Covalent intermolecular cross-linking of collagen is essential for tissue stability. Recent studies have demonstrated that cyclophilin B (CypB), an endoplasmic reticulum (ER)-resident peptidyl-prolyl cis-trans isomerase, modulates lysine (Lys) hydroxylation of type I collagen impacting cross-linking chemistry. However, the extent of modulation, the molecular mechanism and the functional outcome in tissues are not well understood. Here, we report that, in CypB null (KO) mouse skin, two unusual collagen cross-links lacking Lys hydroxylation are formed while neither was detected in wild type (WT) or heterozygous (Het) mice. Mass spectrometric analysis of type I collagen showed that none of the telopeptidyl Lys was hydroxylated in KO or WT/Het mice. Hydroxylation of the helical cross-linking Lys residues was almost complete in WT/Het but was markedly diminished in KO. Lys hydroxylation at other sites was also lower in KO but to a lesser extent. A key glycosylation site, α1(I) Lys-87, was underglycosylated while other sites were mostly overglycosylated in KO. Despite these findings, lysyl hydroxylases and glycosyltransferase 25 domain 1 levels were significantly higher in KO than WT/Het. However, the components of ER chaperone complex that positively or negatively regulates lysyl hydroxylase activities were severely reduced or slightly increased, respectively, in KO. The atomic force microscopy-based nanoindentation modulus were significantly lower in KO skin than WT. These data demonstrate that CypB deficiency profoundly affects Lys post-translational modifications of collagen likely by modulating LH chaperone complexes. Together, our study underscores the critical role of CypB in Lys modifications of collagen, cross-linking and mechanical properties of skin.
Our objective is to provide an in-depth review of the recent technical advances of atomic force microscopy (AFM)-based nanomechanical tests and their contribution to a better understanding and diagnosis of osteoarthritis (OA), as well as the repair of tissues undergoing degeneration during OA progression. We first summarize a range of technical approaches for AFM-based nanoindentation, including considerations in both experimental design and data analysis. We then provide a more detailed description of two recently developed modes of AFM-nanoindentation, a high-bandwidth nanorheometer system for studying poroviscoelasticity and an immunofluorescence-guided nanomechanical mapping technique for delineating the pericellular matrix (PCM) and territorial/interterritorial matrix (T/IT-ECM) of surrounding cells in connective tissues. Next, we summarize recent applications of these approaches to three aspects of joint-related healthcare and disease: cartilage aging and OA, developmental biology and OA pathogenesis in murine models, and nanomechanics of the meniscus. These studies were performed over a hierarchy of length scales, from the molecular, cellular to the whole tissue level. The advances described here have contributed greatly to advancing the fundamental knowledge base for improved understanding, detection, and treatment of OA.
Molecular engineering of biological tissues using synthetic mimics of native matrix molecules can modulate the mechanical properties of the cellular microenvironment through physical interactions with existing matrix molecules, and in turn, mediate the corresponding cell mechanobiology. In articular cartilage, the pericellular matrix (PCM) is the immediate microniche that regulates cell fate, signaling, and metabolism. The negatively charged osmo-environment, as endowed by PCM proteoglycans, is a key biophysical cue for cell mechanosensing. This study demonstrated that biomimetic proteoglycans (BPGs), which mimic the ultrastructure and polyanionic nature of native proteoglycans, can be used to molecularly engineer PCM micromechanics and cell mechanotransduction in cartilage. Upon infiltration into bovine cartilage explant, we showed that localization of BPGs in the PCM leads to increased PCM micromodulus and enhanced chondrocyte intracellular calcium signaling. Applying molecular force spectroscopy, we revealed that BPGs integrate with native PCM through augmenting the molecular adhesion of aggrecan, the major PCM proteoglycan, at the nanoscale. These interactions are enabled by the biomimetic "bottle-brush" ultrastructure of BPGs and facilitate the integration of BPGs within the PCM. Thus, this class of biomimetic molecules can be used for modulating molecular interactions of pericellular proteoglycans and harnessing cell mechanosensing. Because the PCM is a prevalent feature of various cell types, BPGs hold promising potential for improving regeneration and disease modification for not only cartilage-related healthcare but many other tissues and diseases.
