Effects Of Platelet-Rich Plasma On The Adhesion,Proliferation And Osteogenesis Of Bone Marrow Stromal Stem Cells On Bio-Derived Bone Scaffolds

Experiment one-Effect of platelet-rich plasma on adhesion,proliferation and osteogenic differentiation of bone marrow stromal cells on bio-derived bone scaffolds.Objective:To investigate the effects of platelet-rich plasma on the adhesion,proliferation and osteogenic differentiation of bone marrow stromal cells(BMSCs)cultured in vitro on bio-derived bone scaffolds.Methods:MSCs were isolated,cultured,amplified and induced in vitro.The osteogenic phenotype of MSCs was identified by alkaline phosphatase staining and alizarin red staining.Bio-derived bone were prepared and its physicochemical properties were detemined.Rabbit arterial blood was extracted to prepare PRP and its biological activity was determined.50μl of MSCs suspension with a concentration of 1.0 x 107/ml and 50μl PRP was mixed and shaked evenly.The mixed liquid was inoculated on the bio-derived bone scaffold.Then 10μl bovine thrombin solution was added to form bio-derived bone/MSCs/PRP complex.As a control group,50μl MSCs suspension wihh a concentration of 1.0 × 107/ml was inoculated on the bio-derived bone scaffold to form a bio-derived bone/MSCs complex.These complex were cultured under saturated humidity,37℃ and 5%CO2 condition.After 24 hours and 8 days,the effects of PRP on MSCs adhesion and proliferation on bio-derived bone scaffolds were detected by MTT,the effects of PRP on MSCs osteogenic differentiation on bio-derived bone scaffolds were evaluated by detecting alkaline phosphatase activity and osteocalcin content in culture medium.The adhesion,growth and proliferation of MSCs on bio-derived bone scaffolds were observed by scanning electron microscopy.Results:After 24 hours and 8 days of culture in vitro,the optical density of the bio-derived bone/MSCs/PRP group was much higher than that of the bio-derived bone/MSCs group significantly(p<0.05).After 8 days of culture,the ALP activity and OC content in the culture medium of the bio-derived bone/MSCs/PRP group were much higher than those of the bio-derived bone/MSCs group significantly(p<0.05).Scanning electron microscopy showed that the cell scaffold density of the bio-derived bone/MSCs/PRP group was much higher than that of the bio-derived bone/MSCs group significantly after 24 hours culture.The cells of the two groups mostly spread into round,spindle and triangle,and the cells of the bio-derived bone/MSCs/PRP group spread much better than control group.After 8 days of culture in vitro,the cells in both group spread much better than before.Most of the cells were polygonal,flat and wide,with more antennae and higher cell density.In observation,we found that the bio-derived bone/MSCs/PRP group was superior to the control group in both cell density and cell spreading morphology.Conclusions:The vitro culture experiments showed that PRP significantly promoted MSCs adhesion,proliferation and osteogenic differentiation on bio-derived bone scaffolds.Experiment two-Effect of platelet-rich plasma on osteogenesis of bone marrow stromal cells on bio-derived bone scaffolds in vivo.Objective:To evaluate the effect of platelet-rich plasma on osteogenesis of bone marrow stromal stem cells on bio-derived bone scaffolds in vivo.Methods:MSCs were isolated,cultured,amplified and induced in vitro.Bio-derived bone and PRP were prepared.50μl of MSCs suspension with a concentration of 1.0 x 108/ml and 50μl PRP was mixed and shaked evenly.The mixed liquid was inoculated on the bio-derived bone scaffold.Then 10μl bovine thrombin solution was added to form bio-derived bone/MSCs/PRP complex.As a control group,50μl MSCs suspension with a concentration of 1.0 x 107/ml was inoculated on the bio-derived bone scaffold to form a bio-derived bone/MSCs complex.New Zealand white rabbits were selected and bilateral hind limbs were operated under knee to produce metaphyseal bone defect of left and right tibia.The kind of two complexes were implanted into the defect of the left and right tibia at the same time.The left lower limb was the experimental group(group A),the bio-derived bone/MSCs/PRP complex was implanted,and the right lower limb was the control group(group B),and the bio-derived bone/MSCs complex was implanted.Samples were taken at 4 and 8 weeks after operation.The heterotopic osteogenesis was evaluated by general observation,soft tissue observation around bone graft,histological observation and histomorphometric analysis of bone graft.Results:The surface of implanted area was covered by fibrous connective tissue,and no necrosis or hemorrhage occurred.HE staining of soft tissue showed that the surface soft tissue of the implanted area was loose at 4 weeks after operation,and the connective tissue of the implanted surface was periosteoid mature tissue at 8 weeks after operation.The change trend of the experimental group and the control group was basically the same.HE staining and Masson trichrome staining showed that the original bone trabeculae of the implant began to absorb and the pore was enlarged 4 weeks after operation.Block-like woven bone tissue could be seen around the original bone trabeculae,and the amount of new woven bone in the experimental group was significantly higher than that in the control group.At 8 weeks after operation,the trabecular structure of bio-derived bone further absorbed,and the pore increased.At the same time,the new woven bone tissue gradually increased.Both the degradation of bone graft and the formation of new bone and were significantly enhanced.Large new bone sheets appeared and gradually changed into mature bone tissue.The change trend of osteogenesis in the experimental group was faster and more obvious than that in the control group.The speed of bone resorption and bone remodeling in the bio-derived bone/MSCs/PRP composite group was significantly faster than that in the bio-derived bone/MSCs composite group.The results of histomorphometry showed that the new bone mass of bio-derived bone/MSCs/PRP group was significantly higher than that of bio-derived bone/MSCs group(p<0.05).Conclusions:PRP significantly promoted the osteogenesis of bone marrow stromal cells on bio-derived bone scaffolds in vivo.