Tissue-immunoengineering of TMJ neodisc implants - PROJECT SUMMARY Temporomandibular joint (TMJ) disc perforations pose devastating morbidities for millions of Americans. Interventions to slow or prevent progression of this disease do not exist. Toward addressing this as-of-yet intractable problem, tissue-engineered neodisc constructs made with allogeneic costal chondrocytes have shown promise in healing TMJ disc thinning, but larger defects remain a major challenge. Due to the exposed allograft area, larger defects elicit a more severe immune response compared to smaller defects, necessitating novel approaches to engineer TMJ neodisc constructs for large defects typically associated with TMJ disc perforation. Novel tissue-engineering strategies, termed “tissue-immunoengineering” in this proposal, are set forth, where TMJ neodisc constructs are cocultured with macrophages to improve construct functional properties (i.e., biochemical and mechanical properties), toward achieving TMJ disc healing in vivo. Pro-healing and anti- inflammatory M2-polarized macrophages will be examined for their effects on enhancing the robustness and resilience of TMJ neodisc constructs. Prior work for enhancing tissue-engineered constructs has used bioactive agents, such as growth factors and chondrogenic conditioned media, but no studies have introduced coculturing constructs with macrophages to improve TMJ neodisc properties. Macrophages have been shown to direct healing through secretions and their reactions to changes in tissue composition and stiffness during healing. A potential mechanism for the coordination of TMJ disc healing is the bidirectional interaction between macrophages and chondrocytes, resulting in continuous adjustments to changes in the healing microenvironment. Despite their potential, the utility of M2 macrophages, including both pro-healing or M2a (i.e., stimulated with IL-4/IL-13) and anti-inflammatory or M2c (i.e., stimulated with IL-10) subtypes, have not yet been examined for enhancing the suitability of engineered tissues for implantation. Collaboration between the Athanasiou and Liu laboratories recently showed that improving construct functional properties not only results in mechanical durability but, also, excitingly, resistance to immune-mediated degradation. Moreover, exciting new scRNA-seq results from this collaboration are providing insights as to how macrophages and chondrocytes communicate to coordinate tissue formation. Encouraged by these novel and significant results, we propose to test the hypothesis that coculturing TMJ neodisc constructs with M2-polarized macrophages in vitro will increase functional properties of the constructs, thus, improving healing outcomes in large perforation TMJ disc defects in vivo. This overall hypothesis will be examined through three aims: 1) To identify the macrophage origin and in vitro activation conditions that maximize functional properties of TMJ neodisc constructs. 2) To tune the mechanical environment of macrophage-construct direct coculture systems to improve construct functional properties through a) direct mechanical stimulation and b) enhancement of M2 macrophage polarization. 3) To improve the healing of large perforation defects of TMJ discs in a minipig model using a tissue- immunoengineered TMJ neodisc construct. It is anticipated that this proposal will establish a novel and robust tissue-immunoengineering strategy to improve healing of the TMJ disc, with the potential to improve the lives of millions living with TMJ disc perforation.
