An Experimental Study Of Autologous Bone Marrow Mesenchymal Stem Cells In Articular Cartilage Extracellular Matrix To Repair Full-thickness Defects Of Femoral Weight-bearing Articular Cartilage In The Goat

Background: With the development and advancement of medical science, articular cartilage lesion in the early diagnosis and treatment become an important issue, while it is very hard for articular cartilage to spontaneously heal without nerves and blood or lymphatic vessels. However, full thickness osteochondral injury undergoes a fibrocartilaginous repair that does not possess the same biological, biochemical, or biomechanical characteristics of native hyaline cartilage. This fibrocartilagenous repair ultimately failed under normal stresses, leading to degenerative arthritis. Therefore, marrow stimulation techniques (drilling, arthroplasty, microfracture), which were used in clinical treatment, result in fibro- cartilage repair tissue that rapidly deteriorates if subjected to sheer or compressive loads. As a new branch of science, tissue engineering has become the international forefront of biomedical domain, and has been paid more and more attention by researchers. Engineering principles and life sciences technology has been used. Seed cells were integrated to a carrier made by suitable biodegradable materials and been cultured under a biological environment simulated vivo culture conditions to form "cell - materials" polymer. This polymer was used to restitute and repair lesion tissue. As the material continues to degradation and the seed cells continue to mature, the polymer formed new organization which has the same biological activity and function with the normal tissue, and the lesion tissue defects been repaired at the same time. This method breakthrough the traditional treatment model, and provide new ideas and methods for tissue regeneration even organ regeneration. Objective:Chinese goat which has similar anatomic structure with human knee joint was chosen as animal model. This thesis is to study the methods of bone marrow stromal cells (BMSCs) purification, cultivation and amplification in vitro. Evaluation the effect of oriented scaffold derived from BMSC integrated with articular cartilage extracellular matrix (ACECM). Also, we use BMSCs as seed cells to integrate ACECM oriented scaffold, aim to reconstruct articular cartilage for repairing knee joint defects of weight-bearing areas in goat.Methods: (1) BMSCs isolated from goats bone marrow of ilium,and then cultured under osteogenesis induction, lipogenesis induction and chondrogenesis induction to confirm if they were bone marrow stem cells with multi-directional differentiation capability. (2)Combined bone marrow stem cells with normal phenotypic with ECM oriented scaffold and cultured, observed the cell growth within the ECM oriented scaffold, identified the combination effect of cells with EMC oriented scaffold. (3)A full-thickness articular-cartilage defect (6mm in diameter) was created in the femoral condyle of goats. The goats were divided into three groups,①the defects were left empty,②filled with ECM oriented scaffold,②filled with BMSC + ECM oriented scaffold. Results of cartilage repair were assessed after 3,6 months for observation, histological examination, mechanical testing and pathology scoring.Results: (1)Bone marrow stem cells isolated by bone marrow of ilium, cultured under osteogenesis induction, lipogenesis induction and chondrogenesis induction all presented positive when they were stained intensively with Alizarin red,Oil red "O",Safranin"O",Toluidine blue, immunohistochemistry of collagen typeⅡ,respectively. Results confirmed that the amplified cells were bone marrow stem cells with multi-directional differentiation capability. (2) Bone marrow stem cells combined with scaffold cultured for 7 days, more than 95% living cells were observed. Observation with scanning electron microscopy: some cells combined within oriented scaffold stretch to be megaton and had a good adhesiveness to the scaffold after vitro cultured for one week, at the same time with a large number of extracellular matrix secreted. These cells had uniform distribution and spread along with the scaffold orientation structure. (3)Bone marrow mesenchymal stem cells in oriented scaffold driven from articular cartilage extracellular matrix have been used to repair full-thickness defects of femoral weight-bearing articular cartilage in the goat. Applied statistical methods to analyze the consequence of overall observation, organization, mechanics and other experiment results, showed that all groups have different degrees of tissue repair. BMSCs combined with ACECM oriented scaffold was the best way to repair full-thicks cartilage defect in weight-bearing areas of goat knee. The mean difference is significant analyzed by on–way ANOVA between the BMSCs combined with ACECM oriented scaffold group with others.Conclusion :(1) The methods to isolate and culture goat BMSCs have been established successfully.(2) Bone marrow stem cells in the ECM oriented scaffold grew well, with good affinity, differentiation and proliferation along with the channels.(3) Using BMSCs combined with ACECM oriented scaffold, articular cartilage was formed successfully. The tissue has the histological of native cartilage and has the biochemical composition of native cartilage. It can repair cartilage damage of large animals.