Fibrin With Temporomandibular Joint Synovium Derived Mesenchymal Stem Cells In Repairing Of Disc Perforation

Approximately70%of TMJ disorders (TMDs) have TMJ disc displacement, which is associated with degenerative tissue changeIn severe TMDs cases surgical operations are often needed, including repair/remove of the TMJ disc or total joint reconstruction.The in vivo engineering of damaged TMJ disc is a promising strategy to treat patients with disc-related TMDs. The TMJ disc is characterized as fibrocartilage tissue distinct from both hyaline and meniscal cartilages. Previous attempts on the TMJ disc engineering usually used TMJ disc derived cells. They found that this kind of fibrochondrocyte are prone to dedifferentiate during culture, leading to a decrease of ECM synthesis and related gene expression such as collagen type Ⅱ and cartilage oligomeric matrix protein (COMP). Furthermore the use of autologous fibrochondrocyte/chondrocyte is usually limited by donor site scarcity and morbidity. All these drawbacks compromised the use of TMJ derived cells or chondrocytes in repairing of the disc.Synovium derived mesenchymal stem cells (SDSCs) are an attractive cell source for cartilage tissue engineering. SDSCs are able to synthesis COMP, link protein, and sGAG, which demonstrate same properties as chondrocytes. When comparing with other MSCs such as bone marrow mesenchymal stroma cells (BMSCs) and adipose derived stem cells (ADSCs), SDSCs have higher chondrogenic ability and proliferation ability.Likewise, SDSCs can be isolated and cultured from TMJ. In the previous study, the ability of multi-potential differentiation of TMJ-SDSCs has been confirmed. TMJ-SDSCs can be obtained through a minimal invasive arthroscopy to the donor site. It is reasonable to believe that TMJ-SDSCs may benefit the repair of TMJ disc defect.Fibrin gel is a Federal Drug Agency (FDA) approved biological adhesive which is currently being used extensively in wound healing. Fibrin has been proved to be good scaffold for cartilage engineering, for it promotes chondrocyte proliferation and extracellular matrix (ECM) production. However, the intrinsic properties of fibrin gel such as poor mechanical strength and fast degradation make it unsuitable to use along. It is reasonable to expect that scaffold composed of fibrin gel and sponge chitosan may overcome these problems. We hypothesize that incorporating fibrin gel with macroporous chitosan scaffold could enhance TMJ-SDSCs seeding efficiency, cell distribution and ECM production. Meanwhile, fibrin gel may fix the cell/scaffold construct into the site of TMJ disc damage, relay on its bonding characteristic. In this research, we designed an organ culture model to test whether fibrin gel could promote TMJ disc repair using TMJ-SDSCs.To evaluate the in vivo repair ability of SDSCs seeded fibrin/chitosan construct in TMJ disc regeneration, an animal model of TMJ disc perforation should be performed prior to construct implantation. It is difficult and expensive to perform a TMJ disc perforation on living animals, so most studies evaluated their constructs in vitro. In the present study, rat TMJ discs were used as explants to make standardized man-made-perforation. After implanting the cell/scaffold construct into the disc perforation, the TMJ disc explants were subcutaneously embedded in nude mice. Because of the synovial fluid was similar to blood serum, the subcutaneous environment can mimic TMJ cavity environment at some extent.After an embedding period of2-4weeks, the TMJ disc explants were analyzed histologically to evaluate the repair ability of different constructs.Part One:The isolation and multi-directional differentiation of rat Temporomandibular joint derived mesenchymal stem cellsObjectives:To investigate the methods of the isolation, cultivation and ability of multi-directional differentiation of rat Temporomandibular joint derived mesenchymal stem cells (SDSCs).Methods:Synovial tissue was harvested in the TMJ disc of rats. Cells were isolated by explant method and purified by continuous passage. P3SDSCs were collected and observed under microscopy. To evaluate the ability of multi-directional differentiation, SDSCs were induced differentiating into chondrocytes, osteoblasts, and adipocytes. Collagen type Ⅱ immunohistochemical staining, alizarin red staining, and oil red O staining were performed respectively.Results:①The purified SDSCs were obtained and showed spindle fibroblast-like shape after continuous passage.②After cultured in chondrogenic medium, the deposited collagen type Ⅱ was detected by immunohistochemical staining.③After cultured in osteogenic medium, the deposited calcium was detected by alizarin red staining.④After cultured in adipogenic medium, liquid vacuoles was seen in cytoplasm.Conclusion:SDSCs can be isolated from rat TMJ synovial tissue. SDSCs possess the ability of multi-directional differentiation, which can be used as a kind of cell source in cartilage tissue engineering.Part Two:The fabrication of fibrin/chitosan composed scaffoldObjectives:To investigate the fabrication of fibrin/chitosan scaffold for cartilage tissue engineering.Methods:The porous chitosan scaffold was prepared with freeze-drying method. Fibrin gel was produced by mixing equal volume of fibrinogen and thrombin solution. Fibrin/chitosan composed scaffold was fabricated by adding fibrinogen solution to half-dried chitosan scaffold, and then adding equal volume of thrombin solution. After gel coagulation, the surface of fibrin/chitosan scaffold was investigated under scanning electron microscope (SEM).Results:The lyophilized chitosan scaffold exhibited spongy-like microstructure. After mixing fibrinogen with thrombin solution and incubating at37℃for10min, soft, flexible, and transparent gel was formed. Each chitosan scaffold can be combined with50μL of fibrinogen and50μL of thrombin solution to fabricate the fibrin/chitosan composed scaffold. Cross-section SEM images showed the fibrin gel had been filling in the chitosan scaffold. Conclusion:The fibrin/chitosan composed scaffold can be fabricated by adding equal volume of fibrinogen and thrombin solution to pure chitosan scaffold. The scaffold was still spongy-like microstructure, which could be used in tissue engineering.Part Three:In vitro growing characteristic of SDSCs seeded in fibrin/chitosan composed scaffoldObjectives:To investigate the growing characteristic of SDSCs seeded in fibrin/chitosan composed scaffold in vitro.Methods:SDSCs (2x106per scaffold) were seeded into fibrin/chitosan scaffold (Experiment group) and pure chitosan scaffold (Control group). After8hours of cell seeding, unattached cells were collected and counted to evaluate cell seeding efficiency among two groups. The cell quality and distribution was evaluated by FDA/PI staining under confocal laser scanning microscope (CLSM). Cell proliferation ability was evaluated by recording cell numbers among two groups at day1,7,14,21, and28with CCK-8method.Results:Cell seeding efficiency was significantly higher in Experiment group after8hours of cell seeding. CLSM investigation demonstrated more cells were equally distributed among Experiment group at100μm below the scaffold surface. Cell proliferation assay found that at the first7days cells grew equally among two groups; however from day7to28, a significantly higher cell amount was observed in Experiment group.Conclusion:The cell seeding efficiency, distribution and proliferation ability of SDSCs could be enhanced by incorporation the fibrin gel into chitosan scaffold.Part Four:In vitro chondrogenic differentiation of SDSCs in fibrin/chitosan hybrid scaffoldObjectives:To investigate the chondrogenic differentiation ability of SDSCs in fibrin/chitosan scaffold.Methods:SDSCs (2x106per scaffold) were seeded into fibrin/chitosan scaffold (Experiment group) and pure chitosan scaffold (Control group), and cultured with chondrogenic medium for28days. The surface of cell/scaffold constructs were observed under inverted microscope. The cartilage-related gene expression (collagen type Ⅰ and Ⅱ, SOX9) were quantified assayed by Real-time PCR. The GAG/DNA ratio was investigated among two groups to compare the ability of GAG synthesis at day0,14, and28.Results:After chondrogenic induction for28days, more cell/matrix aggregates on the surface of scaffold were observed in Experiment group. The real-time PCR results suggested that a higher expression of collagen type I in Experiment group, while no significant differences were detected on collagen type II and SOX9expression. The GAG/DNA ratio was higher in Experiment group at day14and28comparing with Control group.Conclusion:The fibrin gel could enhance the synthetic ability of cartilaginous ECM for SDSCs which can be used in disc engineering.Part Five:In vivo study on SDSCs seeded fibrin/chitosan constructs in repairing of Temporomandibular joint disc perforationObjectives:To investigate the repair ability of cell/scaffold constructs in animal explant model of disc perforation.Methods:Using customized punch to produce a2mm diameter perforation in rat TMJ disc explant. Scaffolds with or without cell seeded were transplanted into the disc perforation of the explant. After coated with pure fibrin gel, the explants were subcutaneously implanted in nude mice. The samples were evaluated histologically at2or4weeks post implantation. The groups were set as follows:Group SFC (SDSCs+fibrin+chitosan); Group SC (SDSCs+chitosan); Group FC (fibrin+chitosan); Group C:(chitosan only); and Group F:(fibrin only).Results:HE staining showed more cells and ECM accumulation with fewer cavities among the scaffold in SFC group than in SC group. In cell-free groups (Group FC, C, and F), few cells of host oriented migrated into the scaffolds with no ECM deposition. Immunohistochemical assay demonstrated denser staining of collagen type Ⅰ, type Ⅱ, and aggrecan in SFC group than in FC groups. Saranino O/Fast green showed more GAG deposition in SFC group.Conclusion:SDSCs seeded fibrin/chitosan scaffold demonstrated higher repair ability in animal explant model of disc perforation.