BIOCHEMICAL AND HISTOLOGICAL CHARACTERIZATION OF TMJ

Objective of this project is to determine the composition of the the temporal mandibular joint (TMJ) from dissected human cadaver that will be essential for the development of stem cell therapy for individuals with diseased TMJ. This study aims to obtain a better understanding of the components that surround the cells in the TMJ. The components being studied include: collagen, elastin, and sulfated glycosaminoglycans. These molecules surround the cells forming a matrix that hold the TMJ intact. Understanding the exact composition of the TMJ matrix will is key to developing stem cell therapy for TMJ. Methods: Biochemical: Human TMJ discs and attachments were dissected from recently deceased bodies and analyzed both histologically and biochemically. Samples dry weight obtained. Follow up studies include: measuring DNA content, collagen, and sulfated glycosaminoglycans. All these will be done using commercially available kits. Histological: Histological preparations were made with various stains: Alcian Blue for assay acidic glycans; Verhoeff’s Elastic (VEG); and Hematoxylin Eosin (H&E). Results: Elastin composition of human samples show differences compared to previous pig studies. TMJ disc has a denser matrix with less elastic fibers than the TMJ attachments. TMJ attachments have more visible nuclei than disc with H&E staining. TMJ complex is saturated with sulfated GAGs. Conclusion: This study shows that matrix composition of human TMJ may differ from previous pig studies. Further studies from human samples using various ages can better elucidate the matrix composition for targeted human TMJ stem cell therapy. Purpose (a): Objective: Characterizing biochemical and histological composition of the human temporal mandibular joint (TMJ) disc is essential to development of stem cell therapy for diseased TMJ. Components of extracellular matrix (ECM) analyzed are elastin, collagen and sulfated glycosaminoglycan (GAG). Methods (b): Biochemical: Human TMJ discs were dissected from recently deceased bodies and analyzed histologically and biochemically. Five regions of the disc and six distal attachments examined. Following dissection, wet weights recorded. Samples lyophilized to obtain dry weight. Dried samples were digested in a 125mg/mL papain solution overnight at 600C. Follow up studies include: DNA content (measured with the Quant-iT Picrogreen dsDNA Assay Kit (Invitrogen). Following hydrolysis with 4N NaOH for 20 minutes at 110 0C, collagen content to be quantified with modified chloramine-T hydroxyproline assay. Sulfated GAG content will be quantified using Blyscan Glycosaminoglycan Assay Kit (Accurate Chemical and Scientific Corp.). N= 6 samples per group for all biochemical analysis. Histological: Histological preparations were made with various stains: Alcian Blue for assay acidic glycans; Verhoeff’s Elastic (VEG); and Hematoxylin Eosin (H&E). Results (c): Elastin composition of human samples show differences compared to previous porcine studies. TMJ disc has a denser matrix with less elastic fibers than distal attachments. TMJ attachments have more visible nuclei than disc with H&E staining. TMJ complex is saturated with sulfated GAGs. Conclusions (d): This study shows that ECM composition of human TMJ may differ from previous porcine studies. Further studies from human samples using various ages can further elucidate the ECM composition for targeted human TMJ stem cell therapy.