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Sheet of osteoblastic cells combined with platelet-rich fibrin improves the formation of bone in critical-size calvarial defects in rabbits
YBJOM-4732; No. of Pages 6
ARTICLE IN PRESS Available online at www.sciencedirect.com
British Journal of Oral and Maxillofacial Surgery xxx (2015) xxx–xxx
Sheet of osteoblastic cells combined with platelet-rich fibrin improves the formation of bone in critical-size calvarial defects in rabbits Zhifa Wang a,1 , Hanqing Hu b,1 , Zhijin Li a , Yanming Weng c , Taiqiang Dai a , Chunlin Zong a , Yanpu Liu a,∗∗ , Bin Liu d,∗ a
State Key Laboratory of Military Stomatology, Department of Oral and Maxillofacial Surgery, School of Stomatology, Fourth Military Medical University, Xi’an 710032, China b Department of Stomatology, the 94th hospital of PLA, NanChang, Jiangxi Province 330002, China c Department of Oral and Maxillofacial Surgery, Wuhan General Hopital of Guangzhou Military Region, Wuhan 43000, China d State Key Laboratory of Military Stomatology, Department of Oral Biology, School of Stomatology, Fourth Military Medical University, Xi’an 710032, China Accepted 17 December 2015
Abstract Techniques that use sheets of cells have been successfully used in various types of tissue regeneration, and platelet-rich fibrin (PRF) can be used as a source of growth factors to promote angiogenesis. We have investigated the effects of the combination of PRF and sheets of mesenchymal stem cells (MSC) from bone marrow on the restoration of bone in critical-size calvarial defects in rabbits to find out whether the combination promotes bony healing. Sheets of MSC and PRF were prepared from the same donor. We then implanted the combined MSC and PRF in critical-size calvarial defects in rabbits and assessed bony restoration by microcomputed tomography (microCT) and histological analysis. The results showed that PRF significantly increased bony regeneration at 8 weeks after implantation of sheets of MSC and PRF compared with sheets of MSC alone (p = 0.0048). Our results indicate that the combination of sheets of MSC and PRF increases bone regeneration in critical-size calvarial defects in rabbits, and provides a new way to improve skeletal healing. © 2015 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Keywords: Mesenchymal stem cells; Cell sheet; Platelet-rich fibrin; Bone regeneration; Rabbit calvarial critical-size defect
Introduction ∗ Corresponding author at: State Key Laboratory of Military Stomatology, Department of Oral Biology, School of Stomatology, the Fourth Military Medical University, 145 West Changle Road, Xi’an 710032, China. Tel.: +86-29-84776175; Fax: +86-29-84776175. ∗∗ Corresponding author at: State Key Laboratory of Military Stomatology, Department of Oral and Maxillofacial Surgery, School of Stomatology, the Fourth Military Medical University, 145 West Changle Road, Xi’an 710032, China. Tel: +86-29-84772532; Fax: +86-29-83224470. E-mail addresses: [email protected] (Y. Liu), [email protected] (B. Liu). 1 Zhifa Wang and Hanqing Hu contributed equally to this work and should be considered co-first authors.
The technology of engineering bone tissue remains the most promising treatment for the reconstruction of bony defects associated with microvascular flaps from the fibula, scapula, and iliac crest that are required for the treatment of trauma, congenital malformation, and after resection of a tumour.1,2 However, there are still some problems that we need to resolve about tissue-engineered bone grafts.3 For example, the lack of a good vascular supply seriously affects the formation of large areas of engineered bone as it relies on diffusion to supply nutrition as well as to remove any metabolic waste
http://dx.doi.org/10.1016/j.bjoms.2015.12.015 0266-4356/© 2015 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wang Z, et al. Sheet of osteoblastic cells combined with platelet-rich fibrin improves the formation of bone in critical-size calvarial defects in rabbits. Br J Oral Maxillofac Surg (2015), http://dx.doi.org/10.1016/j.bjoms.2015.12.015
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products. Diffusion can sustain cell viability only within a maximum of 200 m into the matrix,3–5 so vascularisation is essential for successful engineering of bone. In recent years, many techniques have been introduced to try and solve the problem of vascularisation including microsurgery, the design and modification of the structure of scaffolds, coculture systems, and incorporation of growth factors.6 Prefabrication of arteriovenous loops and flaps have been the most common microsurgical methods that have been used.7,8 One of the main purposes of the current design of scaffolds is the promotion of vascularisation by modification and sustained release of growth factors.9,10 The coculture of endothelial progenitor cells with mesenchymal stem cells (MSC) has also been reported.3,11 However, these techniques have their own shortcomings and still cannot produce sufficient vascularisation for adequate tissue engineering.12 It is inevitable that growth factors are vital to neovascularisation and the formation of bone so many growth factors have been used in regeneration, including platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor-beta 1 (TGF-1), insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF2), and endothelial cell growth factor (ECGF).13 Fortunately, platelet-rich fibrin (PRF), which is rich in many autologous growth factors such as these, and is capable of releasing them continuously and slowly, offers an appropriate solution for delivery.14 Engineered scaffold-free bone based on sheets of osteogenic MSC alone, without exogenous scaffolds, has also been made successfully.15,16 In a previous study we showed that PRF can considerably increase the osteogenic capacity of sheets of MSC in vitro and at an ectopic site in mice with severe combined immunodeficiency (SCID).17 Based on our previous findings, we investigated the effects of the combination of PRF and sheets of MSC on the restoration of bone in critical-size calvarial defects in rabbits to find out whether such a combination would promote bone healing.
Material and methods
Preparation of PRF PRF was prepared as reported previously.17 Briefly, blood 10 ml was drawn from each rabbit and centrifuged immediately for 10 minutes at 3000 rpm in a laboratory centrifuge (XIANGYI, Hunan, China). The clot was then harvested with tweezers and gently pressed into a membrane between two sterile pieces of gauze. PRF membranes were also prepared for histological examination, scanning EM, and transmission EM (JEM-1230, JEOL, Japan). Protocol A total of 15 New Zealand rabbits (3 months old, weighing about 2.5 kg) were used in the study. The rabbits were randomly divided into 3 groups: in the first the defect was repaired with combined sheets of MSC together with PRF (n=6), in the second (n = 6) with sheets of MSC alone, and in the third (control, n = 3) the defect was left untreated. After anaesthesia a tongue-shaped incision was made to expose the cranium. A full-thickness defect 15 mm in diameter was carefully prepared and treated as described. Native periosteum was removed to exclude any influence on bony regeneration. The scalp was closed with sutures. After 8 weeks rabbits were killed humanely with an overdose of barbiturate (200 mg/kg) given intravenously. MicroCT analysis Each specimen was scanned with a MicroCT system (Inveon, Siemens, Germany; 80 kV, 500 mA, 1200 milliseconds integration time). The scans used a 360◦ radiographic projection (total scan time 30 minutes). 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, and bone volume/total volume (BV/TV), were also calculated.
Cell isolation and culture
Histological analysis of bone
New Zealand rabbits were obtained from the animal holding unit of the Fourth Military Medical University (FMMU), and the protocol was approved by the Institutional Animal Care and Use Committee of FMMU. Briefly, MSC were isolated from the aspirated marrow of the iliums of adult rabbits and cultured in low-glucose Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 1% penicillin/streptomycin (Gibco, Carlsbad, CA, USA), and0.272 g/l L-glutamine (Sigma-Aldrich, St. Louis, MO, USA). Sheets of MSC were then cultured in osteogenic medium for two weeks as reported previously.17 The medium was changed every 2 days. The microstructure was observed by scanning electron microscopy (EM) (S-4800, Hitachi, Japan).
After CT the specimens were divided into two parts, one half of which was prepared and stained with haematoxylin and eosin and the other with Masson’s trichrome, and they were viewed under light microscopy. Sections were selected from each specimen for histomorphometrical examination as reported previously.17 Three high-resolution, low-magnification, micrographs were randomly selected from each section and analysed twice by two unbiased examiners (who were unaware of the experimental conditions) using computer-based image analysis techniques (Leicas Qwin Pro-image Analysis System). The cross-sectional area of mineralised bone and cartilage (blue staining) was measured and expressed as the relative percentage of the total cross-sectional area.
Please cite this article in press as: Wang Z, et al. Sheet of osteoblastic cells combined with platelet-rich fibrin improves the formation of bone in critical-size calvarial defects in rabbits. Br J Oral Maxillofac Surg (2015), http://dx.doi.org/10.1016/j.bjoms.2015.12.015
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Statistical analysis Data were analysed with the help of SPSS for Windows (version 17.0, SPSS, Chicago, IL, USA). Groups were compared using a two-tailed t test after testing by analysis of variance. Probabilities of less than 0.05 were accepted as significant.
Results Characterisation of sheets of osteogenic MSC MSC formed a sheet of cells that could be lifted easily from the culture dish with a cell scraper (Fig.S1 supplemental data). Histologically, the sheet was composed of several layers of cells that had retained their extracellular membrane, and calcium deposits were confirmed by von Kossa staining (Fig. 1). Scanning EM showed mineral-like nodules on the surface of the sheet of cells, and MSC were embedded in their own endogenous extracellular membrane (Fig.S1 supplemental data). Characterisation of PRF A PRF clot was easily obtained when the red corpuscular base and acellular plasma had been removed (Fig.S2, supplemental data). By extracting the fluids trapped within the fibrin matrix we obtained fibrin membranes (Fig. 2), and histological examination showed that they consisted of numerous, densely-arranged, red-stained fibrin bundles (Fig. S2 supplemental data). Scanning EM showed that the fibrin was arranged tightly, and many platelets were embedded in the compact fibrin network of the membranes. Transmission EM verified that fibrin bundles were densely arranged, and every platelet was associated with several fibrin bundles (Fig. S2).
Fig. 1. Multiple layers of cells, extracellular membrane, and mineralisation are evident on von Kossa staining.
Fig. 2. Resilient autologous fibrin membranes were easily obtained by removing the serum from the clot.
Restoration of bone in critical-size calvarial defects Gross examination All animals survived for the duration of the experiment with no complications. There were no wound infections, scalp effusions, disturbed wound healing, haematoma, or inflammation at the surgical site. Palpation of the defects showed that they were almost filled with tight bone-like structures in both the sheets of MSC and in the sheets combined with PRF. MicroCT analysis New bone was formed only at the margins of the defect in the control group (Fig. 3). Both the two experimental groups showed signs of restoration of bone over almost all the defects, but the combination group of sheets of MSC and PRF had more bone (Fig. S3). The 3-dimensional
Fig. 3. Representative microcomputed tomographic image of bony regeneration in the control group.
Please cite this article in press as: Wang Z, et al. Sheet of osteoblastic cells combined with platelet-rich fibrin improves the formation of bone in critical-size calvarial defects in rabbits. Br J Oral Maxillofac Surg (2015), http://dx.doi.org/10.1016/j.bjoms.2015.12.015
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Fig. 5. Masson’s trichrome staining showing that the newly-formed, bonelike tissue in the group with sheets of mesenchymal stem cells and plateletrich fibrin (original magnification × 40). Fig. 4. Representative microcomputed tomographic image of bony regeneration in the group with sheets of mesenchymal stem cells and platelet-rich fibrin.
volume on the MicroCT images also showed that the volume of new bone in the combined group (146.67 (15.36) mm3 was significantly greater than that in the other two groups (p = 0.0035 and p = 0.0026 respectively), and there was also a significant difference between the sheets of MSC alone group (88.33 (7.83) mm3 and the control group (25 (3.65) mm3 ) (p = 0.0007, Fig. 4 and S4). Results were similar for BV/TV, as the BV/TV was higher in the combined group than in the control group (p = 0.0113) and the sheets of MSC alone group (p = 0.0109) (Fig. S4). Histological and histomorphometrical examination There were many interconnected ossified trabeculae and unfinished mineralised pseudocartilaginous structures in the newly-formed construct of the combined group and the sheets of MSC alone group, but there were obvious differences in the newly-formed bone or bone-like tissue between the groups (Fig. S3). In contrast, the control group showed that the area of the defect was almost filled with newly-fibrous tissue and quite a few newly-formed, island–like formations of bone (Fig. S3). Similar results were also found on Masson’s trichrome stain (Fig. 5 and S3). Histomorphometrical examination showed that there were significant differences between the combined (34 (4.65) %) and sheets of MSC alone groups (21.33 (3.24) %) (p = 0.0048; Figs. 5 and 6), and there were also significant differences between the sheets of MSC alone and the control group (7.27 (2.53) %) (p = 0.0025, Fig. 5 and 6).
or additional growth factors, and this approach has several advantages. First, it does not require an exogenous scaffold as a cell carrier. Secondly, MSC were expanded in vitro and harvested together with their self-secreted extracellular membrane and cell-cell connections as a sheet of cells, which retains the integrity of these connections. Thirdly, we used autologous PRF as a source of growth factors, which has excellent biocompatibility and will not be rejected as foreign by the host.18 One of the main problems in fabricating large areas of bone is the difficulty in maintaining the viability of the transplanted cells to ensure that they can actually form bone.11 Fortunately, the “cell sheet” technique, which offers a high density of transplanted cells rich in extracellular membrane with intact cell-cell connections, is a viable approach in tissue engineering.19,20 Sheets of MSC were composed of several layers of cells that had maintained their extracellular membrane (Fig.S1). Scanning EM also showed that distinct mineral-like nodules were present on
Discussion Our results have shown that the combination of sheets of MSC and PRF can be used to repair the bone of critical-size calvarial defects in rabbits without the need for exogenous scaffolds
Fig. 6. The percentage area of new bone and cartilage in the group with sheets of mesenchymal stem cells (MSC) and platelet-rich fibrin PRF was significantly higher than those in the group with sheets of MSC alone, or the control group (*p = 0.0048). There were also significant differences between the group with sheets of MSC alone and the control group (*p = 0.0025).
Please cite this article in press as: Wang Z, et al. Sheet of osteoblastic cells combined with platelet-rich fibrin improves the formation of bone in critical-size calvarial defects in rabbits. Br J Oral Maxillofac Surg (2015), http://dx.doi.org/10.1016/j.bjoms.2015.12.015
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the surface of the cell sheets and the MSC were embedded in the membrane (Fig, S1), which is beneficial for bony formation when transplanted into the body. Some studies have reported that sheets of osteogenic cells can already be used to fabricate scaffold-free bone.12,17,20 Angiogenesis is another key contributor to the systematic growth and regeneration of bone, because sufficient nutritional support is essential.21 Many growth factors have been used in regenerative medicine, 22 but PRF is a secondgeneration form of platelet-rich concentrate that has a stronger and more durable effect on proliferation and differentiation of osteoblasts than platelet-rich plasma in vivo because it contains various growth factors and can release them gradually.23 PRF has also been successfully used clinically for the treatment of periodontal intrabony defects and achieved excellent results.24 We found that PRF improved the osteogenetic ability of sheets of MSC, as shown in Figs.S3 and S4, which confirms our previous result that PRF can increase the osteogenic potential of sheets of MSC in vitro and in SCID mice.17 The reasons that the combination of sheets of MSC and PRF work are that first, sheets of MSC are rich in osteoblasts and undifferentiated mesenchymal stem cells that offer ample source of cells for bony restoration;15 secondly, various growth factors contained in PRF promote angiogenesis, which provides a good vascular supply for bony regeneration;25 and thirdly, the extracellular membrane within the sheets of MSC and the 3-dimensional structures of the PRF offer an excellent microenvironment that facilitates the proliferation, movement, and differentiation of cells.17 The experimental results indicated that PRF significantly promoted bony regeneration of sheets of MSC by 8 weeks after implantation in rabbits, and this may be used clinically in the treatment of delayed bone healing, non-union, and osteonecrosis in the future. However, our study has some limitations, and further work is required. For example, we studied regeneration of only the flat bone in critical-size calvarial defects, and regeneration of long bones should also be studied using the same protocol. In addition, we must better understand the molecular mechanisms of this approach. In summary, we have shown that PRF’s rich fibrin network and supply of growth factors significantly increased bony regeneration of sheets of MSC compared with that of sheets used alone by 8 weeks after implantation in rabbits. Our results indicate that the combination of sheets of MSC together with PRF increases bony regeneration, and provides a promising new way of improving skeletal healing. Conflict of Interest We have no conflicts of interest. Ethics statement/confirmation of patients’ permission This study was reviewed and approved by the Institutional Animal Care and Use Committee at the University, and
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animals were cared for according to established institutional guidelines.
Acknowledgments This work was funded by the National Natural Science Foundation of China (No. 81070820, 31170942).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.bjoms.2015.12.015.
References 1. Lammens J, Laumen A, Delport H, et al. The Pentaconcept in skeletal tissue engineering. A combined approach for the repair of bone defects. Acta Orthop Belg 2012;78:569–73. 2. Cancedda R, Giannoni P, Mastrogiacomo M. A tissue engineering approach to bone repair in large animal models and in clinical practice. Biomaterials 2007;28:4240–50. 3. Yu H, VandeVord PJ, Mao L, et al. Improved tissue-engineered bone regeneration by endothelial cell mediated vascularization. Biomaterials 2009;30:508–17. 4. Shieh SJ, Vacanti JP. State-of-the-art tissue engineering: from tissue engineering to organ building. Surgery 2005;137:1–7. 5. Frerich B, Lindemann N, Kurtz-Hoffmann J, et al. In vitro model of a vascular stroma for the engineering of vascularized tissues. Int J Oral Maxillofac Surg 2001;30:414–20. 6. Santos MI, Reis RL. Vascularization in bone tissue engineering: physiology, current strategies, major hurdles and future challenges. Macromol Biosc 2010;10:12–27. 7. Buehrer G, Balzer A, Arnold I, et al. Combination of BMP2 and MSCs significantly increases bone formation in the rat arterio-venous loop model. Tissue Eng Part A 2015;21:96–105. 8. Kokemuller H, Jehn P, Spalthoff S, et al. En bloc prefabrication of vascularized bioartificial bone grafts in sheep and complete workflow for custom-made transplants. Int J Oral Maxillofac Surg 2014;43:163–72. 9. Duan B, Wang M. Customized Ca-P/PHBV nanocomposite scaffolds for bone tissue engineering: design, fabrication, surface modification and sustained release of growth factor. J Rl Soc Interface 2010;7(suppl 5):S615–29. 10. Laschke MW, Rucker M, Jensen G, et al. Improvement of vascularization of PLGA scaffolds by inosculation of in situ-preformed functional blood vessels with the host microvasculature. Ann Surg 2008;248:939–48. 11. Li G, Wang X, Cao J, et al. Coculture of peripheral blood CD34+ cell and mesenchymal stem cell sheets increase the formation of bone in calvarial critical-size defects in rabbits. Br J Oral Maxillofac Surg 2014;52:134–9. 12. Zhang R, Gao Z, Geng W, et al. Engineering vascularized bone graft with osteogenic and angiogenic lineage differentiated bone marrow mesenchymal stem cells. Artif organs 2012;36:1036–46. 13. Chen FM, Sun HH, Lu H, et al. Stem cell-delivery therapeutics for periodontal tissue regeneration. Biomaterials 2012;33:6320–44. 14. Lundquist R, Dziegiel MH, Agren MS. Bioactivity and stability of endogenous fibrogenic factors in platelet-rich fibrin. Wound Repair Regen 2008;16:356–63. 15. Ma D, Ren L, Liu Y, et al. Engineering scaffold-free bone tissue using bone marrow stromal cell sheets. J Orthop Res 2010;28:697–702.
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16. Akahane M, Shigematsu H, Tadokoro M, et al. Scaffold-free cell sheet injection results in bone formation. J Tissue Eng Regen Med 2010;4:404–11. 17. Wang Z, Weng Y, Lu S, et al. Osteoblastic mesenchymal stem cell sheet combined with Choukroun platelet-rich fibrin induces bone formation at an ectopic site. J Biomed Mater Res B Appl Biomater 2015;103:1204–16. 18. Dohan DM, Choukroun J, Diss A, et al. Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part I: technological concepts and evolution. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101:e37–44. 19. Ma D, Zhong C, Yao H, et al. Engineering injectable bone using bone marrow stromal cell aggregates. Stem Cells Devel 2011;20:989–99. 20. Geng W, Ma D, Yan X, et al. Engineering tubular bone using mesenchymal stem cell sheets and coral particles. Biochem Biophys Res Commun 2013;433:595–601. 21. Kanczler JM, Oreffo RO. Osteogenesis and angiogenesis: the potential for engineering bone. Eur Cell Mater 2008;15:100–14.
22. Kigami R, Sato S, Tsuchiya N, et al. Effect of basic fibroblast growth factor on angiogenesis and bone regeneration in non-critical-size bone defects in rat calvaria. J Oral Sci 2014;56:17–22. 23. He L, Lin Y, Hu X, et al. A comparative study of platelet-rich fibrin (PRF) and platelet-rich plasma (PRP) on the effect of proliferation and differentiation of rat osteoblasts in vitro. Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics 2009;108: 707–13. 24. Shah M, Deshpande N, Bharwani A, et al. Effectiveness of autologous platelet-rich fibrin in the treatment of intra-bony defects: A systematic review and meta-analysis. J Indian Soc Periodontol 2014;18: 698–704. 25. Dohan Ehrenfest DM, Andia I, Zumstein MA, et al. Classification of platelet concentrates (Platelet-Rich Plasma-PRP, Platelet-Rich FibrinPRF) for topical and infiltrative use in orthopedic and sports medicine: current consensus, clinical implications and perspectives. Muscles Ligaments Tendons J 2014;4:3–9.
Please cite this article in press as: Wang Z, et al. Sheet of osteoblastic cells combined with platelet-rich fibrin improves the formation of bone in critical-size calvarial defects in rabbits. Br J Oral Maxillofac Surg (2015), http://dx.doi.org/10.1016/j.bjoms.2015.12.015
ARTICLE IN PRESS Available online at www.sciencedirect.com
British Journal of Oral and Maxillofacial Surgery xxx (2015) xxx–xxx
Sheet of osteoblastic cells combined with platelet-rich fibrin improves the formation of bone in critical-size calvarial defects in rabbits Zhifa Wang a,1 , Hanqing Hu b,1 , Zhijin Li a , Yanming Weng c , Taiqiang Dai a , Chunlin Zong a , Yanpu Liu a,∗∗ , Bin Liu d,∗ a
State Key Laboratory of Military Stomatology, Department of Oral and Maxillofacial Surgery, School of Stomatology, Fourth Military Medical University, Xi’an 710032, China b Department of Stomatology, the 94th hospital of PLA, NanChang, Jiangxi Province 330002, China c Department of Oral and Maxillofacial Surgery, Wuhan General Hopital of Guangzhou Military Region, Wuhan 43000, China d State Key Laboratory of Military Stomatology, Department of Oral Biology, School of Stomatology, Fourth Military Medical University, Xi’an 710032, China Accepted 17 December 2015
Abstract Techniques that use sheets of cells have been successfully used in various types of tissue regeneration, and platelet-rich fibrin (PRF) can be used as a source of growth factors to promote angiogenesis. We have investigated the effects of the combination of PRF and sheets of mesenchymal stem cells (MSC) from bone marrow on the restoration of bone in critical-size calvarial defects in rabbits to find out whether the combination promotes bony healing. Sheets of MSC and PRF were prepared from the same donor. We then implanted the combined MSC and PRF in critical-size calvarial defects in rabbits and assessed bony restoration by microcomputed tomography (microCT) and histological analysis. The results showed that PRF significantly increased bony regeneration at 8 weeks after implantation of sheets of MSC and PRF compared with sheets of MSC alone (p = 0.0048). Our results indicate that the combination of sheets of MSC and PRF increases bone regeneration in critical-size calvarial defects in rabbits, and provides a new way to improve skeletal healing. © 2015 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Keywords: Mesenchymal stem cells; Cell sheet; Platelet-rich fibrin; Bone regeneration; Rabbit calvarial critical-size defect
Introduction ∗ Corresponding author at: State Key Laboratory of Military Stomatology, Department of Oral Biology, School of Stomatology, the Fourth Military Medical University, 145 West Changle Road, Xi’an 710032, China. Tel.: +86-29-84776175; Fax: +86-29-84776175. ∗∗ Corresponding author at: State Key Laboratory of Military Stomatology, Department of Oral and Maxillofacial Surgery, School of Stomatology, the Fourth Military Medical University, 145 West Changle Road, Xi’an 710032, China. Tel: +86-29-84772532; Fax: +86-29-83224470. E-mail addresses: [email protected] (Y. Liu), [email protected] (B. Liu). 1 Zhifa Wang and Hanqing Hu contributed equally to this work and should be considered co-first authors.
The technology of engineering bone tissue remains the most promising treatment for the reconstruction of bony defects associated with microvascular flaps from the fibula, scapula, and iliac crest that are required for the treatment of trauma, congenital malformation, and after resection of a tumour.1,2 However, there are still some problems that we need to resolve about tissue-engineered bone grafts.3 For example, the lack of a good vascular supply seriously affects the formation of large areas of engineered bone as it relies on diffusion to supply nutrition as well as to remove any metabolic waste
http://dx.doi.org/10.1016/j.bjoms.2015.12.015 0266-4356/© 2015 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wang Z, et al. Sheet of osteoblastic cells combined with platelet-rich fibrin improves the formation of bone in critical-size calvarial defects in rabbits. Br J Oral Maxillofac Surg (2015), http://dx.doi.org/10.1016/j.bjoms.2015.12.015
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products. Diffusion can sustain cell viability only within a maximum of 200 m into the matrix,3–5 so vascularisation is essential for successful engineering of bone. In recent years, many techniques have been introduced to try and solve the problem of vascularisation including microsurgery, the design and modification of the structure of scaffolds, coculture systems, and incorporation of growth factors.6 Prefabrication of arteriovenous loops and flaps have been the most common microsurgical methods that have been used.7,8 One of the main purposes of the current design of scaffolds is the promotion of vascularisation by modification and sustained release of growth factors.9,10 The coculture of endothelial progenitor cells with mesenchymal stem cells (MSC) has also been reported.3,11 However, these techniques have their own shortcomings and still cannot produce sufficient vascularisation for adequate tissue engineering.12 It is inevitable that growth factors are vital to neovascularisation and the formation of bone so many growth factors have been used in regeneration, including platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor-beta 1 (TGF-1), insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF2), and endothelial cell growth factor (ECGF).13 Fortunately, platelet-rich fibrin (PRF), which is rich in many autologous growth factors such as these, and is capable of releasing them continuously and slowly, offers an appropriate solution for delivery.14 Engineered scaffold-free bone based on sheets of osteogenic MSC alone, without exogenous scaffolds, has also been made successfully.15,16 In a previous study we showed that PRF can considerably increase the osteogenic capacity of sheets of MSC in vitro and at an ectopic site in mice with severe combined immunodeficiency (SCID).17 Based on our previous findings, we investigated the effects of the combination of PRF and sheets of MSC on the restoration of bone in critical-size calvarial defects in rabbits to find out whether such a combination would promote bone healing.
Material and methods
Preparation of PRF PRF was prepared as reported previously.17 Briefly, blood 10 ml was drawn from each rabbit and centrifuged immediately for 10 minutes at 3000 rpm in a laboratory centrifuge (XIANGYI, Hunan, China). The clot was then harvested with tweezers and gently pressed into a membrane between two sterile pieces of gauze. PRF membranes were also prepared for histological examination, scanning EM, and transmission EM (JEM-1230, JEOL, Japan). Protocol A total of 15 New Zealand rabbits (3 months old, weighing about 2.5 kg) were used in the study. The rabbits were randomly divided into 3 groups: in the first the defect was repaired with combined sheets of MSC together with PRF (n=6), in the second (n = 6) with sheets of MSC alone, and in the third (control, n = 3) the defect was left untreated. After anaesthesia a tongue-shaped incision was made to expose the cranium. A full-thickness defect 15 mm in diameter was carefully prepared and treated as described. Native periosteum was removed to exclude any influence on bony regeneration. The scalp was closed with sutures. After 8 weeks rabbits were killed humanely with an overdose of barbiturate (200 mg/kg) given intravenously. MicroCT analysis Each specimen was scanned with a MicroCT system (Inveon, Siemens, Germany; 80 kV, 500 mA, 1200 milliseconds integration time). The scans used a 360◦ radiographic projection (total scan time 30 minutes). 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, and bone volume/total volume (BV/TV), were also calculated.
Cell isolation and culture
Histological analysis of bone
New Zealand rabbits were obtained from the animal holding unit of the Fourth Military Medical University (FMMU), and the protocol was approved by the Institutional Animal Care and Use Committee of FMMU. Briefly, MSC were isolated from the aspirated marrow of the iliums of adult rabbits and cultured in low-glucose Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 1% penicillin/streptomycin (Gibco, Carlsbad, CA, USA), and0.272 g/l L-glutamine (Sigma-Aldrich, St. Louis, MO, USA). Sheets of MSC were then cultured in osteogenic medium for two weeks as reported previously.17 The medium was changed every 2 days. The microstructure was observed by scanning electron microscopy (EM) (S-4800, Hitachi, Japan).
After CT the specimens were divided into two parts, one half of which was prepared and stained with haematoxylin and eosin and the other with Masson’s trichrome, and they were viewed under light microscopy. Sections were selected from each specimen for histomorphometrical examination as reported previously.17 Three high-resolution, low-magnification, micrographs were randomly selected from each section and analysed twice by two unbiased examiners (who were unaware of the experimental conditions) using computer-based image analysis techniques (Leicas Qwin Pro-image Analysis System). The cross-sectional area of mineralised bone and cartilage (blue staining) was measured and expressed as the relative percentage of the total cross-sectional area.
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Statistical analysis Data were analysed with the help of SPSS for Windows (version 17.0, SPSS, Chicago, IL, USA). Groups were compared using a two-tailed t test after testing by analysis of variance. Probabilities of less than 0.05 were accepted as significant.
Results Characterisation of sheets of osteogenic MSC MSC formed a sheet of cells that could be lifted easily from the culture dish with a cell scraper (Fig.S1 supplemental data). Histologically, the sheet was composed of several layers of cells that had retained their extracellular membrane, and calcium deposits were confirmed by von Kossa staining (Fig. 1). Scanning EM showed mineral-like nodules on the surface of the sheet of cells, and MSC were embedded in their own endogenous extracellular membrane (Fig.S1 supplemental data). Characterisation of PRF A PRF clot was easily obtained when the red corpuscular base and acellular plasma had been removed (Fig.S2, supplemental data). By extracting the fluids trapped within the fibrin matrix we obtained fibrin membranes (Fig. 2), and histological examination showed that they consisted of numerous, densely-arranged, red-stained fibrin bundles (Fig. S2 supplemental data). Scanning EM showed that the fibrin was arranged tightly, and many platelets were embedded in the compact fibrin network of the membranes. Transmission EM verified that fibrin bundles were densely arranged, and every platelet was associated with several fibrin bundles (Fig. S2).
Fig. 1. Multiple layers of cells, extracellular membrane, and mineralisation are evident on von Kossa staining.
Fig. 2. Resilient autologous fibrin membranes were easily obtained by removing the serum from the clot.
Restoration of bone in critical-size calvarial defects Gross examination All animals survived for the duration of the experiment with no complications. There were no wound infections, scalp effusions, disturbed wound healing, haematoma, or inflammation at the surgical site. Palpation of the defects showed that they were almost filled with tight bone-like structures in both the sheets of MSC and in the sheets combined with PRF. MicroCT analysis New bone was formed only at the margins of the defect in the control group (Fig. 3). Both the two experimental groups showed signs of restoration of bone over almost all the defects, but the combination group of sheets of MSC and PRF had more bone (Fig. S3). The 3-dimensional
Fig. 3. Representative microcomputed tomographic image of bony regeneration in the control group.
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Fig. 5. Masson’s trichrome staining showing that the newly-formed, bonelike tissue in the group with sheets of mesenchymal stem cells and plateletrich fibrin (original magnification × 40). Fig. 4. Representative microcomputed tomographic image of bony regeneration in the group with sheets of mesenchymal stem cells and platelet-rich fibrin.
volume on the MicroCT images also showed that the volume of new bone in the combined group (146.67 (15.36) mm3 was significantly greater than that in the other two groups (p = 0.0035 and p = 0.0026 respectively), and there was also a significant difference between the sheets of MSC alone group (88.33 (7.83) mm3 and the control group (25 (3.65) mm3 ) (p = 0.0007, Fig. 4 and S4). Results were similar for BV/TV, as the BV/TV was higher in the combined group than in the control group (p = 0.0113) and the sheets of MSC alone group (p = 0.0109) (Fig. S4). Histological and histomorphometrical examination There were many interconnected ossified trabeculae and unfinished mineralised pseudocartilaginous structures in the newly-formed construct of the combined group and the sheets of MSC alone group, but there were obvious differences in the newly-formed bone or bone-like tissue between the groups (Fig. S3). In contrast, the control group showed that the area of the defect was almost filled with newly-fibrous tissue and quite a few newly-formed, island–like formations of bone (Fig. S3). Similar results were also found on Masson’s trichrome stain (Fig. 5 and S3). Histomorphometrical examination showed that there were significant differences between the combined (34 (4.65) %) and sheets of MSC alone groups (21.33 (3.24) %) (p = 0.0048; Figs. 5 and 6), and there were also significant differences between the sheets of MSC alone and the control group (7.27 (2.53) %) (p = 0.0025, Fig. 5 and 6).
or additional growth factors, and this approach has several advantages. First, it does not require an exogenous scaffold as a cell carrier. Secondly, MSC were expanded in vitro and harvested together with their self-secreted extracellular membrane and cell-cell connections as a sheet of cells, which retains the integrity of these connections. Thirdly, we used autologous PRF as a source of growth factors, which has excellent biocompatibility and will not be rejected as foreign by the host.18 One of the main problems in fabricating large areas of bone is the difficulty in maintaining the viability of the transplanted cells to ensure that they can actually form bone.11 Fortunately, the “cell sheet” technique, which offers a high density of transplanted cells rich in extracellular membrane with intact cell-cell connections, is a viable approach in tissue engineering.19,20 Sheets of MSC were composed of several layers of cells that had maintained their extracellular membrane (Fig.S1). Scanning EM also showed that distinct mineral-like nodules were present on
Discussion Our results have shown that the combination of sheets of MSC and PRF can be used to repair the bone of critical-size calvarial defects in rabbits without the need for exogenous scaffolds
Fig. 6. The percentage area of new bone and cartilage in the group with sheets of mesenchymal stem cells (MSC) and platelet-rich fibrin PRF was significantly higher than those in the group with sheets of MSC alone, or the control group (*p = 0.0048). There were also significant differences between the group with sheets of MSC alone and the control group (*p = 0.0025).
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the surface of the cell sheets and the MSC were embedded in the membrane (Fig, S1), which is beneficial for bony formation when transplanted into the body. Some studies have reported that sheets of osteogenic cells can already be used to fabricate scaffold-free bone.12,17,20 Angiogenesis is another key contributor to the systematic growth and regeneration of bone, because sufficient nutritional support is essential.21 Many growth factors have been used in regenerative medicine, 22 but PRF is a secondgeneration form of platelet-rich concentrate that has a stronger and more durable effect on proliferation and differentiation of osteoblasts than platelet-rich plasma in vivo because it contains various growth factors and can release them gradually.23 PRF has also been successfully used clinically for the treatment of periodontal intrabony defects and achieved excellent results.24 We found that PRF improved the osteogenetic ability of sheets of MSC, as shown in Figs.S3 and S4, which confirms our previous result that PRF can increase the osteogenic potential of sheets of MSC in vitro and in SCID mice.17 The reasons that the combination of sheets of MSC and PRF work are that first, sheets of MSC are rich in osteoblasts and undifferentiated mesenchymal stem cells that offer ample source of cells for bony restoration;15 secondly, various growth factors contained in PRF promote angiogenesis, which provides a good vascular supply for bony regeneration;25 and thirdly, the extracellular membrane within the sheets of MSC and the 3-dimensional structures of the PRF offer an excellent microenvironment that facilitates the proliferation, movement, and differentiation of cells.17 The experimental results indicated that PRF significantly promoted bony regeneration of sheets of MSC by 8 weeks after implantation in rabbits, and this may be used clinically in the treatment of delayed bone healing, non-union, and osteonecrosis in the future. However, our study has some limitations, and further work is required. For example, we studied regeneration of only the flat bone in critical-size calvarial defects, and regeneration of long bones should also be studied using the same protocol. In addition, we must better understand the molecular mechanisms of this approach. In summary, we have shown that PRF’s rich fibrin network and supply of growth factors significantly increased bony regeneration of sheets of MSC compared with that of sheets used alone by 8 weeks after implantation in rabbits. Our results indicate that the combination of sheets of MSC together with PRF increases bony regeneration, and provides a promising new way of improving skeletal healing. Conflict of Interest We have no conflicts of interest. Ethics statement/confirmation of patients’ permission This study was reviewed and approved by the Institutional Animal Care and Use Committee at the University, and
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animals were cared for according to established institutional guidelines.
Acknowledgments This work was funded by the National Natural Science Foundation of China (No. 81070820, 31170942).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.bjoms.2015.12.015.
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