An Experimental Study On Tissue-engineered Bone Constructed With Mesenchymal Stem Cells And Calcium Sulfate/Demineralized Bone Matrix Scaffold And Repairing Bone Defects In Rat

Objective: To culture rat Mesenchymal stem cells (MSCs) in vitro and investigate in vitro effects of transforming growth factor-β₁(TGF-β₁) and basic fibroblast growth factor (bFGF) on the proliferation and osteogenic differentiation of MSCs. Then to construct the tissue-engineered bone with the calcium sulfate/demineralized bone matrix(DBM) and to test the ability to promote repair of rat radius segmental bone defects. Methods: (1)MSCs were isolated and cultured according to their adhesive property, and purified by passages culture in vitro. MSCs were observed by means of growth curve, cell surface molecules CD31, CD44, CD45 and CD90 on the third passage MSCs were analyzed by flow cytometry technique, and were culture in a conditional medium including dexamethasone, vitamine C and β-glycerophosphate, then evaluate ALP activity and Alkaline Phosphatase staining. At last observed the change after osteogenic differentiation by transmission electron microscope. (2)The influence of proliferation on MSCs caused by different doses TGF-β₁ and bFGF was observed by MTT test. Expressions of alkaline ghosphatase(ALP) , Osteocalcin and Osteopontin were tested through RT-PCR. (3)After cultured and amplified and osteogenic differentiation, MSCs were combined with calcium sulfate/DBM at the density 2×10⁶/ml to construct the tissue-engineered bone. The MSCs and calcium sulfate/DBM combination was cultured in vitro for 5d and tested under scanning electron microscope and each sample was implanted into 20 rat subcuraneously, calcium sulfate/DBM as the control. Specimens were harvested after 4, 8weeks for X-ray and histological observation. (4)Sixty adult SD rat were created the radius segmental bone defect(5mm length) and randomly assigned to one of the four groups. One group include fifteen rat and treatment as below: group A: calcium sulfate/DBM+MSCs(treatment by TGF-β₁ and bFGF); group B: calcium sulfate/DBM+MSCs(without treatment by TGF-β₁ and bFGF); group C: calcium sulfate/DBM; group D: without treatment as control. Samples were taken 4, 8 week after operation. The curative effect was evaluated by radiographic examination, histologic analysis and biomechanical test.Result: (1)The growth characteristic of cultured MSCs were steady before the 9th passage. The biological features of MSCs have no changes after freezing and resuscitation. The 3th passage MSCs express CD90, CD44; CD45 and CD31 is negative. After osteogenic differentiation Alkaline Phosphatase staining is positive, ALP activity is increased and the cells began to generate large amount of enlarged endoplasmic reticulum, Golgi complexes and mitochondria, with immature cell nuclei. (2)Low concentration TGF-β₁ and bFGF can stimulate the proliferation of MSCs; in contrast, high concentration TGF-β₁ and bFGF suppress the proliferation of MSCs. Expression of alkaline ghosphatase (ALP) , Osteocalcin(OCN) and Osteopontin (OPN) of MSCs were increased in addition to low concentration TGF-β₁ and bFGF. (3)At day 5 of culture, MSCs and calcium sulfate/DBM combined well, the cell matrix was productive and full of the scaffold. The osteocytes were found inside in the scaffold in the experimental groups, while no osteocytes in the control groups. (4)In animal experiment, Group A and group B conducted a far more extensive bone formation than did group C and group D, especially group A, over a 8 week period. But atrophic nonunion occurred in the group D, and only a small amount of trabecular bone formed at the cut ends of the cortex of the host bone in this group. The biomechanical test demonstrated that Group A and group B were remarkably high to group C and group D.Conclusion: MSCs are ideal autogenous source of seed cells for bone tissue engineering. Low concentration TGF-β₁ and bFGF are effective induction factor for MSCs. Both of them can stimulate the proliferation and differentiation of the MSCs cultured in vitro. Calcium sulfate/DBM can be used as a alternative scaffold for seeded cells. The -tissue-engineered bone potentially could be used clinically in the treatment of extensive long bone defects.