High spatial resolution analysis using automated indentation mapping differentiates biomechanical properties of normal vs. degenerated articular cartilage in mice.

Characterizing the biomechanical properties of articular cartilage is crucial to understanding processes of tissue homeostasis vs. degeneration. In mouse models, however, limitations are imposed by their small joint size and thin cartilage surfaces. Here we present a three-dimensional (3D) automated surface mapping system and methodology that allows for mechanical characterization of mouse cartilage with high spatial resolution. We performed repeated indentation mappings, followed by cartilage thickness measurement via needle probing, at 31 predefined positions distributed over the medial and lateral femoral condyles of healthy mice. High-resolution 3D x-ray microscopy (XRM) imaging was used to validate tissue thickness measurements. The automated indentation mapping was reproducible, and needle probing yielded cartilage thicknesses comparable to XRM imaging. When comparing healthy vs. degenerated cartilage, topographical variations in biomechanics were identified, with altered thickness and stiffness (instantaneous modulus) across condyles and within anteroposterior sub-regions. This quantitative technique comprehensively characterized cartilage function in mice femoral condyle cartilage. Hence, it has the potential to improve our understanding of tissue structure-function interplay in mouse models of repair and disease.

Proteoglycan 4 (PRG4) treatment enhances wound closure and tissue regeneration.

The wound healing response is one of most primitive and conserved physiological responses in the animal kingdom, as restoring tissue integrity/homeostasis can be the difference between life and death. Wound healing in mammals is mediated by immune cells and inflammatory signaling molecules that regulate tissue resident cells, including local progenitor cells, to mediate closure of the wound through formation of a scar. Proteoglycan 4 (PRG4), a protein found throughout the animal kingdom from fish to elephants, is best known as a glycoprotein that reduces friction between articulating surfaces (e.g. cartilage). Previously, PRG4 was also shown to regulate the inflammatory and fibrotic response. Based on this, we asked whether PRG4 plays a role in the wound healing response. Using an ear wound model, topical application of exogenous recombinant human (rh)PRG4 hastened wound closure and enhanced tissue regeneration. Our results also suggest that rhPRG4 may impact the fibrotic response, angiogenesis/blood flow to the injury site, macrophage inflammatory dynamics, recruitment of immune and increased proliferation of adult mesenchymal progenitor cells (MPCs) and promoting chondrogenic differentiation of MPCs to form the auricular cartilage scaffold of the injured ear. These results suggest that PRG4 has the potential to suppress scar formation while enhancing connective tissue regeneration post-injury by modulating aspects of each wound healing stage (blood clotting, inflammation, tissue generation and tissue remodeling). Therefore, we propose that rhPRG4 may represent a potential therapy to mitigate scar and improve wound healing.

Synovial mesenchymal progenitor derived aggrecan regulates cartilage homeostasis and endogenous repair capacity.

Aggrecan is a critical component of the extracellular matrix of all cartilages. One of the early hallmarks of osteoarthritis (OA) is the loss of aggrecan from articular cartilage followed by degeneration of the tissue. Mesenchymal progenitor cell (MPC) populations in joints, including those in the synovium, have been hypothesized to play a role in the maintenance and/or repair of cartilage, however, the mechanism by which this may occur is unknown. In the current study, we have uncovered that aggrecan is secreted by synovial MPCs from healthy joints yet accumulates inside synovial MPCs within OA joints. Using human synovial biopsies and a rat model of OA, we established that this observation in aggrecan metabolism also occurs in vivo. Moreover, the loss of the "anti-proteinase" molecule alpha-2 macroglobulin (A2M) inhibits aggrecan secretion in OA synovial MPCs, whereas overexpressing A2M rescues the normal secretion of aggrecan. Using mice models of OA and cartilage repair, we have demonstrated that intra-articular injection of aggrecan into OA joints inhibits cartilage degeneration and stimulates cartilage repair respectively. Furthermore, when synovial MPCs overexpressing aggrecan were transplanted into injured joints, increased cartilage regeneration was observed vs. wild-type MPCs or MPCs with diminished aggrecan expression. Overall, these results suggest that aggrecan secreted from joint-associated MPCs may play a role in tissue homeostasis and repair of synovial joints.

Understanding cartilage protection in OA and injury: a spectrum of possibilities.

BACKGROUND: Osteoarthritis (OA) is a prevalent musculoskeletal disease resulting in progressive degeneration of the hyaline articular cartilage within synovial joints. Current repair treatments for OA often result in poor quality tissue that is functionally ineffective compared to the hyaline cartilage and demonstrates increased failure rates post-treatment. Complicating efforts to improve clinical outcomes, animal models used in pre-clinical research show significant heterogeneity in their regenerative and degenerative responses associated with their species, age, genetic/epigenetic traits, and context of cartilage injury or disease. These can lead to variable outcomes when testing and validating novel therapeutic approaches for OA. Furthermore, it remains unclear whether protection against OA among different model systems is driven by inhibition of cartilage degeneration, enhancement of cartilage regeneration, or any combination thereof. MAIN TEXT: Understanding the mechanistic basis underlying this context-dependent duality is essential for the rational design of targeted cartilage repair and OA therapies. Here, we discuss some of the critical variables related to the cross-species paradigm of degenerative and regenerative abilities found in pre-clinical animal models, to highlight that a gradient of regenerative competence within cartilage may exist across species and even in the greater human population, and likely influences clinical outcomes. CONCLUSIONS: A more complete understanding of the endogenous regenerative potential of cartilage in a species specific context may facilitate the development of effective therapeutic approaches for cartilage injury and/or OA.

Evolutionary Selection and Constraint on Human Knee Chondrocyte Regulation Impacts Osteoarthritis Risk.

During human evolution, the knee adapted to the biomechanical demands of bipedalism by altering chondrocyte developmental programs. This adaptive process was likely not without deleterious consequences to health. Today, osteoarthritis occurs in 250 million people, with risk variants enriched in non-coding sequences near chondrocyte genes, loci that likely became optimized during knee evolution. We explore this relationship by epigenetically profiling joint chondrocytes, revealing ancient selection and recent constraint and drift on knee regulatory elements, which also overlap osteoarthritis variants that contribute to disease heritability by tending to modify constrained functional sequence. We propose a model whereby genetic violations to regulatory constraint, tolerated during knee development, lead to adult pathology. In support, we discover a causal enhancer variant (rs6060369) present in billions of people at a risk locus (GDF5-UQCC1), showing how it impacts mouse knee-shape and osteoarthritis. Overall, our methods link an evolutionarily novel aspect of human anatomy to its pathogenesis.

Isolation and Characterization of an Adult Stem Cell Population from Human Epidural Fat.

STUDY DESIGN: Isolation and characterization of human epidural fat (HEF) stem/progenitor cells. OBJECTIVE: To identify a progenitor population within HEF and to determine if they meet the minimal criteria of a mesenchymal stem cell (MSC). SUMMARY OF BACKGROUND DATA: The biological function, if any, has yet to be determined for HEF. The presence of MSCs within HEF may indicate a regenerative potential within the HEF. METHODS: HEF was isolated from 10 patients during elective spinal surgery. HEF cells were differentiated along osteo-, adipo-, and chondrogenic lineages, with differentiation analyzed via qPCR and histology. The cell surface receptor profile of HEF cells was examined by flow cytometry. HEF cells were also assayed through the collagen contraction assay. Prx1 CreERT2GFP:R26R TdTomato MSC lineage-tracking mice were employed to identify EF MSCs in vivo. RESULTS: HEF cell lines were obtained from all 10 patients in the study. Cells from 2/10 patients demonstrated full MSC potential, while cells from 6/10 patients demonstrated progenitor potential; 2/10 patients presented with cells that retained only adipogenic potential. HEF cells demonstrated MSC surface marker expression. All patient cell lines contracted collagen gels. A Prx1-positive population in mouse epidural fat that appeared to contribute to the dura of the spinal cord was observed in vivo. CONCLUSIONS: MSC and progenitor populations are present within HEF. MSCs were not identified in all patients examined in the current study. Furthermore, all patient lines demonstrated collagen contraction capacity, suggesting either a contaminating activated fibroblast population or HEF MSCs/progenitors also demonstrating a fibroblast-like phenotype. In vivo analysis suggests that these cell populations may contribute to the dura. Overall, these results suggest that cells within epidural fat may play a biological role within the local environment above providing a mechanical buffer.

17-DMAG regulates p21 expression to induce chondrogenesis in vitro and in vivo.

Cartilage degeneration after injury affects a significant percentage of the population, including those that will go on to develop osteoarthritis (OA). Like humans, most mammals, including mice, are incapable of regenerating injured cartilage. Interestingly, it has previously been shown that p21 (Cdkn1a) knockout (p21-/-) mice demonstrate auricular (ear) cartilage regeneration. However, the loss of p21 expression is highly correlated with the development of numerous types of cancer and autoimmune diseases, limiting the therapeutic translation of these findings. Therefore, in this study, we employed a screening approach to identify an inhibitor (17-DMAG) that negatively regulates the expression of p21. We also validated that this compound can induce chondrogenesis in vitro (in adult mesenchymal stem cells) and in vivo (auricular cartilage injury model). Furthermore, our results suggest that 17-DMAG can induce the proliferation of terminally differentiated chondrocytes (in vitro and in vivo), while maintaining their chondrogenic phenotype. This study provides new insights into the regulation of chondrogenesis that might ultimately lead to new therapies for cartilage injury and/or OA.

Increased levels of p21((CIP1/WAF1)) correlate with decreased chondrogenic differentiation potential in synovial membrane progenitor cells.

Cartilage injuries are a major concern in the field of orthopedics. They occur following trauma, as well as from a variety of pathological conditions including Osteoarthritis (OA). Although cartilage does not exhibit robust endogenous repair, it has been demonstrated that modulating the activity of p21 can increase the regenerative abilities of cartilage in vitro and in vivo. Since the synovial membrane is abundant with mesenchymal progenitor cells (MPCs) capable of differentiating into cartilage both in vitro and in vivo, we examined if p21 expression levels varied between MPCs derived from normal vs. OA knee joints. Analysis of p21 at the mRNA and protein levels within normal and OA MPCs demonstrated differential levels of expression between these two groups, with OA MPCs having higher p21 expression levels. The higher levels of p21 in OA MPCs are also correlated with a decreased chondrogenic differentiation capacity and synovial inflammation, however, there was no evidence of senescence in the OA cells. The results of this study suggest that cell cycle regulation in MPCs may be altered in OA and that modulation of this pathway may have therapeutic potential once the mechanism by which this regulates stem/progenitor cells is better understood.