Function Of Skeletal Matrix Genes

National Institute Of Dental & Craniofacial Research

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Abstract

The role of Biglycan in bone formation, maturation and healing. To determine what role Bgn may play in early stages of bone development, scRNA seq analysis was performed on E16.5 day-old bones and revealed there were less mature osteoblasts and more immature embryonic fibroblast cells in the Bgn KO compared to WT bones, suggesting that Bgn is needed for early stages of bone development. To test the possibility that changes in cell populations at these early changes could eventually affect adult bone micro CT was performed on 6-week old femurs and showed they had a reduction in cortical thickness and trabecular number compared to WT controls. The structural integrity of the Bgn KO bones was also found to be compromised judged by second harmonic generation microscopy that showed irregular structures and spacing between the collagen fibers of the Bgn KO bones. These structural changes were coupled with a reduction in the hardness of the Bgn KO bones. To test whether Bgn could also affect bone healing we next examined bone regeneration in fractured bones. Bone healing begins with an inflammatory response at the fracture site, followed by activation and proliferation of skeletal stem cells in the periosteum. Bgn KO mice showed reduced inflammation and macrophage infiltration around the fracture site. In addition, RNAseq analysis showed an enhanced expression of genes involved in suppressing inflammation. Since inflammation is important to trigger the activation and proliferation of the skeletal progenitors in the periosteum, we next measured the periosteum after fracture and found that the Bgn KO periosteum did not expand to the same extent as the WT, leading to reduced callus formation up to 8 weeks post fracture. Immunohistochemistry and RNA analysis were performed and showed that Bgn expression is greatly enhanced in the periosteum in response to injury providing further evidence of its importance in this regenerative tissue. Since periosteal-derived cells (PDCs) have an important role in fracture healing, we then investigated their function further. Our data indicates that Bgn is needed for conversion of the PDCs into cartilage which is a critical phase that precedes bone formation. Collectively, our study identifies Bgn as an influencing factor in PDCs activation during bone development and bone regeneration after fracture. Type VI collagen regulates endochondral ossification in the temporomandibular joint The temporomandibular joint (TMJ) is one of the most common tissues affected by osteoarthritis (OA) which can cause chronic pain and mandibular dysfunction ultimately affecting the quality of life. TMJ-OA is manifested by degenerative changes in the TMJ condyle including erosion, flattening, subchondral cysts, and osteophyte formation; however, there is no effective regenerative treatment because the mechanisms regulating the TMJ development remain unclear. Endochondral ossification is a biological process which is tightly regulated by numerous growth factors and their signaling pathways. During endochondral ossification, chondrocytes in the TMJ undergo a multi-step process of differentiation. Based on the location and proliferative and differentiative state, chondrocytes are classified into a superficial zone, proliferative zone, transitional zone and hypertrophic zone, each characterized by their unique morphology and specific gene expression patterns. In vivo lineage tracing studies showed that chondrocytes in the TMJ cartilage can transdifferentiate into bone cells during subchondral bone formation and endochondral ossification. Thus, elucidating the mechanisms regulating chondrocyte differentiation is important to understand endochondral ossification, and it could provide new mechanistic insights to develop novel ways to regenerate the damaged condyle. The TMJ cartilage contains an extracellular matrix (ECM) composed of collagen, non-collagenous proteins and proteoglycans that all play an important role in facilitating interactions between cells and signaling molecules, and controlling chondrocyte differentiation during endochondral ossification. Type VI collagen is one of the major components of the ECM in the TMJ cartilage and is composed of three alpha chains 1(VI), 2(VI), 3(VI). If any one of the alpha chains is deficient, the type VI collagen triplex is not secreted or incorporated into the ECM. Type VI collagen is both a structural and a signaling molecule, interacting with other matrix molecules to organize ECM structure and modulate biological processes. Although type VI collagen is highly expressed in the TMJ cartilage ECM, its exact function remains unclear. Previous studies examined the role of Type VI collagen on cartilage using Type VI collagen 1 chain deficient mice and found there was a significant difference in skeletal development in knee and hip joints. Based on these findings we predicted it is possible that type VI collagen could have a role regulating skeletal development in other joints. In this study, we focused on the role of type VI collagen in endochondral ossification in the TMJ and found that there was a decreased bone volume/ tissue volume (BV/TV) in the condyle of mice deficient in the 2 chain of type VI collagen (Col62-KO). This was accompanied by a large bone marrow space, arising from abnormal chondrocyte differentiation. Immunohistochemical analysis demonstrated abnormal Runx2 expression in the Col62-KO chondrocytes leading to an altered activation of the Smad1/5/8 signaling pathway. We propose that loss of 2(VI) results in reduced endochondral ossification, caused by abnormal regulation of key signaling pathways that are important for chondrocyte differentiation.