Construction And Mechanical Properties Of Self-assembly Constructs Derived From BMSCs

Similar to articular cartilage, the TMJ disc lacks self-regenerative capacity. Currently, surgery is considered for patients suffering from significant persistent pain and joint dysfunction. Therefore, it is essential to develop functional temporomandibular joint disc replacement in the future. The mature development of tissue engineering provides a possibility for the regeneration of functional temporomandibular joint disc tissue. Seed cells, one of the three basic element in tissue engineering, are important to reconstruction of TMJ disc tissue. Due to the terminally differentiation of the TMJ disc cells and the characteristics of rapid loss the cartilage phenotype when cultured in vitro, the use of TMJ disc cells directly is usually limited by donor site scarcity when reparativing the defective of lost tissues. As the multiple differentiation of SMCs, the researchers tried to induce the cells by taking various means including physics, biology and chemistry. In this study, two system were used to induce and construct self-assembly fibrocartilage model of goat Bone marrow mesenchymal stem cells. Furthermore, mechanical properties of the constructs was improved to provide the basis for the future research. This study proved that both the two system have similar mechanical properties and provide reference for the improvement of the performance of the tissue engineering in the later stage. This study conclude 3 sections:section 1. Isolation of goat bone marrow-derived mesenchymal stem cells: a comparison between two protocols on growth proliferation and biological characteristics;Objective: To investigate the two protocols of goat bone marrow-derived mesenchymal stem cells(gBMSCs) isolation: percoll gradient density separation protocols and holo-bone marrow adherence on purity and biological characteristics in vitro, and to provide large numbers of cells for subsequent experiments.Methods: Bone marrow was sterilely harvested from the thighbones of goat aged about 3-5 months, gBMSCs were isolated by both 2 protocols. The shapes of cells were observed by inverted phase contrast microscopes. The colony forming unit-fibroblast(CFU-F) assay was performed to test those self-renewing. Moreover, the growth curve of passage(P)3 cells was drawn and surface marker expression of P3 cells was investigated by flow cytometry(FCM).Results: The primary and passaged cells were spindle-shaped and grew in colonies. The growth curve of cultured g BMSCs by both two protocols was like “S” shape. The results of CFU-F assay demonstrated that cells harvestd by holo-bone marrow adherence possessed higher self-renewal capacities. Two kinds of cells were uniform in surface marker expression. Both CD34 and CD44 were positive expression and CD45 was negative expression.Conclusions: Though cells at P0 retrieved with the percoll protocol had lower proliferation when compared with the holo-bone marrow adherence, there was no significant difference between protocols at P3 except self-renewal capacity.section 2. Effect of transforming growth factor β1, bone morphogenetic protein 2 on Self-assembly tissue engineering fibrocartilage model of goat bone marrow-derived mesenchymal stem cells;Objective: To induce and construct self-assembly fibrocartilage model of gBMSCs, to observe the biological characteristics of the induced constructs and tested for its mechanical properties, further to explore a new way for tissue engineering of the temporomandibular joint disc.Methods: gBMSCs were isolated by holo-bone marrow adherence, RT-q PCR was used for detecting the expression level of cartilage related genes. 5.5×106 cells were seeded in each agarose well within the mold, the induced medium(10ng/ml TGF-β1, 500ng/ml BMP-2, 10-7M DEX, 1×ITS, 50 mg/l ascorbic acid, 40 μg/ml proline, 100 μg/ml pyruvic acid, 100 U/ml penicillin/100 mg/ml streptomycin)was changed every day. The constructs were cultured for 42 d in vitro. The morphology, composition and mechanical properties of the constructs were evaluated at14 d, 28 d and 42 d.Result: RT-qPCR was used to detect cartilage gene relative expression(ColⅠ, ColⅡ, ColⅩ, SOX9, GAG)after 14 days of induction, the induced group was significantly higher than that of the control group(P<0.01). In contrast, Oct-4 expression was decreased to 19% of control group(P<0.01) in the induction group. Compared with the conltrol group, the Safranin-O staining of the induced goup was showed positive at 14 d, 28 d and 42 d, which showed the constructs can produce GAG after cultured by induced medium. Immunohistochemistry staining of ColⅠ was examined brown particles in induced group, which showed constructs can produce ColⅠby induced medium as the main component of the primary natural articular disc. Compression experiments showed that the compression modulus of the control group was less than the induced group at each time(P<0.05), for the induction group, the constructs of 42 d were maximum during this period.Conclusions: Fibrocartilage model of gBMSCs was induced and constructed successfully, the constructs after induced can produce extracellular matrix which was similar to native temporomandibular joint disc, and the compression modulus was maximum after cultured at 42 d in vitro.Section 3. Mechanical properties of self-assembly constructs derived from BMSCs;Objective: To improve and enhance the mechanical properties of self-assembly constructs derived from BMSCs, to provide the construction of cells derived tissues and mechanical parameters for the later use.Methods: The induced group(10ng/mlTGF-β1, 500ng/ml BMP-2, 10-7M DEX, 1×ITS, 50 mg/l ascorbic acid, 40 μg/ml proline, 100 μg/ml pyruvic acid, 100 U/ml penicillin/100 mg/ml streptomycin) and the co-culture group(gBMSCs:TMJ disc cells=2:1) were constructed respectivly. They were cultured for 42 d in viro, then the morphology, composition, profiles of the constructs, and mechanical properties of the constructs were evaluated.Results: the observation results of stereo microscope showed that except the negative control group, others were in rules of sphere, with smooth and continuous surface. Compared with the negative control group, histological and immunohistochemical staining of other groups were positive, among them the induced constructs were the most positive. This means that all of the constructs except the negative control group can secrete GAG and collagen I, and the induced constructs were higher than the co-culture constructs. SEM results showed that cells in the constructs were synaptic contacts with each other, Cells were ordered stacking arrangement, large numbers of fibre aligned in a certain direction was found in induced group, cells were mostly stratified and overlapping. Short and thin fiber could be found in co-culture group, cells were often distributed in layered structure. Results of mechanical test showed that the compressive properties of both the induced group and the co-culture group was higher than positive control group(P<0.05), and the induced group was significantly higher than that of compressive properties of co-culture group(P<0.05).Conclusion: Constructs made by co-culture system were similar to the disc cells constructs in electron microscopic structure and mechanical properties. The mechanical properties of the induced contructs was better when compared with the co-culture contructs, but both of them were less than the TMJ disc tissue.