, which is a new tissue engineering technique, recreates a biological microenvironment similar to that of a regenerative milieu. Cell sheets that are fabricated from various types of cells including MSC, endothelial cells, hepatocytes, and macrophages have been studied.11–13 The technology has been proved to be effective for harvesting cells together with their endogenous extracellular matrix so that adhesion molecules on the surface of the cell and cell–cell interactions remain intact.14 The aims of the present study were to: coculture PBCD34+ cells with MSC in vitro, and assemble the cocultured cell sheet with hydroxyapatite to find out whether this would increase the formation of bone in healing calvarial criticalsize defects in rabbits.
Materials and methods Coculture of PB-CD34+ cells and MSC Total mononuclear cells were separated from the rabbits’ peripheral blood using Histopaque-1077 (Sigma–Aldrich, St. Louis, MO, USA). PB-CD34+ cells were isolated from mononuclear cells by flow cytometry using a conjugated CD34 antibody (PE-Cy5, Bioss Inc., Woburn, MA, USA). Bone marrow was aspirated from the bones of the rabbits’ hind limbs under sterile conditions. MSC were isolated from aspirated bone marrow by the Percoll density gradient centrifugation method and cultured in ␣-minimum essential medium (␣-MEM; Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% foetal bovine serum (Gibco BRL, Gaithersburg, MD, USA), l-glutamine 2 mmol (Invitrogen, Carlsbad, CA, USA), and 1% penicillin/streptomycin (Gibco BRL, Gaithersburg, MD, USA) in a humidified atmosphere of 5% carbon dioxide. A total of 1 × 105 MSC (passage 3) was plated into each well of a 6-well plate and cultured in ␣-MEM supplemented with 10% foetal bovine serum, l-glutamine 2 mmol, and 1% penicillin/streptomycin for one day. PB-CD34+ cells were then seeded on to the same plate at a density of 5 × 105 cells per well. The medium was not changed for the first 7 days, but was changed 2–3 times during the second week. After 14 days’ culture, non-adherent cells were removed. We used immunofluorescence to find out whether PB-CD34+
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Fig. 1. The composite hydroxyapatite/cell sheet transplanted into the calvarial defect.
cells existed in adherent cells. Cells were examined under a fluorescence microscope (BX-51, Olympus, Japan). In vitro experiments Osteogenic differentiation in vitro To induce differentiation of osteoblasts, cocultured cells, and MSC alone, were cultured in ␣-MEM supplemented with 10% foetal bovine serum, dexamethasone 100 nmol, ascorbic acid 50 g/ml, and -glycerophosphate 5 mmol. The medium was not changed for the first 7 days, but was changed 2–3 times during the second week. Alkaline phosphatase staining and activity were measured with an alkaline phosphatase colour development kit (Beyotime, Shanghai, China) and an alkaline phosphatase detection kit (Jiancheng Bioengineering, Nanjing, China) after 14 days in culture according to the manufacturers’ protocols. Construction of cell sheets The method for preparation of the cell sheets has been reported previously.15 Cocultured cells and MSC alone were cultured with ␣-MEM supplemented with 10% foetal bovine serum, dexamethasone 10 nmol, and ascorbic acid 50 g/ml until they reached confluence (about day 14). The medium was changed every 2 days. In vivo experiments Surgical protocol A total of 15 New Zealand rabbits (aged 3 months, weight about 2.6 kg) were used in the study. All protocols were approved by the Animal Welfare Committee of the Fourth Military Medical University. Commercially available hydroxyapatite (Sigma–Aldrich Co., Saint Louis, MO, USA) was sterilised by 60 Co irradiation. For ectopic transplantation, hydroxyapatite 100 mg was wrapped in a piece of cell sheet to assemble a composite. The rabbits were randomly divided into 3 groups: in the first the defect was repaired by
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cocultured cell composites (n = 6); in the second (n = 6) the defect was repaired by MSC composites; and in the third (control) group (n = 3) the defect was left untreated. After anaesthesia a tongue-shaped incision was made to expose the cranium. A 15-mm diameter full-thickness defect was carefully prepared and then treated as shown (Fig. 1). Native periosteum was removed to exclude its possible influence on bony regeneration. The scalp was closed with sutures. At 8 weeks rabbits were killed humanely with an intravenous overdose of barbiturate (200 mg/kg). MicroCT analysis Each specimen was scanned with a microCT system (Inveon, Siemens, Germany; 80 kV, 500 mA, 1200 ms integration time). The scans used a 360◦ radiographic projection (total scan time 30 min). Scanning images were switched into 3-dimensional volumes (21 m resolution) using Cobra software (Siemens reconstruction software). The volume of new bone in the defect site and bone volume/total volume (BV/TV) were also calculated. Histological analysis of bone After microCT scanning, each specimen was fixed in 95% ethanol and embedded in methyl methacrylate. Specimens were then cut and ground to sections 30-m thick. The slides were prepared and stained with Van Gieson reagents. Formation of new bone within the defect was calculated histomorphometrically using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA). The percentage area of new bone was obtained by dividing the bony area by the area of the whole defect. Statistical analysis
Fig. 2. Representative microcomputed tomographic image of bony regeneration in the animals treated with mesenchymal stem cells alone.
formed. Intact cell sheets could be detached with a cell scraper and further manipulated for wrapping with hydroxyapatite.
In vivo experiments Gross morphology of bone regeneration in rabbits All rabbits recovered well from the anaesthetic and the operation. Palpation of the defects showed that they were filled with tight bone-like structures in both the coculture and MSC groups. There were no obvious macroscopic differences between the 2 groups.
Data were analysed with the help of SPSS 17.0 software (SPSS, Chicago, IL, USA). Groups were compared using Student’s t test after testing by analysis of variance. Probabilities of less than 0.05 were accepted as significant.
Results In vitro experiments Osteogenic differentiation in vitro The osteogenic differentiation capacity was studied after 14 days of culture under osteogenic culture conditions. The morphology of the cells became more polygonal and cell clusters were formed when osteogenic supplements were added to the cultures. The alkaline phosphatase activity in cocultured cells was significantly higher than that in MSC (p = 0.012). Fabrication of cell sheets In both the cocultured cells and MSC there was an increase in the synthesis of extracellular matrix and dense cell sheets had
Fig. 3. Representative microcomputed tomographic image of bony regeneration in the animals treated with the coculture.
G. Li et al. / British Journal of Oral and Maxillofacial Surgery 52 (2014) 134–139
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Table 1 Comparisons of new bone volume (mm2 ), bone volume/total volume (BV/TV, %), and the extent of new bone formation (%) among the 3 groups. Groups
New bone volume (mm2 )
BV/TV (%)
Extent of new bone formation (%)
Control Mesenchymal stem cells Coculture
34.76(7.83) (P = 0.001) 90.26(10.51) (P = 0.017) 138.82(12.28)
3.93(0.51) (P = 0.002) 10.22(1.38) (P = 0.026) 16.72(1.87)
5.4(1.7) (P = 0.001) 16.7(2.9) (P = 0.023) 27.5(4.1)
p value
Fig. 4. Representative Van Gieson stain of bony regeneration in the animals treated with mesenchymal stem cells alone. Blue arrows indicate the site of the defect (Van Gieson stain, original magnification 5×).
MicroCT results New bone was formed only at the margins of the defect in the control group. Both the MSC and coculture groups showed signs of bony repair all over the defect, but the coculture group had a larger area of bone (Figs. 2 and 3). The 3-dimensional volume on the microCT images showed that there were significant differences in the volume of bone between the coculture and MSC groups (p = 0.017). There were also significant differences between that in the coculture group and the control group (p = 0.001, Table 1). Similar results were also found for BV/TV (Table 1). Histological results Representative histological images are shown in Figs. 4 and 5. The defects in the coculture group showed bony bridging and new bone formation throughout the defect, but the formation of bone was sparse in the MSC group. The extent of new bony formation in each group was calculated and there was a significantly larger area of new
bone in the coculture group than in the MSC and control groups (p = 0.023, 0.001, Table 1).
Discussion One of the main problems when repairing large craniofacial defects is the difficulty in maintaining the viability of the cells within the bone-grafted tissue to ensure its survival and the eventual restoration of the defect. Angiogenesis is a key contributor to the systematic growth and repair of bone.16 Therapeutic neovascularisation induced by endothelial progenitor or CD34+ cells has been tested in ischaemic diseases, and promising outcomes reported.6,17 Apart from the potential for vasculogenic induction, PB-CD34+ cells are capable of differentiation into osteoblasts in vitro.8–10 Mifune et al.10 also reported that PB-CD34+ cells could be induced to adherent cells without losing their haematopoietic stem cell markers. These findings suggest that PB-CD34+
Fig. 5. Representative Van Gieson stain of bony regeneration in the animals treated with the coculture. Blue arrows indicate the site of the defect (Van Gieson stain, original magnification 5×).
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cells may have the potential to differentiate into not only haematopoietic and endothelial lineages but also into mesenchymal lineages, including osteogenic cells. On the basis of these studies we postulated that coseeding of PB-CD34+ cells with MSC would lead to the efficient formation of bone in ectopic implants. Our results have shown that the cocultured cells could facilitate formation of bone in vitro and in vivo. In the in vitro experiments we showed that the harvested cells contained PB-CD34+ cells and MSC after 14 days of coculture. Meanwhile cocultured cells promoted osteogenic differentiation more than MSC, which was confirmed by the presence of osteoblast-like cells and higher alkaline phosphatase activity. In the in vivo experiment, consistent results were also found in healing the critical-size defects in the rabbits. The microCT images from the calvarial defect in the control rabbits showed that the defects did not heal on their own, although there were signs of partial ossification in a centripetal fashion from the rim. In the two experimental groups, bone regenerated from several small regions within the central portions of the defects that were covered by the transplanted cell sheets. However, quantification of the microCT data showed that there was about 1.5 times more incorporation of the cocultured cell composite in the area of newly-formed bone and BV/TV than in the MSC composites. The quantification of histological staining also showed that there were significant differences in the extent of new bony formation between the coculture and the MSC groups. We hypothesised that various cytokines and growth factors secreted from cocultured cells may have exerted a greater paracrine effect than those from MSC for intrinsic angio-osteogenesis. We used a model of calvarial critical-size defects in rabbits because it is a well-documented model for the evaluation of bony healing.18 The patterns of bony accretion and overall bone mass in rabbits during skeletal maturation are similar to those in humans.19 We used a 15-mm defect and defined a critical-size defect using the criteria of Hollinger and Kleinschmidt.20 To create a more challenging environment for bony generation we also resected the periosteum, and a cell sheet technique was used to harvest cells and produce tissue-engineered bone. The use of cell sheets could significantly increase cell retention, stimulate greater vascular density, and improve the graft-host cell connection in the transplanted area.21 Finally, degradable hydroxyapatite scaffolds were used to promote bony regeneration22 and maintain the initial shape and volume of the cell sheet, which usually shrank spontaneously after detachment. Our study has several limitations. The use of unlabelled cells did not allow us to track and compare the contribution of transplanted cells. We also did no biomechanical testing because the cranium is a non-load-bearing area and the samples were limited. We plan to apply these techniques and tests in further studies. In addition, intensive studies are still required to elucidate the mechanism for promoting the ossification of cocultured cells.
Although there are several shortcomings we can conclude that the use of the cocultured cell composite (with hydroxyapatite) could promote bony regeneration more efficiently than MSC composite alone in a model of a calvarial critical-size defect in rabbits.
Acknowledgments The study is supported by National Natural Science Foundation of China (No. 31070873 and No. 81170938). We are grateful to Prof. Yan Jin from The Center for Tissue Engineering in Fourth Military Medical University for his excellent technical assistance.
References 1. Sotiropoulou PA, Perez SA, Salagianni M, et al. Characterization of the optimal culture conditions for clinical scale production of human mesenchymal stem cells. Stem Cells 2006;24:462–71. 2. Shieh SJ, Vacanti JP. State-of-the-art tissue engineering: from tissue engineering to organ building. Surgery 2005;137:1–7. 3. Yu H, Vandevord PJ, Gong W, et al. Promotion of osteogenesis in tissueengineered bone by pre-seeding endothelial progenitor cells-derived endothelial cells. J Orthop Res 2008;26:1147–52. 4. Koob S, Torio-Padron N, Stark GB, et al. Bone formation and neovascularization mediated by mesenchymal stem cells and endothelial cells in critical-sized calvarial defects. Tissue Eng Part A 2011;17: 311–21. 5. Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275:964–7. 6. Taguchi A, Soma T, Tanaka H, et al. Administration of CD34+ cells after stroke enhances neurogenesis via angiogenesis in a mouse model. J Clin Invest 2004;114:330–8. 7. Popa ER, Harmsen MC, Tio RA, et al. Circulating CD34+ progenitor cells modulate host angiogenesis and inflammation in vivo. J Mol Cell Cardiol 2006;41:86–96. 8. Chen JL, Hunt P, McElvain M, et al. Osteoblast precursor cells are found in CD34+ cells from human bone marrow. Stem Cells 1997;15:368–77. 9. Matsumoto T, Kawamoto A, Kuroda R, et al. Therapeutic potential of vasculogenesis and osteogenesis promoted by peripheral blood CD34-positive cells for functional bone healing. Am J Pathol 2006;169:1440–57. 10. Mifune Y, Matsumoto T, Kawamoto A, et al. Local delivery of granulocyte colony stimulating factor-mobilized CD34-positive progenitor cells using bioscaffold for modality of unhealing bone fracture. Stem Cells 2008;26:1395–405. 11. Nakamura A, Akahane M, Shigematsu H, et al. Cell sheet transplantation of cultured mesenchymal stem cells enhances bone formation in a rat nonunion model. Bone 2010;46:418–24. 12. L’Heureux N, Paquet S, Labbe R, et al. A completely biological tissueengineered human blood vessel. FASEB J 1998;12:47–56. 13. Yang J, Yamato M, Kohno C, et al. Cell sheet engineering: recreating tissues without biodegradable scaffolds. Biomaterials 2005;26:6415–22. 14. Ma D, Yao H, Tian W, et al. Enhancing bone formation by transplantation of a scaffold-free tissue-engineered periosteum in a rabbit model. Clin Oral Implants Res 2011;22:1193–9. 15. Shimizu T, Yamato M, Isoi Y, et al. Fabrication of pulsatile cardiac tissue grafts using a novel 3-dimensional cell sheet manipulation technique and temperature-responsive cell culture surfaces. Circ Res 2002;90:e40–8. 16. Kanczler JM, Oreffo RO. Osteogenesis and angiogenesis: the potential for engineering bone. Eur Cell Mater 2008;15:100–14.
G. Li et al. / British Journal of Oral and Maxillofacial Surgery 52 (2014) 134–139 17. Kawamoto A, Tkebuchava T, Yamaguchi J, et al. Intramyocardial transplantation of autologous endothelial progenitor cells for therapeutic neovascularization of myocardial ischemia. Circulation 2003;107:461–8. 18. Schmitz JP, Hollinger JO. The critical size defect as an experimental model for craniomandibulofacial nonunions. Clin Orthop Relat Res 1986;(205):299–308. 19. Djasim UM, Wolvius EB, van Neck JW, et al. Recommendations for optimal distraction protocols for various animal models on the basis of a systematic review of the literature. Int J Oral Maxillofac Surg 2007;36:877–83.
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20. Hollinger JO, Kleinschmidt JC. The critical size defect as an experimental model to test bone repair materials. J Craniofac Surg 1990;1: 60–8. 21. Wang CC, Chen CH, Lin WW, et al. Direct intramyocardial injection of mesenchymal stem cell sheet fragments improves cardiac functions after infarction. Cardiovasc Res 2008;77:515–24. 22. Boyne PJ, Rothstein SS, Gumaer KI, et al. Long-term study of hydroxylapatite implants in canine alveolar bone. J Oral Maxillofac Surg 1984;42:589–94.
British Journal of Oral and Maxillofacial Surgery 52 (2014) 134–139
Coculture of peripheral blood CD34+ cell and mesenchymal stem cell sheets increase the formation of bone in calvarial critical-size defects in rabbits Guanghui Li a,b,1 , Xi Wang c,1 , Jian Cao d , Zhaoyu Ju a , Dongyang Ma d , Yanpu Liu a,∗∗ , Junrui Zhang a,∗ a
Department of Oral and Maxillofacial Surgery, School of Stomatology, Fourth Military Medical University, Xi’an 710032, China Department of Stomatology, PLA 252 Hospital, Baoding 071000, China c Department of Orthodontics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China d Department of Oral and Maxillofacial Surgery, Lanzhou General Hospital, Lanzhou Command of PLA, Lanzhou 730000, China b
Accepted 11 October 2013 Available online 6 November 2013
Abstract The reconstruction of large bony defects remains a clinical challenge, and angiogenesis and neovascularisation are being given more attention in bone tissue engineering. In this study we cocultured peripheral blood CD34+ cells (PB-CD34+ cells), an endothelial progenitor cell/haematopoietic stem cell-enriched population, with bone marrow-derived mesenchymal stem cells (MSC) to investigate their potential for bony regeneration. Cocultured cells showed better osteogenic differentiation than MSC alone in vitro. The cocultured cells and MSC sheets were also composited with hydroxyapatite and implanted in calvarial critical-size defects in rabbits. The rabbits were killed before microcomputed tomographic (MicroCT) and histological analysis. The results showed that cocultured cell composites had promoted bony regeneration more efficiently by 8 weeks after implantation. Our results indicate that the coculture of PB-CD34+ cells and MSC increases bony regeneration in calvarial critical-size defects in rabbits, and provide a new promising therapeutic strategy to aid skeletal healing. © 2013 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Keywords: Peripheral blood CD34+ cells; Mesenchymal stem cells; Co-culture; Bone regeneration; Rabbit calvarial critical-size defect; Cell sheet transplantation
Introduction ∗
Corresponding author at: Department of Oral and Maxillofacial Surgery, School of Stomatology, Fourth Military Medical University, #145 Changlexi Road, Xi’an, Shannxi 710032, China. Tel.: +86 29 84772534; fax: +86 29 83224470. ∗∗ Corresponding author at: Department of Oral and Maxillofacial Surgery, School of Stomatology, Fourth Military Medical University, #145 Changlexi Road, Xi’an, Shannxi 710032, China. Tel.: +86 29 84772532; fax: +86 29 83224470. E-mail addresses: [email protected] (Y. Liu), [email protected] (J. Zhang). 1 Guanghui Li and Xi Wang contributed equally to the research, and should be regarded as joint first author.
The reconstruction of large bony defects caused by tumours, trauma, or inflammation remains a major clinical challenge. Tissue engineering is a promising technique for bony regeneration, in which osteogenic cells are combined with scaffolds to construct tissue-engineered bone. Mesenchymal stem cells (MSC) have gained much interest because of their therapeutic potential.1 However, the size of the regenerated bone is often limited by lack of vessels, and this prevents sufficient nutritional support getting to the entire bone graft. For efficient bony regeneration by tissue engineering, rapid neovascularisation of the implanted graft is essential.2
0266-4356/$ – see front matter © 2013 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bjoms.2013.10.004
G. Li et al. / British Journal of Oral and Maxillofacial Surgery 52 (2014) 134–139
Some research workers have recently studied the coimplantation or potential coculture of endothelial progenitor cells with MSC for bone tissue engineering,3,4 and shown that they can form a prevascular network during coculture and increase osteoblastic activity when the 2 types of cell are in direct contact. CD34+ cells that contain intensive endothelial progenitor and haematopoietic stem cells5 are capable of inducing neovascularisation in vivo.6,7 CD34+ cells have also been reported to differentiate into osteoblasts in vitro.8–10 These findings strongly suggest that CD34+ cells have therapeutic potential for both vasculogenesis and osteogenesis. Cell sheet engineering, which is a new tissue engineering technique, recreates a biological microenvironment similar to that of a regenerative milieu. Cell sheets that are fabricated from various types of cells including MSC, endothelial cells, hepatocytes, and macrophages have been studied.11–13 The technology has been proved to be effective for harvesting cells together with their endogenous extracellular matrix so that adhesion molecules on the surface of the cell and cell–cell interactions remain intact.14 The aims of the present study were to: coculture PBCD34+ cells with MSC in vitro, and assemble the cocultured cell sheet with hydroxyapatite to find out whether this would increase the formation of bone in healing calvarial criticalsize defects in rabbits.
Materials and methods Coculture of PB-CD34+ cells and MSC Total mononuclear cells were separated from the rabbits’ peripheral blood using Histopaque-1077 (Sigma–Aldrich, St. Louis, MO, USA). PB-CD34+ cells were isolated from mononuclear cells by flow cytometry using a conjugated CD34 antibody (PE-Cy5, Bioss Inc., Woburn, MA, USA). Bone marrow was aspirated from the bones of the rabbits’ hind limbs under sterile conditions. MSC were isolated from aspirated bone marrow by the Percoll density gradient centrifugation method and cultured in ␣-minimum essential medium (␣-MEM; Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% foetal bovine serum (Gibco BRL, Gaithersburg, MD, USA), l-glutamine 2 mmol (Invitrogen, Carlsbad, CA, USA), and 1% penicillin/streptomycin (Gibco BRL, Gaithersburg, MD, USA) in a humidified atmosphere of 5% carbon dioxide. A total of 1 × 105 MSC (passage 3) was plated into each well of a 6-well plate and cultured in ␣-MEM supplemented with 10% foetal bovine serum, l-glutamine 2 mmol, and 1% penicillin/streptomycin for one day. PB-CD34+ cells were then seeded on to the same plate at a density of 5 × 105 cells per well. The medium was not changed for the first 7 days, but was changed 2–3 times during the second week. After 14 days’ culture, non-adherent cells were removed. We used immunofluorescence to find out whether PB-CD34+
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Fig. 1. The composite hydroxyapatite/cell sheet transplanted into the calvarial defect.
cells existed in adherent cells. Cells were examined under a fluorescence microscope (BX-51, Olympus, Japan). In vitro experiments Osteogenic differentiation in vitro To induce differentiation of osteoblasts, cocultured cells, and MSC alone, were cultured in ␣-MEM supplemented with 10% foetal bovine serum, dexamethasone 100 nmol, ascorbic acid 50 g/ml, and -glycerophosphate 5 mmol. The medium was not changed for the first 7 days, but was changed 2–3 times during the second week. Alkaline phosphatase staining and activity were measured with an alkaline phosphatase colour development kit (Beyotime, Shanghai, China) and an alkaline phosphatase detection kit (Jiancheng Bioengineering, Nanjing, China) after 14 days in culture according to the manufacturers’ protocols. Construction of cell sheets The method for preparation of the cell sheets has been reported previously.15 Cocultured cells and MSC alone were cultured with ␣-MEM supplemented with 10% foetal bovine serum, dexamethasone 10 nmol, and ascorbic acid 50 g/ml until they reached confluence (about day 14). The medium was changed every 2 days. In vivo experiments Surgical protocol A total of 15 New Zealand rabbits (aged 3 months, weight about 2.6 kg) were used in the study. All protocols were approved by the Animal Welfare Committee of the Fourth Military Medical University. Commercially available hydroxyapatite (Sigma–Aldrich Co., Saint Louis, MO, USA) was sterilised by 60 Co irradiation. For ectopic transplantation, hydroxyapatite 100 mg was wrapped in a piece of cell sheet to assemble a composite. The rabbits were randomly divided into 3 groups: in the first the defect was repaired by
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cocultured cell composites (n = 6); in the second (n = 6) the defect was repaired by MSC composites; and in the third (control) group (n = 3) the defect was left untreated. After anaesthesia a tongue-shaped incision was made to expose the cranium. A 15-mm diameter full-thickness defect was carefully prepared and then treated as shown (Fig. 1). Native periosteum was removed to exclude its possible influence on bony regeneration. The scalp was closed with sutures. At 8 weeks rabbits were killed humanely with an intravenous overdose of barbiturate (200 mg/kg). MicroCT analysis Each specimen was scanned with a microCT system (Inveon, Siemens, Germany; 80 kV, 500 mA, 1200 ms integration time). The scans used a 360◦ radiographic projection (total scan time 30 min). Scanning images were switched into 3-dimensional volumes (21 m resolution) using Cobra software (Siemens reconstruction software). The volume of new bone in the defect site and bone volume/total volume (BV/TV) were also calculated. Histological analysis of bone After microCT scanning, each specimen was fixed in 95% ethanol and embedded in methyl methacrylate. Specimens were then cut and ground to sections 30-m thick. The slides were prepared and stained with Van Gieson reagents. Formation of new bone within the defect was calculated histomorphometrically using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA). The percentage area of new bone was obtained by dividing the bony area by the area of the whole defect. Statistical analysis
Fig. 2. Representative microcomputed tomographic image of bony regeneration in the animals treated with mesenchymal stem cells alone.
formed. Intact cell sheets could be detached with a cell scraper and further manipulated for wrapping with hydroxyapatite.
In vivo experiments Gross morphology of bone regeneration in rabbits All rabbits recovered well from the anaesthetic and the operation. Palpation of the defects showed that they were filled with tight bone-like structures in both the coculture and MSC groups. There were no obvious macroscopic differences between the 2 groups.
Data were analysed with the help of SPSS 17.0 software (SPSS, Chicago, IL, USA). Groups were compared using Student’s t test after testing by analysis of variance. Probabilities of less than 0.05 were accepted as significant.
Results In vitro experiments Osteogenic differentiation in vitro The osteogenic differentiation capacity was studied after 14 days of culture under osteogenic culture conditions. The morphology of the cells became more polygonal and cell clusters were formed when osteogenic supplements were added to the cultures. The alkaline phosphatase activity in cocultured cells was significantly higher than that in MSC (p = 0.012). Fabrication of cell sheets In both the cocultured cells and MSC there was an increase in the synthesis of extracellular matrix and dense cell sheets had
Fig. 3. Representative microcomputed tomographic image of bony regeneration in the animals treated with the coculture.
G. Li et al. / British Journal of Oral and Maxillofacial Surgery 52 (2014) 134–139
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Table 1 Comparisons of new bone volume (mm2 ), bone volume/total volume (BV/TV, %), and the extent of new bone formation (%) among the 3 groups. Groups
New bone volume (mm2 )
BV/TV (%)
Extent of new bone formation (%)
Control Mesenchymal stem cells Coculture
34.76(7.83) (P = 0.001) 90.26(10.51) (P = 0.017) 138.82(12.28)
3.93(0.51) (P = 0.002) 10.22(1.38) (P = 0.026) 16.72(1.87)
5.4(1.7) (P = 0.001) 16.7(2.9) (P = 0.023) 27.5(4.1)
p value
Fig. 4. Representative Van Gieson stain of bony regeneration in the animals treated with mesenchymal stem cells alone. Blue arrows indicate the site of the defect (Van Gieson stain, original magnification 5×).
MicroCT results New bone was formed only at the margins of the defect in the control group. Both the MSC and coculture groups showed signs of bony repair all over the defect, but the coculture group had a larger area of bone (Figs. 2 and 3). The 3-dimensional volume on the microCT images showed that there were significant differences in the volume of bone between the coculture and MSC groups (p = 0.017). There were also significant differences between that in the coculture group and the control group (p = 0.001, Table 1). Similar results were also found for BV/TV (Table 1). Histological results Representative histological images are shown in Figs. 4 and 5. The defects in the coculture group showed bony bridging and new bone formation throughout the defect, but the formation of bone was sparse in the MSC group. The extent of new bony formation in each group was calculated and there was a significantly larger area of new
bone in the coculture group than in the MSC and control groups (p = 0.023, 0.001, Table 1).
Discussion One of the main problems when repairing large craniofacial defects is the difficulty in maintaining the viability of the cells within the bone-grafted tissue to ensure its survival and the eventual restoration of the defect. Angiogenesis is a key contributor to the systematic growth and repair of bone.16 Therapeutic neovascularisation induced by endothelial progenitor or CD34+ cells has been tested in ischaemic diseases, and promising outcomes reported.6,17 Apart from the potential for vasculogenic induction, PB-CD34+ cells are capable of differentiation into osteoblasts in vitro.8–10 Mifune et al.10 also reported that PB-CD34+ cells could be induced to adherent cells without losing their haematopoietic stem cell markers. These findings suggest that PB-CD34+
Fig. 5. Representative Van Gieson stain of bony regeneration in the animals treated with the coculture. Blue arrows indicate the site of the defect (Van Gieson stain, original magnification 5×).
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cells may have the potential to differentiate into not only haematopoietic and endothelial lineages but also into mesenchymal lineages, including osteogenic cells. On the basis of these studies we postulated that coseeding of PB-CD34+ cells with MSC would lead to the efficient formation of bone in ectopic implants. Our results have shown that the cocultured cells could facilitate formation of bone in vitro and in vivo. In the in vitro experiments we showed that the harvested cells contained PB-CD34+ cells and MSC after 14 days of coculture. Meanwhile cocultured cells promoted osteogenic differentiation more than MSC, which was confirmed by the presence of osteoblast-like cells and higher alkaline phosphatase activity. In the in vivo experiment, consistent results were also found in healing the critical-size defects in the rabbits. The microCT images from the calvarial defect in the control rabbits showed that the defects did not heal on their own, although there were signs of partial ossification in a centripetal fashion from the rim. In the two experimental groups, bone regenerated from several small regions within the central portions of the defects that were covered by the transplanted cell sheets. However, quantification of the microCT data showed that there was about 1.5 times more incorporation of the cocultured cell composite in the area of newly-formed bone and BV/TV than in the MSC composites. The quantification of histological staining also showed that there were significant differences in the extent of new bony formation between the coculture and the MSC groups. We hypothesised that various cytokines and growth factors secreted from cocultured cells may have exerted a greater paracrine effect than those from MSC for intrinsic angio-osteogenesis. We used a model of calvarial critical-size defects in rabbits because it is a well-documented model for the evaluation of bony healing.18 The patterns of bony accretion and overall bone mass in rabbits during skeletal maturation are similar to those in humans.19 We used a 15-mm defect and defined a critical-size defect using the criteria of Hollinger and Kleinschmidt.20 To create a more challenging environment for bony generation we also resected the periosteum, and a cell sheet technique was used to harvest cells and produce tissue-engineered bone. The use of cell sheets could significantly increase cell retention, stimulate greater vascular density, and improve the graft-host cell connection in the transplanted area.21 Finally, degradable hydroxyapatite scaffolds were used to promote bony regeneration22 and maintain the initial shape and volume of the cell sheet, which usually shrank spontaneously after detachment. Our study has several limitations. The use of unlabelled cells did not allow us to track and compare the contribution of transplanted cells. We also did no biomechanical testing because the cranium is a non-load-bearing area and the samples were limited. We plan to apply these techniques and tests in further studies. In addition, intensive studies are still required to elucidate the mechanism for promoting the ossification of cocultured cells.
Although there are several shortcomings we can conclude that the use of the cocultured cell composite (with hydroxyapatite) could promote bony regeneration more efficiently than MSC composite alone in a model of a calvarial critical-size defect in rabbits.
Acknowledgments The study is supported by National Natural Science Foundation of China (No. 31070873 and No. 81170938). We are grateful to Prof. Yan Jin from The Center for Tissue Engineering in Fourth Military Medical University for his excellent technical assistance.
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