Repair Of Articular Cartilage Defects With SOX9 Gene Modified Bone Marrow Derived Mesenchymal Stem Cells

Articular cartilages are hyaline cartilage covering the surface of joints, absorbing mechanic shocks and providing almost frictionless motion between the articular surfaces of diarthrodial joints. Chondrocytes are the only type of cells residing in cartilage and are trapped in their lacuna formed by extracellular matrix. Because of their avascular nature, cartilage shows very poor capacity of self-repair; most of the injuries are maintained for years and can eventually lead to further degeneration. Joint pain caused by cartilage degeneration is one of the major reasons of disability in the population of elder populations, while cartilage trauma is one of the most common sport injuries in young patients. Although many repair techniques have been proposed over the past decades, none has successfully regenerated long-lasting hyaline cartilage tissue to replace damaged cartilage. The repair of articular cartilage remains one of the most challenging areas in orthopaedic researches.Tissue engineering is a newly developed technique which combines the life science and engineering approaches, using in vitro amplification cells loaded onto biomaterials to build new live tissues. Recently, with more successful reports, tissue engineering demonstrated tremendous clinical potential for regeneration of hyaline-like cartilage tissue and treatment of chondral lesions.As the only type of cell in the cartilage, chondrocytes were firstly researched for repairing cartilage injuries. Brittberg et al first report that they successfully repair the full thickness defects of knee joint with hyaline-like cartilage using patients'autologous chondrocytes. However more researches show that chondrocytes are not ideal for tissue engineering, for the concerns of limited donor cells for transplantation and the dedifferentiation when cultured in vitro. So it is still necessary to find alternative cells for cartilage tissue engineering.Stem cells are cells in an"immature"or undifferentiated situation. They have high self renewal capacity and are capable of differentiate into several type of cells. The ethics concerns with ES cells limited their researches and usage. As there are few ethics concerns, more researches are now focusing on adult stem cells. Mesenchymal stem cells (MSCs) are one of the adult stem cells residing in many tissues. MSCs have multi-lineage potentials and can differentiate into osteoblasts, chondrocytes and adipocytes etc. Among MSCs, the bone marrow derived mesenchymal stem cells (BMSCs), which reside in the bone marrow tissue, can be easily obtained with minor injuries, sounds be an ideal source of MSCs.Gene transfer technology has opened novel treatment avenues toward the treatment of damaged musculoskeletal tissues. Gene transfer provides the capability to achieve sustained, localized presentation of bioactive molecules or gene products to sites of cartilage damage that could improve cartilage healing. Many investigators are investigating the feasibility of using genetically engineering chondrocytes or chondrocyte precursors for tissue-engineering applications. By using transgenic techniques, the cells can produce essential growth factors to stimulate chondrogenesis of mesenchymal stem cells or promote the synthesis of cartilageous matrix. As growth factors have limited bioavailability and stability in vivo, transfection of the cells with target gene encoding the growth factors should be a sound way to improve tissue engineering. Initial experiments have showed that overexpression of growth factors include TGF-β, IGF-1, and BMP-2 etc by transduced chondrocytes or mesenchymal cells have enhanced the repair of cartilage injuries. But more researches showed that the overexpression of growth factors may have some adverse effects such as degeneration of cartilage and forming of osteoarthritis.Sox9 is the key transcription factor during chondrogenesis. During the embryo skeleton development, Sox9 play an important role for the aggregate and differentiation of mesenchymal stem cells. Sox9 is essential for chondrocyte differentiation and cartilage formation. SOX9 enhances the promoter activity of type II collagen and aggrecan thus promote the chondrogenesis of MSCs. Although there are many researches on the structure and function of Sox9, most of them are focused on the cartilage formation. There is limited knowledge of Sox9 on the adult MSCs cultured in vitro. Based on the previous studies of Sox9, our hypothesis is that the Sox9 gene may have same chondrogenesis effect on adult MSCs in vitro. Using Sox9 overexpressing MSCs may promote the repair of cartilage injuries without the adverse effects of growth factors.The purpose of our study are aimed at①to establish a high efficacy method to isolate the BMSCs from bone marrow tissues and expand the BMSCs in vitro;②to investigate the influence of extensive subcultivation on the multiple differentiation potentials of BMSCs;③to amplify and purify the recombinant Sox9 plasmid and transfected BMSCs using a liposome vector;④to observe the influence of Sox9 overexpression on the chondrogenesis of BMSCs;⑤to repair articular cartilage defects with Sox9 gene modified BMSCs embedded in alginate.The whole project was divided into five partsChapter I Isolation and observation of rabbit bone marrow stem cellsObjective To establish a high efficacy method to isolate the rabbit BMSCs from bone marrow tissues and observe the biological characteristics of the cells cultured in vitro.Methods Bone marrow stem cells of New Zealand Rabbit were obtained and purified by gradient centrifuge and adhesion culture in vitro. The cells were analysised for their proliferation ability and growth kinetics. Flow cytometry were used for the analysis of surface antigens and cell cycle distributions.Results After the density gradient isolation and selective adhere culture procedure; Most of the isolated cells were round mononuclear cells with over 95% of viability After 3 times of medium changing, most of the unattached haematopoietic cells were washed away. After passaged into new flasks, the sub-cultured cells proliferated much more quickly than the primary culture During the first 8 subcultivation passaging period, the morphology of the cells are similar, most of the cells are uniformly spindle-like shaped. The expanded cells of the first 8 passages grow faster than the primary culture and experienced similar growth kinetics. The growth speed was much slower for late passaged cells of passage 10. The doubling time of the cells from Passage 10 are longer that that of Passage 3, 5, 8. Flow cytometry assay revealed that after 3 passaging process, the BMSCs were quite homogenous, with 91.7% of the cultured cells positive for mesenchymal adhesion molecules CD44 and only 1.5% of the cells expressing the haematopoietic antigens CD45.Conclusions The combination of density gradient centrifugation and selective adhere culture is a highly efficacy method to isolate BMSCs from bone marrow. The first 8 passaged cells have great capacity of proliferation and could be used for the further tissue engineering study.Chapter II The study of influence of extensive subcultivation on the multiple differentiation potentials of BMSCsObjective To further confirm the BMSCs by multilineage differentiation potentials and investigate the influence of extensive subcultivation on the multiple differentiation potentials of BMSCs.Methods The passage 3 and passage 8 of the isolated cells were used for multiple differentiations into osteoblasts, chondrocyte and adipocyte with different induction mediums respectively. Histology, immunohistology, biochemistry assay, RT-PCR and Western blot were used for the tests of multilineage differentiations.Results The cells from both passage 3 and passage 8 could be induced into osteoblasts, adipocytes and chondrocytes in vitro. There were no differences on the osteogenic and adipogenic potentials between two passages, but there was a decrease on the chondrogenic potential of BMSCs from passage 3 to passage 8.Conclusions Despite there were no changes of osteogenic and adipogenic potentials during the in vitro subcultivation, there was a decrease of the chondrogenic potential in late passaged cells comparing to the early passaged cells. So, for cartilage repairing, to achieve good results, it is better to use the early passage BMSCs than the later ones.Chapter III The preparation recombinant eukaryotic expression Sox9 plasmids and the transfection of BMSCsObjective To amplify and purify the recombinant Sox9 plasmid and transfected BMSCs using a liposome method.Methods Recombinant eukaryotic expression Sox9 plasmid were amplified, purified and then confirmed by restriction digestion and gene sequencing. Rabbit BMSCs were transfected by Sox9 plasmids and control EGFP plasmids. The expression of the exogenous gene was confirmed by RT-PCR and Western blot. The gene transfection efficiency was tested by GFP expressing cells using flow cytometry assay and fluoresce microscopic observation.Results The plasmids were purified after the preparation process with a consistent OD260/OD280 between 1.7~1.8. BMSCs were successfully transfected with Sox9 gene and verified by RT-PCR and Western blot analysis. The transfection efficiency was about 26%.Conclusions BMSCs can be transfected by recombinant eukaryotic expression Sox9 plasmids using the liposome method. The Sox9 gene could express in BMSCs and produced their coding proteins.Chapter IV The influence of overexpression Sox9 on the chondrogenesis of BMSCs Objective To observe the influence of Sox9 overexpression on the chondrogenesis of BMSCsMethods BMSCs were transfected by recombinant eukaryotic expression Sox9 plasmid using liposome vectors and the stable expressing cells were screened by geneticin selections. Gene transfected cells were analysised by the growth kinetic and cell cycle assay. The chondrogenesis of gene modified BMSCs were analysised by RT-PCR, western blot, immunohistology and GAG assay.Results The overexpression of Sox9 gene had no effects of cell proliferation and cell cycle distribution. The BMSCs went into chondrogenic differentiation after transfected by Sox9 gene. The transfected cells began to express chondrogenic marker molecules and synthesis more GAG than the control groups.Conclusions Overexpression of exogenous Sox9 gene has no effect on the growth kinetics of BMSCs while trigger the chondrogenic differentiation process of BMSCs.Chapter V Repair cartilage defects with Sox9 gene modified MSCs embedded in AlginateObjective To repair articular cartilage defects with Sox9 gene modified BMSCs embedded in alginate.Methods Cartilage defects were made in the medial condylar of femur.48 knee joints of 24 rabbits were randomly divided into 3 groups. In the experiment group (group I), the defects were fill with Sox9 gene modified MSCs embedded in alginate, in the experiment control group (group II) the defects were filled with MSCs embedded in alginate and the defects of empty control group (group III) were filled with alginate alone. The animals were sacrificed at 6 weeks or 12 weeks after the implantation. The samples were evaluated macroscopically and histologically. A semi-quantity scoring system was also used to evaluate the repairing. Results The defects in group I were filled with hyaline like cartilage tissue which showed smooth surface and good integrating with the adjacent cartilage, both at 6 and12 weeks postoperatively; while most of the defects in group II were filled with fibrocartilage regenerate tissue and most of the defects in group III were filled with fibrous tissues. The histological scoring system showed that the cartilage repair of experiment groups are better than the two control groups with statistical significances.Conclusions Sox9 gene modified MSCs can enhance the repair of cartilage defects. Gene enhanced tissue engineering may be a promising way for the treatment of cartilage injuries.SummariesThe combination of density gradient centrifugation and selective attachment culture may be an efficient way to isolate and purify BMSCs from bone marrow. The BMSCs cultured in vitro shows multi-lineage potentials of differentiation. The chondrogenic potential of late passage BMSCs are poor than the early passage cellsBMSCs can be transfected by recombinant Sox9 plasmid using liposome vectors. The overexpression of exogenous Sox9 gene can trigger the chondrogenic differentiation of BMSCs.Sox9 gene modified BMSCs embedded in alginate may regenerate hyaline like cartilage. Gene enhanced tissue engineering may be a promising way to repair cartilage defects.