The functional properties of tendon require an extracellular matrix (ECM) rich in elongated collagen fibrils in parallel register. We sought to understand how embryonic fibroblasts elaborate this exquisite arrangement of fibrils. We show that procollagen processing and collagen fibrillogenesis are initiated in Golgi to plasma membrane carriers (GPCs). These carriers and their cargo of 28-nm-diam fibrils are targeted to previously unidentified plasma membrane (PM) protrusions (here designated “fibripositors”) that are parallel to the tendon axis and project into parallel channels between cells. The base of the fibripositor lumen (buried several microns within the cell) is a nucleation site of collagen fibrillogenesis. The tip of the fibripositor is the site of fibril deposition to the ECM. Fibripositors are absent at postnatal stages when fibrils increase in diameter by accretion of extracellular collagen, thereby maintaining parallelism of the tendon. Thus, we show that the parallelism of tendon is determined by the late secretory pathway and interaction of adjacent PMs to form extracellular channels.

Human bone marrow stem cells (hMSCs) have been shown to differentiate in vitro into a number of cell lineages and are a potential autologous cell source for the repair and replacement of damaged and diseased musculoskeletal tissues. hMSC differentiation into chondrocytes has been described in highdensity cell pellets cultured with specific growth and differentiation factors. We now describe how culture of hMSCs as a shallow multicellular layer on a permeable membrane over 2-4 weeks resulted in a much more efficient formation of cartilaginous tissue than in established chondrogenic assays. In this format, the hMSCs differentiated in 14 days to produce translucent, flexible discs, 6 mm in diameter by 0.8 -1 mm in thickness from 0.5 ؋ 10 6 cells. The discs contained an extensive cartilage-like extracellular matrix (ECM), with more than 50% greater proteoglycan content per cell than control hMSCs differentiated in standard cell pellet cultures. The disc constructs were also enriched in the cartilage-specific collagen II, and this was more homogeneously distributed than in cell pellet cultures. The expression of cartilage matrix genes for collagen type II and aggrecan was enhanced in disc cultures, but improved matrix production was not accompanied by increased expression of the transcription factors SOX9, L-SOX5, and SOX6. The fast continuous growth of cartilage ECM in these cultures up to 4 weeks appeared to result from the geometry of the construct and the efficient delivery of nutrients to the cells. Scaffold-free growth of cartilage in this format will provide a valuable experimental system for both experimental and potential clinical studies.

Pseudoachondroplasia (PSACH) is one of the more common skeletal dysplasias and results from mutations in cartilage oligomeric matrix protein (COMP). Most COMP mutations identified to date cluster in the TSP3 repeat region of COMP and the mutant protein is retained in the rough endoplasmic reticulum (rER) of chondrocytes and may result in increased cell death. In contrast, the pathomolecular mechanism of PSACH resulting from C-terminal domain COMP mutations remain largely unknown. This study describes the generation and analysis of a murine model of mild PSACH resulting from a p.Thr583Met mutation in the C-terminal globular domain (CTD) of COMP. Mutant animals are normal at birth, but grow slower than their wild-type littermates and by 9 weeks of age they have mild short-limb dwarfism. Furthermore, by 16 months of age mutant animals exhibit severe degeneration of articular cartilage, which is consistent with early onset osteoarthritis seen in PSACH patients. In the growth plates of mutant mice the chondrocyte columns are sparser and poorly organized. Mutant COMP is secreted into the extracellular matrix, but its localization is disrupted along with the distribution of several COMP-binding proteins. Although mutant COMP is not retained within the rER there is an unfolded protein/cell stress response and chondrocyte proliferation is significantly reduced, while apoptosis is both increased and spatially dysregulated. Overall, these data suggests a mutation in the CTD of COMP exerts a dominant-negative effect on both intra- and extracellular processes. This ultimately affects the morphology and proliferation of growth plate chondrocytes, eventually leading to chondrodysplasia and reduced long bone growth.