Influence Of Platelet-rich Plasma On Osteogenenic Differentiation Of Bone Marrow Stromal Cells And Bone Formation In Porous Coral

Bone tissue engineering offers a promising new approach for bone repair. Compared to traditional autograft and allograft procedures, bone tissue engineering techniques based on autogenous cell/tissue transplantation would eliminate problems of donor scarcity, supply limitation, and pathogen transfer and immune rejection. Therefore, it has become a rapidly expanding research area since the emergence of the concept of tissue engineering.Engineering bone typically uses an artificial extracellular matrix (or scaffold), osteoblasts or cells that can become osteoblasts, and growth factors that promote cell recruitment, growth differentiation and mineralized bone tissue formation. The scaffold for cell seeding should be porous so that it provides a large internal surface for adhesion and migration of cells and makes it easy for the exchange of nutrients and metabolic waste. Natural coral, which is biocompatible and osteoconductive, has pore sizes and architecture similar to those of human bone. It is an excellent material for supporting marrow stromal cells (MSCs) or osteoblasts attachment, proliferation, and differentiation while gradually being degraded and finally replaced by new bone. Platelet-rich plasma (PRP) is an autologous source of various growth factors that is obtained by sequestering and concentrating freshly drawn venous blood. When activated with thrombin and calcium chloride, the platelets in the PRP delivered a high concentration of growth factors into the recipient bed, which are all involved in reparative processes such as bone healing.MSCs have a high proliferative capacity and the ability to differentiate into osteoblasts, chondrocytes, and adipocytes. MSCs can be loaded onto biomaterials to create a bioactive composite. Adding expanded MSCs to biomaterials in vivo could significantly improve bone formation. However,the influence of PRP on expanded MSCs for osteogenesis and bone formation remains to be elucidated.Our first hypothesis was that the combination of expanded MSCs with PRP in resorbable natural coral could promote osteogenesis and enhance bone formation. We further hypothesized that cell-sheet could be growed by combined MSCs and PRP and bone could be engineered in vivo by using the cell-sheet in combination with coral scaffold.Experiment 1 Influence of platelet-rich plasma on proliferation and differentiation of MSCs in coral scaffold in vitroPurpose: To investigate the Influence of PRP on proliferation and differentiation of MSCs in coral scaffold in vitro.Method: A suspension of 5×106 MSCs in 50μl medium were dissolved in 50μl of PRP. The mixture of MSCs and PRP was spread on a coral disc with diameter of 8 mm and thickness of 2 mm, so coral/MSCs/PRP composite was obtained. A suspension of MSCs in 50μl medium was spread on a coral disc to obtain coral/MSCs composite. 50μl of PRP was added on a coral disc to obtain coral/PRP composite. Coral/MSCs/PRP composite was cultured in a humidified incubator for 8 and 14 days. The medium was changed every other day. Coral/MSCs composite and coral/PRP composite used as control. Then three types of composites were processed for to evaluate ALP activity, osteocalcin and proliferation of MSCs in the constructs. The composites were processed also for to evaluate adhesion, growth and proliferation of MSCs on coral scaffold by scanning electron microscope.Results: The ALP activity and content of osteocalcin were significantly higher in the samples of coral/MSCs/PRP group compared to that of coral/MSC group at 8 and 14 days in vitro (p<0.05). A comparison of the results of MSCs proliferation showed that cell growth increases as a function of PRP in coral scaffolds. Scanning electron micrographs show the coral scaffold had interconnected porous networks, more cells adhered to the scaffolds of coral/MSCs/PRP than to that of coral/MSCs, and the MSCs were homogeneously distributed throughout the scaffolds. Some asteroid MSCs are bound by the fibrin network, and these cells are believed to be in an active proliferating phase. A lot of floccular extracellular matrix was evident, implying the presence of active secreting MSCs. SEM analysis confirmed the extensive growth of MSCs in the scaffolds of coral/MSCs/PRP over the 14 days.Conclusion: These in vitro study results suggest that MSCs may be encouraged to proliferate and osteogenically differentiate by PRP in the coral scaffold, leading to increased osteogenesis in vitro.Experiment 2 Influence of platelet-rich plasma on ectopic bone formation of bone marrow stromal cells in porous coralPurpose: To evaluate the effect of PRP on ectopic bone formation of MSCs in porous coral.Methods: Natural coral disks with diameter of 8 mm and thickness of 2 mm were used in this study. A suspension of 5×10~6 MSCs in 50μl medium were dissolved in 50μl of PRP. The mixture of MSCs and PRP was spread on a coral disc. The constructs were implanted into the dorsal subcutaneous area of athymic mice. Coral scaffolds seeded with MSCs alone or added with PRP alone acted as control. The ectopic bone formation was investigated by gross examination, histological observation and Histomorphometric analyses 4 and 8 weeks after operation.Results: Gross examination showed that three types of constructs retained their original shape. The tissue formed in the coral/MSCs/PRP or coral/MSCs constructs was pink and hard to the touch with surgical forceps, a feature absent in the coral/PRP constructs. There was a trend of progressively increased pink and hardness between coral/MSCs and coral/MSCs/PRP constructs. New bone and/or cartilage formation could be observed in specimens from both coral/MSCs/PRP group and coral/MSCs group in ectopic sites, and osteogenesis followed the pattern of endochondral bone formation. Histomorphometric analyses showed enhanced cartilage and/or bone formation in coral/MSCs/PRP group 4 and 8 weeks after implantation, compared with the coral/MSCs group. In contrast, no bone or cartilage formation could be observed in coral/PRP group.Conclusion: Combination of MSCs with PRP in porous coral could improve increased ectopic bone formation.Experiment 3 Treatment of rabbit calvaria defects with bone marrow stromal cells in combination with platelet-rich plasma in porous coral Purpose: To evaluate local bone formation following surgical implantation of MSCs in combination with PRP in porous coral using a critical-size rabbit calvaria defect model.Methods: Natural coral disks with diameter of 15 mm and thickness of 2 mm were used in this study. A suspension of 3.3×107 MSCs in 300μl medium were dissolved in 300μl of PRP. The mixture of MSCs and PRP was spread on a coral disc. The construct was implanted into a critical-size 15mm rabbit calvaria defect. Coral disc alone or autograft used as control. The bone formation was investigated by gross examination, X-ray, histological observation and Histomorphometric analyses 6 and 12 weeks after operation.Results: Six weeks after grafting, new bone was distributed throughout the coral scaffolds in specimens from the test group, and new bone appeared only in the periphery region of the coral scaffold from the coral group. Histomorphometric data revealed a significantly higher bone area in test group than in the coral group (p<0.05). Twelve weeks after grafting, the bone defects of the test group were repaired fully with bone, when those of the coral group were repaired partly with bone and fibrous tissue was evident in the central region of defects. The test group had evident advantage over the coral group in term of bone regeneration (p<0.05). There was no statistically significant difference in bone formation between the test group and autograft group at 12 weeks post-surgery (p>0.05).Conclusion: Combination of MSCs with PRP in porous coral could enhance the bone healing considerably.Experiment 4 Cell-sheet constructed by combined marrow stromal cells and Platelet-rich plasma and its ectopic bone formation Purpose: To investigate method of growing cell-sheet by combined MSCs and PRP and its ectopic bone formation.Methods: A suspension of 5×10~6 MSCs in 500μl medium were dissolved in 500μl of PRP. The mixture of MSCs and PRP was plated in one well of 6-well plate and cultured in a humidified incubator for 3 weeks. The culture media was standard DMEM for first week and then was changed to osteogenic DMEM for next two weeks. The medium was changed every other day. MSCs sheet was formed and could detached intact from the substratum using a cell scraper. The sheet was processed for to evaluate its construction by scanning electron microscope. The sheet was then wrapped around the coral scaffold, and the sheet–scaffold construct was implanted into the dorsal subcutaneous area of athymic mice. Coral scaffolds acted as control. The ectopic bone formation was investigated by histological observation 4 and 8 weeks after operation.Results: The sheet comprising multilayered spindle-like osteoblasts possessed good mechanical properties. New bone and/or cartilage formation could be observed both inside and outside the scaffold in specimens from the sheet–scaffold construct group in ectopic sites 4 and 8 weeks after implantation, and osteogenesis followed the pattern of endochondral bone formation. In contrast, no bone or cartilage formation could be observed in coral group.Conclusion: The method of growing cell-sheet by combined MSCs and PRP was convenient and simple, and bone graft could be engineered through combination of the sheet and coral scaffold.