MSC Sheet Combined With Platelet Rich Plasma Gel/calcium Phospate Nanoparticles For Bone Regeneration

IntroductionThe reconstruction of fracture and bone defects caused by trauma, tumors or infections often requires surgical treatment in clinical practice. In many treatments, autogenous cancellous bone graft is considered the gold standard. However; the cancellous bone graft has some shortcomings, such as donor site morbidity, source limitation, increased pain and operation risk for patients. In the repair of long bone defects, the problem appears more serioius, which undoubtedly limited the application of autogenous cancellous bone graft. The presence of infection caused by exogenous diseases and risk of immune responses can not be ignored for allogeneic boe graft. In recent years, the method of tissue engineering to repair or reconstruction of the bone defects is becoming a research hotspot, which is based on three elements, namely the seed cells, growth factors and biomaterial scaffolds. Stem cells have the potential of multilineage differentiation and have been widely used in bone tissue engineering. The obtaination of bone marrow derived mesenchymal stem cells is simple and convenient.Therefore, bone marrow derived MSCs is the ideal seed cells. In addition, previous studies have indicated that stem cells cultured at high density can form cell sheet structure, namely MSC sheet. Growth factors can induce the directional differentiation of MSCs in vitro and promote the repair of bone defects in vivo. However; the growth factor is expensive (such as BMP-2). Therefore, seeking to a drug which can play the same effect of growth factors or autologous source of growth factors is very significant. Researches showed that simvastatin could stimulate BMP-2secretion of endogenous cells; autologous platelet-rich plasma (PRP) contains a variety of growth factors. As for biological material, calcium sulfate due to its excellent biocompatibility and osteoconductivity, calcium phosphate due to its same composition of bone tissue, the two kinds of material has been widely used in bone tissue engineering. Therefore; in the present study, we investigated the effect of simvastatin and PRP on MSCs in vitro; and evaluated the potential clinical applications of MSC sheet, simvastatin-loaded calcium sulfate scaffold, MSC sheet combined with PRP gel/calcium phosphate nanoparticles on bone regeneration.This study contains five parts:(1) Simvastatin locally applied from simvastatin-loaded calcium sulphate scaffolds improves bone regeneration comparable to rhBMP-2application;(2) The effect of simvastatin on the proliferation, differentiation of rat bone marrow derived MSCs;(3) Transplantation of angiogenesis and osteogenesis MSC sheet promoted fracture healing;(4) The effects of platelet rich plasma/calcium phosphate nanoparticles on the proliferation and differentiation of rat MSCs;(5) MSC sheet combined’ with PRP gel/calcium phosphate nanoparticles for bone defects repair.Part Ⅰ Simvastatin locally applied from simvastatin-loaded calcium sulphate scaffolds improves bone regeneration comparable to rhBMP-2applicationAim:This study aimed to investigate whether simvastatin can be efficiently released from simvastatin-loaded calcium sulphate (CS) scaffolds in vitro and locally applied simvastatin can promote bone regeneration in vivo.Methods and results:0.5mg simvastatin was incooporated into CS scaffold of different weight (160mg,480mg). Simvastatin was highly efficient released from simvastatin-loaded CS at the onset and stable release was maintained. Thiry-six complete1.2-cm bone defects were created in ulna of rabbits and treated with CS, simvastatin-loaded CS or rhBMP-2-loaded CS. The defects treated with rhBMP-2-loaded CS and simvastatin-loaded CS showed significantly higher scores by X-ray analysis and more bone formation determined by histology than CS group (p<0.05). No significant differences in X-ray score and bone formation were found between groups of rhBMP-2-loaded CS and simvastatin-loaded CS (p>0.05).Conclusions:The present study revealed that simvastatin could stimulate bone regeneration when it was locally released from CS scaffolds into bone defects and that the beneficial effect of simvastatin was similar to the effect of rhBMP-2. It meant that the simvastatin-loaded CS scaffolds might have great potential in clinical bone repair.Part II The effect of simvastatin on the proliferation, differentiation of rat bone marrow derived mesenchymal stem cellsAim:This study aimed to investigate the effect of simvastatin on the proliferation, differentiation of rat BMSCs.Methods and results:The proliferation of MSCs cocultured with different concentrations of simvastatin for1,3, and7were detected by MTT. The results showed that simvastatin concentration higher than1μM inhibited the proliferation of MSCs. Based on the result of MTT,0.01μM,0.1μM,1μM simvastatin were cocultured with MSCs. MSCs cocultured without simvastatin served as control. After3and7days, the gene expressions of BMP-2, ALP, OCN, OPG and VEGF were detected. The results showed that the gene expressions of MSCs induced by simvastatin were significantly higher than those in control group. Moreover, with the increase of simvastatin concentration, the gene expressions upregulated. MSCs can form MSC sheet when MSCs were cultured with high density. The gene expressions of BMP-2, ALP, OCN, OPG and VEGF of MSC sheet were detected after MSC sheet were cocultured with1μM simvastatin for7days. Untreated MSC sheet served as control. The results showed that the gene expressions of BMP-2, ALP, OCN, OPG and VEGF of MSC sheet cocultured with simvastatin were significantly higher than those in MSC sheet (control).Conclusions:Low concentration of simvastatin can promote the osteogenic differentiation and angiogenesis of MSCs and MSC sheet. Thus MSC sheet with osteogenesis and angiogenesis may have great potential in bone tissue engineering. Part Ⅲ Transplantation of angiogenesis and osteogenesis MSC sheet promoted fracture healingAim:This study aimed to investigate the effects of angiogenesis and osteogenesis MSC sheet on fracture healing.Methods and results:Rat bone marrow derived MSCs were cultured in high density and form MSC sheet. MSC sheet induced by1μM simvastatin for7days was used for in vivo transplantation. In vivo, an osteotomy model was made in rat tibia and fractured tibias of20rats were treated with simvastatin-induced MSC sheet or untrated. Tibias were harvested at2or8weeks and underwent X-ray, micro-CT and histological analysis. After2and8weeks, X ray scores in MSC sheet group were significantly higher than those of the control group. The histology and micro-CT showed that the fracture site in MSC sheet group was partially bridged at2weeks and complete bone union was obtained at8weeks. While the fracture site in control group was filled with fibrous tissue at2weeks and showed nonunion at8weeks.Conclusions:The tibia fracture was completely cured by transplantation of osteogenesis and angiogenesis MSC sheet. Such MSC sheet transplantation may contribute to the treatment of fractures, nonunion bone defects in clinic.Part IV The effects of platelet rich plasma/calcium phosphate nanoparticles on the proliferation and differentiation of rat MSCsAim:This study aimed to investigate the effects platelet rich plasma or with calcium phosphate nanoparticles on the proliferation and differentiation of rat MSCsMethods and results:Calcium phosphate nanoparticles (about140nm in diameter) were fabricated and platelet rich plasma was extracted from heart blood of rats. Different concentrations of calcium phosphate nanoparticles (0.001%,0.01%, and0.1%) were cocultured with MSCs. After1,3and7days, different concentrations of calcium phosphate nanoparticles on the proliferation of MSCs was detected by MTT. The results showed that calcium phosphate nanoparticles had a concentration dependent inhibition on the proliferation of MSCs. calcium phosphate nano particle higher than0.001%inhibited the proliferation of MSCs. Based on the results of MTT,0.001%concentration of calcium phosphate nanoparticles or/with PRP were cocultured with MSCs for7days. Untreated MSCs served as control. The gene expressions of ALP and OCN were compared. The results showed that the gene expressions of ALP and OCN in calcium phosphate nanoparticles group were significantly higher than those in the control group; alizarin red staining showed calcified nodule of MSCs cultured with calcium phosphate nanoparticles. When the PRP (10μL) combined with calcium phosphate particles were cocultured with MSCs the gene expressions significantly increased and the calcified nodule was more positive.Conclusions:Calcium phosphate nanoparticles had a concentration dependent manner on the proliferation of MSCs. Calcium phosphate nanoparticles can induce the osteogenic differentiation of MSCs; PRP combined with calcium phosphate nanoparticles can synergistically promote the osteogenic differentiation of MSCs.PartⅤ MSC sheet combined with PRP gel/calcium phosphate nanoparticles for bone defects repair.Aim:This study aimed to investigate whether incorporated the power of mesenchymal stem cell (MSC) sheet to platelet-rich plasma (PRP) gel/calcium phosphate particles can promote bone formation in femoral bone defects of rats.Methods and results:Calcium phosphate nanoparticles (about140nm in diameter) were fabricated and platelet rich plasma was extracted from heart blood of rats.10μL PRP,10μL activator (100U/ml containing bovine thrombin10%calcium chloride solution) and10mg calcium phosphate particles and mixed, and then placed in37℃incubator to r form a gel/particles mixture. MSC sheet wrapped gel/particles is called MSC sheet wrapped PRP gel/calcium phosphate particles mixtures; MSC sheet wrapped separate calcium phosphate particles is called MSC sheet/calcium phosphate particle mixture. In vivo, thirty2.5×5mm bone defects were randomly treated with calcium phosphate particles, PRP gel/calcium phosphate particles, MSC sheet/calcium phosphate particles or MSC sheet/PRP gel/calcium phosphate particles or untreated (n=6/group). Additional four bone defects were treated with chloromethyl-benzamidodialkylcarbocyanine (CM-Dil) labeled MSC sheet/PRP gel/calcium phosphate particles and observed by small animal in vivo fluorescence imaging system to trace the implanted MSCs. The implanted MSCs were detectable at2weeks after surgery. At4weeks after implantation, the defects treated with MSC sheet/PRP gel/calcium phosphate particles showed significantly more bone formation than other four groups.Conclusions:Incorporation of MSC sheet to PRP gel/calcium phosphate particles greatly promoted bone regeneration. MSC sheet and PRP gel can provide cells and growth factors; thus enhancing bone regeneration. Such MSC sheet and tissue engineering strategies offer therapeutic opportunities for promoting bone defects repair in clinical situations.