Osteocytic Pericellular Matrix (PCM): Accelerated Degradation under In Vivo Loading and Unloading Conditions Using a Novel Imaging Approach

The proteoglycan-containing pericellular matrix (PCM) controls both the biophysical and biochemical microenvironment of osteocytes, which are the most abundant cells embedded and dispersed in bones. As a molecular sieve, osteocytic PCMs not only regulate mass transport to and from osteocytes but also act as sensors of external mechanical environments. The turnover of osteocytic PCM remains largely unknown due to technical challenges. Here, we report a novel imaging technique based on metabolic labeling and “click-chemistry,” which labels de novo PCM as “halos” surrounding osteocytes in vitro and in vivo. We then tested the method and showed different labeling patterns in young vs. old bones. Further “pulse-chase” experiments revealed dramatic difference in the “half-life” of PCM of cultured osteocytes (~70 h) and that of osteocytes in vivo (~75 d). When mice were subjected to either 3-week hindlimb unloading or 7-week tibial loading (5.1 N, 4 Hz, 3 d/week), PCM half-life was shortened (~20 d) and degradation accelerated. Matrix metallopeptidase MMP-14 was elevated in mechanically loaded osteocytes, which may contribute to PCM degradation. This study provides a detailed procedure that enables semi-quantitative study of the osteocytic PCM remodeling in vivo and in vitro.

Direct Perfusion Improves Redifferentiation of Human Chondrocytes in Fibrin Hydrogel with the Deposition of Cartilage Pericellular Matrix

Articular cartilage has limited potential for self-repair, and cell-based strategies combining scaffolds and chondrocytes are currently used to treat cartilage injuries. However, achieving a satisfying level of cell redifferentiation following expansion remains challenging. Hydrogels and perfusion bioreactors are known to exert beneficial cues on chondrocytes; however, the effect of a combined approach on the quality of cartilage matrix deposited by cells is not fully understood. Here, we combined soluble factors (BMP-2, Insulin, and Triiodothyronine, that is, BIT), fibrin hydrogel, direct perfusion and human articular chondrocytes (HACs) to engineer large cartilage tissues. Following cell expansion, cells were embedded in fibrin gels and cultivated under either static or perfusion conditions. The nature of the matrix synthesized was assessed by Western blotting and immunohistochemistry. The stability of cartilage grafts and integration with native tissue were also investigated by subcutaneous implantation of human osteochondral cylinders in nude mice. Perfusion preconditioning improved matrix quality and spatial distribution. Specifically, perfusion preconditioning resulted in a matrix rich in type II collagen but not in type I collagen, indicating the reconstruction of hyaline cartilage. Remarkably, the production of type VI collagen, the main component of the pericellular matrix, was also increased, indicating that chondrocytes were connecting to the hyaline matrix they produced.

mTOR repression in response to amino acid starvation promotes ECM degradation through MT1-MMP endocytosis arrest

Under conditions of starvation, normal and tumor epithelial cells can rewire their metabolism towards the consumption of extracellular matrix-derived components as nutrient sources. The mechanism of pericellular matrix degradation by starved cells has been largely overlooked. Here we show that matrix degradation by breast and pancreatic tumor cells and patient-derived xenograft explants increases by one order of magnitude upon amino acid and growth factor deprivation. In addition, we found that collagenolysis requires the invadopodia components, TKS5 and the transmembrane metalloproteinase, MT1-MMP, which are key to the tumor invasion program. Increased collagenolysis is controlled by mTOR repression upon nutrient depletion or pharmacological inhibition by rapamycin. Our results reveal that starvation hampers clathrin-mediated endocytosis, resulting in MT1-MMP accumulation in arrested clathrin-coated pits. Our study uncovers a new mechanism whereby mTOR repression in starved cells leads to the repurposing of abundant plasma membrane clathrin-coated pits into robust ECM-degradative assemblies.

Phenotypic Differences in Adult and Fetal Dermal Fibroblast Responses to Mechanical Tension

ABSTRACTThe regenerative wound healing phenotype observed in the fetal wounds is characterized by a unique hyaluronan rich wound microenvironment. Fetal fibroblasts produce a pericellular matrix which is abundant in high molecular weight HA. In vivo, fetal skin has negligible resting tension and heals regeneratively when small wounds are made. However, critically large fetal wounds heal with increased scar. We hypothesize that higher mechanical tension will differentially alter fibroblast-mediated HA metabolism in adult and fetal fibroblasts, leading to a profibrotic fetal fibroblast phenotype. C57BL/6J fetal (FFB, E14.5) and adult (AFB, 8 wk) dermal fibroblasts were cultured on silicone membranes +/-10% static strain (1, 3, 6, and 12h). Monolayers were analyzed via PCR for HA-synthesis (HAS1-3), HA-remodeling (HYAL1-2, KIAA1199), and HA-receptor (CD44) genes, normalized to static FFB. Total HA was analyzed by gel electrophoresis at 12h. Data is reported as mean+/-SD (n=3), with p-values calculated by two-way ANOVA (post-hoc Tukey’s test). FFB pericellular matrix was reduced after tension conditions, and HA profile shifted from HMW-HA to LMW-HA. Under static conditions, AFB had increased expression of HAS 1 and 2, as well as increased expression of KIA1199, HYAL1 and 2 when compared to FFB. Overall, tension resulted in an increase in HAS1, HAS3, and HYAL1 expression in FFB, and decreased HAS2 but increased HYAL1 for AFB under tension. Staining for cytoskeletal components demonstrates that tension results in organization of F-actin and aSMA in FFB to resemble AFB. Expression of aSMA is increased in AFB with tension, while CD26 expression is increased in FFB with tension. These insights into the intrinsic differences between regenerative FFB and fibrotic AFB may yield targets to attenuate fibrosis.