The pilot study of fibrin with temporomandibular joint derived synovial stem cells in repairing TMJ disc perforation

Abstract

TMJ disc related diseases are difficult to be cured due to the poor repair ability of the disc. TMJ-SDSCs were ideal cell sources for cartilage tissue engineering which have been widely used in hyaline cartilage regeneration. Fibrin gel has been demonstrated as a potential scaffold for neocartilage formation. The aim of this study was to repair the TMJ disc perforation using fibrin/chitosan hybrid scaffold combined with TMJ-SDSCs. Rat TMJ-SDSCs were cultured on hybrid scaffold or pure chitosan scaffolds. The cell seeding efficiency, distribution, proliferation, and chondrogenic differentiation capacity were investigated. To evaluate the in vivo repair ability of cell/scaffold construct, rat TMJ disc explants were punched with a defect to mimic TMJ disc perforation. Cell seeded scaffolds were inserted into the defect of TMJ disc explants and then were implanted subcutaneously in nude mice for 4 weeks. Results demonstrated that fibrin may improve cell seeding, proliferation, and chondrogenic induction in vitro. The in vivo experiments showed more cartilage ECM deposition in fibrin/chitosan scaffold, which suggested an enhanced reparative ability. This pilot study demonstrated that the regenerative ability of TMJ-SDSCs seeded in fibrin/chitosan scaffold could be applied for repairing TMJ disc perforation.

Figure 1

The experiment design and animal models for repairing TMJ disc perforation.

Figure 2

(a) Rat TMJ discs were obtained from mandibular condyle (C: condyle and D: TMJ disc); (b) TMJ disc perforation of 2 mm in diameter was surgically made by a puncher; (c) scaffolds with or without cells (arrow) were inserted into the perforation of disc and then coated the explants with pure fibrin before subcutaneous implantation (D: TMJ disc and F: fibrin).

Figure 3

Morphologic features of two scaffolds. SEM results showed more cavities among pure chitosan scaffold (b-1), while the cavities were filled with fibrin in fibrin/chitosan scaffold (a-1). CLSM results showed that more vital cells (green) existed in fibrin/chitosan scaffold (a-2) than in chitosan scaffold (b-2), although the number of dead cells (red) was relatively higher in hybrid scaffold ((a-3) and (b-3)). Scale bar is equal to 300 μm.

Figure 4

Biocompatible and biochemical results of TMJ-SDSCs seeded in two scaffolds. Columns and error bars represent means and SD. Cell adhesion (a), GAG/DNA ratio (c), and relative expression of Col I (d) were significantly improved in fibrin/chitosan scaffold. Asterisks (∗) indicate significant difference from control (P < 0.05), based on the post hoc analysis comparing each individual group. Cell proliferation results (b) demonstrated significant difference (P < 0.05) in the two-factor ANOVA (fibrin and time duration).

Figure 5

HE staining of TMJ disc explants. (a-1)–(e-1) are gross views of TMJ explants. Scale bar is equal to 1000 μm; (a-2)–(d-2) are magnifications of the conjunction between constructs and native disc tissue; (a-3)–(d-3) are magnifications of the central part of scaffold; scale bar is equal to 100 μm.

Figure 6

Safranin O/Fast Green staining of each group. Synthesized sGAG were stained red in Groups A and B. Chitosan scaffold was stained green and cell nuclei of cells were stained black. No sign of red staining in Group C indicated that fibrin gel was unable to be stained with safranin O. Scale bar is equal to 100 μm.

Figure 7

IHC staining of collagen type I ((a-1)–(d-1)) and type II ((a-2)–(d-2)). (a) Group A explants; (b) Group B explants; (c) negative control of primary antibody; (d) native rat TMJ disc. These results confirmed that TMJ-SDSCs may synthesis both collagen types I and II after chondrogenic induction. (d-1) and (d-2) confirmed that collagen type I was the major component of native TMJ disc. Scale bar is equal to 100 μm.