The Construction And Empirical Study Of An Injectable Bone Tissue Engineering Combining Platelet-rich Plasma

Objective1.To study the feasibility of chitosan-beta-tricalcium phosphate (beta-TCP) used as an injectable scaffold material to deliver bone marrow mesenchymal stem cells (MSCs) or platelet-rich plasma(PRP) in vitro, and evaluate the biocompatibility of the biomaterial.2.By studing the direct effect of PRP on osteogenic differentiation of MSCs in vitro, and the effect of PRP withdrawal on proliferation and osteogenic differentiation of MSCs, to explore mechanicsm of action of PRP promoting bone regeneration.3.To prepare the model of tibia bone hole defect of Chinese goat which can be suitable to study injectable tissue engineering bone in vivo.4. To study the effect of a novel injectable bone tissue engineering bone- injectable scaffold material chitosan- beta-TCP combining MSCs and PRP on repairing tibia bone defect of Chinese goat.Methods 1.MSCs from bone marrow of Chinese goat were cultured and induced by dexamethasone(DEX), the third passage of which were used to culture with chitosan-beta-TCP, with the cells cultured with pure fetal calf serum(FCS) as the control group. The morphology and proliferation of the cells were observed by inverted phase contrast microscope. MTT assay was used to examine the effects of chitosan-beta-TCP on MSCs viability. MSCs were mixed with chitosan-beta-TCP to generate MSCs/chitosan-beta -TCP composite with final cellular numbers of 1×10~7. The morphology of the cells on the surface of chitosan-beta-TCP was observed by scanning electronmicroscope (SEM). The third passage of MSCs were cultured in vitro and randomly divided into the PRP/chitosan-beta-TCP group, chitosan-beta-TCP group and blank control group respectively. The morphology and proliferation of MSCs were observed by inverted phase contrast microscope. MTT assay was used to evaluate the effects of PRP in Chitosan-beta -TCP on proliferation of MSCs.2.Human bone marrow mesenchymal stem cells (HMSCs) or MSCs of Chinese goat were divided into pure fetal calf serum (FCS) culture group, dexamethasone (DEX) group and PRP group respectively. Alkaline phosphate (ALP) staining and calcium depositions staining were essayed. In addition, mRNA expression of ALP (alkaline phosphatase), OC (Osteocalcin), Coll-Ⅰ(Collagen typeⅠ), ON (osteonectin), Cbfal (core binding factor alpha 1 ) and TGF-β1 (transforming growth factor betal) were used to essay the effect of PRP on gene expression of HMSCs.3.HMSCs treated by PRP or untreated were divided to two groups: fetal calf serum group (FCS group), PRP group(HMSCs treated by PRP). MTT assay was used to evaluate cell proliferation at day 2,4,6,8. HMSCs treated by PRP or untreated were divided to three groups: FCS group, PRP group, dexamethasone group (HMSCs treated by PRP were exposed to dexamethasone, DEX group). ALP staining, calcium staining and gene expression of ALP, OC, Coll-Ⅰ, ON, Cbfal and TGF-β1 were used to evaluate the effect of PRP on osteogenic differatiation of MSCs.4.Circular hole bone defect, the diameter was 1.2cm, was made under tibial medial plateau of Chinese goat. X ray, histological assay, and image analysis were used to evaluate the validity of the model as bone defect model of limbs suited to study injectable bone.5.The model the studies was 1.2cm diameter circular hole tibia bone defect of Chinese goat. 30 Chinese goats were divied into 5 groups: balnk group: nothing was embeded in bone defect; simple material group: the material embeded in bone defect was chitosan-beta-TCP; PRP group: the material was chitosan-beta-TCP combining PRP; MSCs group: the material was chitosan-beta-TCP combining MSCs; PRP/MSCs group: the material was chitosan-beta-TCP combining MSCs and PRP. At 4, 8 weeks after operation, the samples were observed and histological analysis and image analysis were used to evaluate the effect of bone regeneration.Results1.MSCs cocultured with Chitosan-beta-TCP exhibited good growth as observed under inverted phase contrast microscope, without significant difference from the control group. The OD value of the control group and Chitosan-beta-TCP group was 0.120±0.027,0.404±0.041,0.902±0.097,1.197±0.074; 0.116±0.021,0.372±0.051,0.878±0.064,1.139±0.094 at 2,4,6,8 days respectively. MTT assay showed no significant difference in the number of cells between Chitosan-beta-TCP (Mean=0.626) and the blank control group(Mean=0.656) (F = 2.02, P=0.157). Under SEM, the MSCs showed good adhesion to Chitosan-beta-TCP with obvious proliferation.2.MSCs cocultured with PRP/Chitosan-beta -TCP exhibited good growth as observed under inverted phase contrast microscope, significantly different from other two groups. MTT assay showed The OD value of the control blank group, simple Chitosan-beta-TCP group and PRP/Chitosan-beta -TCP group was 0.124±0.019,0. 424±0.029,0. 922±0.044,1. 233±0.084; 0.118±0.024,0.398±0.054,0.887±0.111,1.193±0.118; 0.130±0.023,0.491±0.062,1.087±0.081,1.384±0.092 at 2,4,6,8 days respectively.There was significant difference in the number of cells of all groups (F=17.227, P=0.000). The number of cells PRP/Chitosan-beta -TCP group (Mean=0.733) was more than other two groups. While, there was no significant difference between chitosan- beta-TCP group(Mean=0.649) and the blank control group(Mean=0.676).3.PRP and DEX had opposite effect on osteogenic differentiation of MSCs compared to FCS culture. PRP decreased the number of ALP-positive cell at 7days and calcium depositions at 19 days of MSCs of human or Chinese goat, and down-regulated ALP, OC mRNA expression of HMSCs. While DEX increased the number of ALP-positive cell and calcium depositions, up-regulated ALP, OC mRNA expression. PRP can up-regulated TGF-β1 mRNA expression of HMSCs, but DEX down-regulated TGF-β1 mRNA expression.4.The OD value of the FCS group and PRP group was 0.149±0.039,0.405±0.063,0.933±0.048,1.161±0.057; 0.152±0.028,0.397±0.035,0.964±0.033,1.103±0.061 at 2,4,6,8 days respectively. Cell proliferation of PRP group (Mean=0.654) was similar to FCS group(Mean=0.662)(F=0.311, P=0.580). There was no obvious difference of the number of ALP-positive cells between PRP and FCS group at 7 days, while the DEX increased the number of ALP-positive cells at days. There was also no obvious difference of mineral deposition between PRP and FCS group at 19 days, with few calcium nodes. Compared to FCS group, treatment of HMSCs with DEX markedly increased mineral deposition with many more obvious calcium nodes at 19 days. DEX resulted in an earlier expression of OC at day 7 compared to FCS, subsequently, DEX increased OC mRNA expressions during other culture period. At same time, DEX up-regulated ALP mRNA expression during the culture period. Compared to FCS group, PRP increased TGF-β1 mRNA expressions, but OC and ALP mRNA expressions were similar. During the different time points, the Cbfal, ON, COL-ⅠmRNA expressions had not significant changes between three groups.5.The X ray showed that at 4 weeks, there were no obvious dense region in bone defect zone, and at 8 weeks, only a few dense region close to normal bone borderline, however, there were still no dense region in center zone of bone defect. Histological slice showed, at week 4, there were few irregular new osteoids at only partial boundary of normal bone, while there were no new bone tissues at central zone; and at 8 weeks, new osteoids at boundary of normal bone were more than those at 4 weeks, and there was stille no new bone tissue at most parts of normal bone borderline, the tissues of central zone of bone defect were fibrous tissues. Image analysis showed that the areas of the new bone tissue was 8.79±3.63%,at 4weeks and about 15.41±4.21% at 8 weeks,6.At 4 weeks, all the bone defects were covered by thin fibrous tissue. The tissues of bone defect zone of balnk group were flabbier than simple material, the degree of hardness of both PRP and MSCs group was higher than simple material group, while, the degree of hardness of PRP/MSCs was the highest. At 8 weeks, the degree of hardness of bone defect zone of balnk group increased, but still was the lowest between five groups. PRP/MSCs group was still the highest. On the surface of bone defect zone of PRP and the MSCs group, continuous new bones and unabsorb material could be seen. The surface of bone defect zone of PRP/MSCs group were coverd by continuous new bones, like normal bone without bone defect from gross appearance. Histological slice showed the bone defect of the blank group was never restored, and the bone defect was invaded mostly by fibrous tissue with only few osteoid at boundary of normal bone. There were few osteoid at boundary of normal bone, and small punctiform or lamellar new bone tissues at the bone defect zone of simple material group. However, up to the 8th week after operation, there was still no obvious new bone tissue at the center zone of bone defect. The osteoid at boundary of normal bone of MSCs/PRP group obviously increased compare to other groups at the 4th or 8th week after operation respectively. The new bone tissues of bone defect were punctiform or lamellar new bone tissues, in which the propotion of big lamellar new bone tissue obviously increased. Image analysis showed that the areas of balnk group, simple material group, PRP group, MSCs group, PRP/MSCs group were 8.79+3.63,14.49+3.72,24.18+5.38,24.42+5.10,31.10+3.49 at 4 weeks and 15.41+4.21,25.56+5.37,30.71+4.39,33.97+4.45,48.60+5.97 at 8 weeks respectively. There was significant difference among the areas of bone regeneration of all groups (F=60.376, P=0.000). The effect of bone regeneration of PRP/MSCs group (Mean=39.85%) was the best. The effect of simple material group (Mean=19.92%) was better than balnk group (Mean=12.10%). There was no significant differentce between PRP groups (Mean=27.44%) and MSCs groups (Mean=29.20%), the effect of which was better than simple material group or blank group respectively.Conclusion1.MSCs can grow and proliferate well on the compound BMSCs/chitosan-beta-TCP and Chitosan-beta-TCP has good biocompatibility with MSCs in vitro, which may be used as a good scaffold material for injectable tissue engineering bone.2.PRP in injectable scaffolds material chitosan-beta-TCP can promote MSCs proliferation, which can be used as growth factor resource of chitosan-beta- TCP.3.The direct effect of PRP is inhibiting osteogenic differentiation of MSCs. HMSCs treated by PRP can restore to the normal proliferation rate, and PRP is safe when been used in vivo. The ability of osteogenic differentiation of HMSCs treated by PRP is similar to normal HMSCs and DEX can induced HMSCs treated by PRP osteogenic differentiation. PRP can increase TGFβ1 expression of HMSCs and HMSCs treated by PRP can still sustain higher TGF-β1 expression than normal HMSCs, this may be one of reasons that PRP can promote bone tissue regeneration.4.To study the bone repairing ability of injectable bone on bone defect of limbs, the model of tibia bone hole defect of Chinese caprine is suitable. The preparation the model is easy without plate; one side of hole bone defect is closed, which facilitates the application of injectable bone; the reliability of the model is good with few areas of bone regeneration; the zone of bone defect locates weight loading bone, the local condition of bone regeneration and mechanics condition are all suitable to the need of clinical research for bone defect of limbs.5.The effect of chitosan- beta-TCP combining MSCs and PRPon repairing bone defect of Chinese goat was the best, which had good foreground of clinical application.