Engineering Human TMJ Discs with Protein-Releasing 3D-Printed Scaffolds

The temporomandibular joint (TMJ) disc is a heterogeneous fibrocartilaginous tissue positioned between the mandibular condyle and glenoid fossa of the temporal bone, with important roles in TMJ functions. Tissue engineering TMJ discs has emerged as an alternative approach to overcoming limitations of current treatments for TMJ disorders. However, the anisotropic collagen orientation and inhomogeneous fibrocartilaginous matrix distribution present challenges in the tissue engineering of functional TMJ discs. Here, we developed 3-dimensional (3D) printed anatomically correct scaffolds with region-variant microstrand alignment, mimicking anisotropic collagen alignment in the TMJ disc and corresponding mechanical properties. Connective tissue growth factor (CTGF) and transforming growth factor beta 3 (TG933) were then delivered in the scaffolds by spatially embedding CTGF- or TG933-encapsulated microspheres (pS) to reconstruct the regionally variant fibrocartilaginous matrix in the native TMJ disc. When cultured with human mesenchymal stem/progenitor cells (MSCs) for 6 wk, 3D-printed scaffolds with CTGF/TG933-pS resulted in a heterogeneous fibrocartilaginous matrix with overall distribution of collagen-rich fibrous structure in the anterior/posterior (AP) bands and fibrocartilaginous matrix in the intermediate zone, reminiscent of the native TMJ disc. High dose of CTGF/TG933-pS (100 mg pS/g of scaffold) showed significantly more collagen 11 and aggrecan in the intermediate zone than a low dose (50 mg pS/g of scaffold). Similarly, a high dose of CTGF/TG933pS yielded significantly higher collagen 1 expression in the AP bands compared with the low-dose and empty pS. From stress relaxation tests, the ratio of relaxation modulus to instantaneous modulus was significantly smaller with CTGF/TG933-pS than empty pS. Similarly, a significantly higher coefficient of viscosity was achieved with the high dose of CTGF/TG933-pS compared with the low-dose and empty PS, suggesting the dose effect of CTGF and TG933 on fibrocartilage formation. Together, our findings may represent an efficient approach to engineering the TMJ disc graft with anisotropic scaffold microstructure, heterogeneous fibrocartilaginous matrix, and region-dependent viscoelastic properties.