Research On Cartilage Repair Using An In Vitro Generated Scaffold-free Tissue-engineered Construct Derived From Human Mesenchymal Stem Cells Induced By Growth Differentiation Factor 5

PartⅠThe experimental study of growth differentiation factor 5 promotes the differentiation of mesenchymal stem cells into chondrocytesObjective To explore the effects and the best-induced concentration of growth differentiation factor 5 promotes the differentiation of human bone marrow-derived mesenchymal stem cells (BMSCs) into chondrocytes.Methods BMSCs were cultivated and purified with the methods of whole bone marrow culture. Flow cytometer were performed to detect the cell surface marker of CD45, CD 14, CD44, CD29 and CD105. Differentiation into dipocytes and osteoblasts was, respectively, identified by Oil Red 0 stain and alizarin red stain. BMSCs were induced with different GDF-5 concentrations (0,50, and 100 ng/ml) for three weeks. Morphological changes of cells were observed. The mRNA quantities of ColⅡand Aggrecan expressed by the induced MSCs of each groups were detected with RT-PCR. Immunohistochemistry method was performed to detect the expressrion of ColⅡ, and the proteoglycan synthesized were detected with alcian blue staining and Safranin O staining.Rusult Primary BMSCs proliferated in visible colonies with spindle-shaped whirl pool. BMSCs expressed CD2、CD44 and CD 105 strongly, but CD 14 and CD45 were expressed negatively. Under the suitable nutrient condition, BMSCs could be induced to differentiate into osteoblasts, and adipocytes. After 7 days of induction with 100 ng/ml GDF-5, the BMSCs developed from spindle-shaped to polygon or elliptic shape. The expression of collagenⅡhas a statistically significant difference between other groups(P<0.05).The expression of aggrecan were significant differences among other groups(P<0.05), except that no significant difference was evident between the CM group and the 10 ng/mL group(P >0.05). After 21 days of induction with 100 ng/ml GDF-5. The results of ColⅡImmunohistochemistry staining, alcian blue staining and safranin staining were positive.Conclusion BMSCs were harvested with the method of whole bone marrow culture.100 ng/ml GDF-5 can effectively promote the differentiation of adult BMSCs to chondrocytes.PartⅡBMSCs Induced by Growth Differentiation factor-5 Self-assembled into Tissue Engineered CartilageObjective To investigate the feasibility of constructing tissue-engineered cartilage derived from human mesenchymal stem cells induced by growth differentiation factor 5, and to explore the optimum method for self-assembled tissue engineered.Methods BMSCs were isolated by whole bone marrow culture method and identified. BMSCs at Passage 4 were used in this study. The experiment was divided into four groups: BMSCs directly self-assembled in cartilaginous differentiation media (CM1); BMSCs directly self-assembled in cartilaginous differentiation media supplemented with 100 ng/ml GDF-5(D1); BMSCs was induced by cartilaginous differentiation media supplemented with 100 ng/ml GDF-5 for 3 weeks, and the predifferentiated BMSCs were digested and suspended in cartilaginous differentiation media (CM2) or in cartilaginous differentiation media supplemented with 100 ng/ml GDF-5(D2) to self-assembled. After 6 weeks, the gross appearance, histological structure and the expression of collagen typeⅡ,Ⅹ, GAG were observed. The content of GAG, total collagen and living cells was detected by biochemical analysis.Rusult At 6 weeks in vitro culture, all the constructs had cartilage-like appearance. With all the constructs having the same cell-seeding density, D2 constructs had the highest diameter, water content, dry weight and wet weight. The histological result revealed that the expression of collagen typeⅡand GAG was strongest of all while collagen typeⅩhad weakest expression in D2 constructs. And the biochemical result showed that D2 constructs had highest content of GAG, total collagen and living cells.Conclusion In the presence of GDF-5, BMSCs can self-assembled into a scaffold-free tissue-engineered construct with hyaline-like appearance, histological, biochemical, and molecular biological properties. Moreover, the predifferentiated BMSCs with GDF-5 can self-assemble into the construct that was more approaching to native cartilage.PartⅢBiological redevelopment of self-assembled tissue-engineered cartilage in nude miceObjective To construct engineered cartilage in vitro by the technique of self-assembly, combined with growth differentiation factor 5 (GDF-5) and to compare cartilage-related bio-characteristics of engineered cartilage before and after in vivo implantation and investigate the feasibility of clinical application.Methods BMSCs were isolated by whole bone marrow culture method. BMSCs at Passage 4 were induced with chondrogenic medium containing 100 ng/ml GDF-5 for 3 weeks.3 weeks later, the cells were suspended and then inoculated into each well of 2% agarose-coated 48-well plates at a density of 1×107/ml. After 6 weeks’in vivo culture, some constructs were implanted subcutaneously into nude mice and harvested at 6 weeks of post-implantation. Gross observation, glycosaminoglycan (GAG) quantification, histology, immunohistochemistry and RT-PCR were used to compare cartilage-related bio-characteristics of engineered cartilage before and after in vivo implantation.Results At 6 weeks in vitro culture, the self-assembled cartilage-like constructs were generated from pre-differentiated BMSCs, with a weak intension. At 6 weeks of post-implantation, self-assembled engineered constructs still maintained their cartilage-like appearance, the GAG content of in vivo specimens were higher than that of in vitro specimens with significant difference(P<0.05). Histologically, the mature cartilage lacuna can be observed, the cartilage matrix, aggrecan and CollagenⅡstaining of in vivo specimens were stronger than that of in vitro specimens, the expression of collagenⅡand aggrecan mRNA was detected by RT-PCR.Conclusion The self-assembled engineered cartilage can maintain fine cartilage characteristics in subcutaneous environment. Moreover, in vivo implantation may promote biological redevelopment and further maturation of engineered cartilage.Part IVIn vitro Cartilage repair using Self-assembled tissue-engineered constructsObjective To explore the feasibility of in vitro Cartilage repair using Self-assembled tissue-engineered constructs derived from BMSCs Induced by GDF-5, and study its outcome. Methods Human bone marrow mesenchymal stem cells were isolated by whole bone marrow culture method. Human bone marrow mesenchymal stem cells at Passage 4 were induced with cartilaginous differentiation media supplemented with 100 ng/ml GDF-5 for 3 weeks. After 3 weeks, the cells were suspended and then inoculated into each well of 2% agarose-coated 48-well plates at a density of 1×10/ml, then 400μl of cell suspension was pipetted into each well. After another 3 week’s self-assembly, specimen were observed by gross appearance and histology while biological properties were detected by biochemical assays. Other specimen was implanted into an in vitro cartilage defect model and cultured for another 3 weeks. Then the repair effects were observed while biological properties were compared with those in pre-implantation.Rusult At 3 weeks culture in vitro, the self-assembled cartilage-like constructs were generated from pre-differentiated BMSCs. The cartilage-specific matrix, aggrecan and CollⅡ, was detected by histology staining. After three-week’s culture in vitro, gross appearance and histology staining showed that full-thickness articular cartilage defects had been repaired. The integration occurred between the normal cartilage and engineered cartilage. Histology staining showed that the cartilage-specific matrix, aggrecan and CollⅡ, was still positively expressed. Immunohistochemical staining detected fibronectin existed abundant in the interface.However, the result of biochemical quantification showed cell number, GAG and total collagen contents of post-implantated specimen were significantly decreased compared with those of pre-implantated specimen (P<0.05), which was consistent with the result of RT-PCR.Conclusion The self-assembled tissue-engineered cartilage can effectively repaired in vitro articular cartilage defects with a stable cartilageous phenotype.