Cartilage, the tissue that provides a lubricious surface in diarthrodial joints, does not regenerate when injured or diseased. No treatments for cartilage degeneration succeed at regenerating native cartilage, and most fail in the long term. Tissue-engineered cartilage is a promising therapeutic for cartilage repair and regeneration; this is particularly pertinent for people who have ailments of the temporomandibular joint (TMJ) cartilages — up to 8-16% of the population. However, a deep understanding of the extracellular matrix (ECM) of tissue-engineered cartilage is limited by a dearth of analytic techniques for collagens, the proteins that play essential functional roles in cartilage ECM. Through novel liquid chromatography–mass spectrometry (LC-MS) methods, the global objectives of this work were to: 1) develop methods for the quantification of collagen subtypes and crosslinks, 2) interrogate the temporal changes in collagen subtype and crosslink profiles of native cartilage and tissue-engineered neocartilage during tissue development, and 3) investigate the collagen subtypes and crosslinks of cartilage regeneration through the use of tissue-engineered neocartilage implants in a large animal model.Toward addressing the dearth of techniques for collagen subtype quantification, novel LC-MS methods were developed to quantify individual collagen subtypes and crosslinks in biological samples, and bottom-up proteomics techniques were used to quantify all proteins in tissue ECM, including collagen subtypes, using the cartilages of the Yucatan minipig as a model. The high-throughput LC-MS methods for collagen subtype and crosslink quantification are low in cost and operator time and can be translated to any facility that has access to a triple quadrupole mass spectrometer. These novel collagenomic techniques are widely applicable to researchers studying tissue characterization, disease states, and tissue engineering of all collagenous tissues, including cartilage, skin, bone, ligament, tendon, and many others.
The novel collagenomic methods were applied to study the development of native and engineered cartilages. In addition to well-known developmental changes in native cartilage such as total collagen and DNA contents, trends in the collagen subtype and crosslink profiles throughout maturation of knee cartilage were elucidated. The self-assembling process of tissue engineered cartilage was also examined with collagenomic techniques, and many similarities in development between the self-assembled cartilage and native cartilage, such as an increase in collagen type II, were determined. These trends showed evidence that self-assembled engineered cartilage recapitulates aspects of the development of native cartilages.
Toward developing a deeper understanding of cartilage regeneration with self-assembled neocartilage implants, Yucatan minipig TMJ disc perforation models were evaluated. Small perforation (3-mm) and large perforation (6-mm) defects were regenerated with self-assembled neocartilage implants and evaluated at 24 weeks and 8 weeks, respectively. In both in vivo studies, treatment with self-assembled neocartilage implants resulted in regenerated fibrocartilaginous repair tissue, an important step toward translating self-assembled cartilage implants to clinical usage. Quantitative collagenomics was used to show that the repair tissue of empty defects in the small perforation group had a collagen subtype profile similar to that of scar tissue (more collagen type III and less collagen type I) than repair tissue of implant-treated TMJ discs, which had a collagen subtype profile of native TMJ disc tissue.
Overall, this work is highly significant in that it introduces novel methods for collagen subtype and crosslink quantification, which are applicable not only to cartilage, but to a wide breadth of tissue fields. Because of the critical roles of major and minor collagens in providing structure to tissues, collagenomics techniques are particularly relevant to researchers studying tissue characterization, disease states, structure-function relationships, and tissue engineering of cartilage, bone, heart valve, ligament, tendon, blood vessels, and skin. Applying collagenomics techniques to the development of native and engineered cartilages allows for a deeper understanding of the self-assembling process of cartilage tissue engineering beyond traditional benchtop biochemical assays. Finally, showing the fibrocartilaginous collagenomic profile of regenerated cartilage tissue in a large animal model is a major milestone toward translating tissue-engineered cartilage to the clinic for human usage, which can drastically improve the quality of life in millions of patients who suffer from TMJ disorders.
