Aggrecan and Hyaluronan: The Infamous Cartilage Polyelectrolytes - Then and Now.

Cartilages are unique in the family of connective tissues in that they contain a high concentration of the glycosaminoglycans, chondroitin sulfate and keratan sulfate attached to the core protein of the proteoglycan, aggrecan. Multiple aggrecan molecules are organized in the extracellular matrix via a domain-specific molecular interaction with hyaluronan and a link protein, and these high molecular weight aggregates are immobilized within the collagen and glycoprotein network. The high negative charge density of glycosaminoglycans provides hydrophilicity, high osmotic swelling pressure and conformational flexibility, which together function to absorb fluctuations in biomechanical stresses on cartilage during movement of an articular joint. We have summarized information on the history and current knowledge obtained by biochemical and genetic approaches, on cell-mediated regulation of aggrecan metabolism and its role in skeletal development, growth as well as during the development of joint disease. In addition, we describe the pathways for hyaluronan metabolism, with particular focus on the role as a "metabolic rheostat" during chondrocyte responses in cartilage remodeling in growth and disease.Future advances in effective therapeutic targeting of cartilage loss during osteoarthritic diseases of the joint as an organ as well as in cartilage tissue engineering would benefit from 'big data' approaches and bioinformatics, to uncover novel feed-forward and feed-back mechanisms for regulating transcription and translation of genes and their integration into cell-specific pathways.

The gut microbiota may be a novel pathogenic mechanism in loosening of orthopedic implants in rats.

Particles released from implants cause inflammatory bone loss, which is a key factor in aseptic loosening, the most common reason for joint replacement failure. With the anticipated increased incidence of total joint replacement in the next decade, implant failure will continue to burden patients. The gut microbiome is increasingly recognized as an important factor in bone physiology, however, its role in implant loosening is currently unknown. We tested the hypothesis that implant loosening is associated with changes in the gut microbiota in a preclinical model. When the particle challenge caused local joint inflammation, decreased peri-implant bone volume, and decreased implant fixation, the gut microbiota was affected. When the particle challenge did not cause this triad of local effects, the gut microbiota was not affected. Our results suggest that cross-talk between these compartments is a previously unrecognized mechanism of failure following total joint replacement.

Calcinosis in Systemic Sclerosis: Updates in Pathophysiology, Evaluation, and Treatment.

PURPOSE OF REVIEW: Calcinosis is a common complication of systemic sclerosis with no known effective pharmacologic therapy. We reviewed the literature regarding systemic sclerosis-related calcinosis as well as other disorders of biomineralization in order to identify targets of future study for calcinosis. RECENT FINDINGS: Patients with systemic sclerosis-related calcinosis demonstrate systemic abnormalities in mineralization pathways, including decreased levels of the mineralization inhibitor inorganic pyrophosphate. Insights from other mineralization disorders suggest that local and systemic phosphate metabolism pathways involving the ABCC6, ENPP1, and NT5E genes play a critical role in regulation of ectopic calcification. Knockout models of these genes may lead to an appropriate murine model for study of calcinosis. Poly(ADP-ribose) polymerase (PARP) enzymes may also play a critical role in hydroxyapatite nucleation and warrant future study in systemic sclerosis. Study of local and systemic mineralization pathways, particularly phosphate metabolism pathways and PARP enzymes, should provide greater insight into the pathogenesis of systemic sclerosis-related calcinosis.

Gene expression profiling of progenitor cells isolated from rat rotator cuff musculotendinous junction.

BACKGROUND: Rotator cuff tendon tears are typically degenerative and usually affect the region of tendon insertion on bone. The remnant torn tendon is degenerative and may not be an ideal source for progenitor cells for cell-based therapies. Therefore, the aim of this study was to determine if musculotendinous junction (MTJ), which is adjacent to tendon would be a viable alternate source of progenitor stem cells. We also sought to study the gene expression profile MTJ progenitors and compare it with progenitors isolated from RC tendon, RC muscle and other existing tissue sources (bone marrow, adipose tissue, and Achilles tendon). METHODS: Rotator cuff tendon (RCT), muscle (RCM), and RCMTJ as well as Achilles tendon (AT) tissues were harvested from healthy male Lewis rats and progenitor cultures were established from these tissues and also from bone marrow and adipose tissue. Quantitative RT-PCR was performed on RNA extracts from intact tissues and progenitor cells using a custom array for the mesenchymal stem cell (MSC) differentiation marker genes. The gene expression profile of MSC differentiation markers within four tissues types, six progenitor cells, and between tissue and their corresponding progenitors were compared. RESULTS: Progenitors cells can be isolated from rat rotator cuff musculotendinous tissue and their pattern of MSC gene expression was similar to the rotator cuff tendon progenitors for majority of the genes tested. However, there were significant differences between the MSC gene expression patterns of RCMTJ and RCM progenitors. Furthermore, there were differences in gene expression between the RCMTJ tissue and its progenitor cells with respect to MSC differentiation markers. The gene expression pattern of RCMTJ tissue was similar to RCM tissue with respect to markers of chondrogenesis, myogenesis, tenogenesis, and MSC specific markers. CONCLUSION: We demonstrate that the musculotendinous junction contains distinct set of progenitor cells and their MSC gene expression pattern is similar to rotator cuff tendon progenitors. RCMTJ progenitors will be an attractive option for cell-based regenerative treatment of chronic rotator cuff tears.

TGF-b1 or hypoxia enhance glucose metabolism and lactate production via HIF1A signaling in tendon cells.

Purpose/Aim of the study: Healthy tendons are maintained in homeostasis through controlled usage of glucose for energy and redox equilibrium. Tendon cell stress imposed by overuse injury or vascular insufficiency is accompanied by activation of wound healing pathways which facilitate an adaptive response and the restoration of homeostasis. To understand this response at the gene expression level we have studied the in vivo effects of injected TGF-ß1 in a murine model of tendinopathy, as well as treatment of murine tendon explants with either TGF-ß1 or hypoxia in vitro. METHODS AND RESULTS: We provide evidence (from expression patterns and immunohistochemistry) that both in vivo and in vitro, the stress response in tendon cells may be metabolically controlled in part by glycolytic reprogramming. A major feature of the response to TGF-ß1 or hypoxia is activation of the Warburg pathway which generates lactate from glucose under normoxia and thereby inhibits mitochondrial energy production. CONCLUSIONS: We discuss the likely outcome of this major metabolic shift in terms of the potential benefits and damage to tendon and suggest how incorporation of this metabolic response into our understanding of initiation and progression of tendinopathies may offer new opportunities for diagnosis and the monitoring of therapies.

Contrast-enhanced micro-computed tomography of compartment and time-dependent changes in femoral cartilage and subchondral plate in a murine model of osteoarthritis.

A lack of understanding of the mechanisms underlying osteoarthritis (OA) progression limits the development of effective long-term treatments. Quantitatively tracking spatiotemporal patterns of cartilage and bone degeneration is critical for assessment of more appropriately targeted OA therapies. In this study, we use contrast-enhanced micro-computed tomography (µCT) to establish a timeline of subchondral plate (SCP) and cartilage changes in the murine femur after destabilization of the medial meniscus (DMM). We performed DMM or sham surgery in 10-12-week-old male C57Bl/6J mice. Femora were imaged using µCT after 0, 2, 4, or 8 weeks. Cartilage-optimized scans were performed after immersion in contrast agent CA4+. Bone mineral density distribution (BMDD), cartilage attenuation, SCP, and cartilage thickness and volume were measured, including lateral and medial femoral condyle and patellar groove compartments. As early as 2 weeks post-DMM, cartilage thickness significantly increased and cartilage attenuation, SCP volume, and BMDD mean significantly decreased. Trends in cartilage and SCP metrics within each joint compartment reflected those seen in global measurements, and both BMDD and SCP thickness were consistently greater in the lateral and medial condyles than the patellar groove. Sham surgery also resulted in significant changes to SCP and cartilage metrics, highlighting a potential limitation of using surgical models to study tissue morphology or composition changes during OA progression. Contrast-enhanced µCT analysis is an effective tool to monitor changes in morphology and composition of cartilage, and when combined with bone-optimized µCT, can be used to assess the progression of degenerative changes after joint injury.

Adamts5 deletion blocks murine dermal repair through CD44-mediated aggrecan accumulation and modulation of transforming growth factor ß1 (TGFß1) signaling.

ADAMTS5 has been implicated in the degradation of cartilage aggrecan in human osteoarthritis. Here, we describe a novel role for the enzyme in the regulation of TGFß1 signaling in dermal fibroblasts both in vivo and in vitro. Adamts5(-/-) mice, generated by deletion of exon 2, exhibit impaired contraction and dermal collagen deposition in an excisional wound healing model. This was accompanied by accumulation in the dermal layer of cell aggregates and fibroblastic cells surrounded by a pericellular matrix enriched in full-length aggrecan. Adamts5(-/-) wounds exhibit low expression (relative to wild type) of collagen type I and type III but show a persistently elevated expression of tgfbRII and alk1. Aggrecan deposition and impaired dermal repair in Adamts5(-/-) mice are both dependent on CD44, and Cd44(-/-)/Adamts5(-/-) mice display robust activation of TGFß receptor II and collagen type III expression and the dermal regeneration seen in WT mice. TGFß1 treatment of newborn fibroblasts from wild type mice results in Smad2/3 phosphorylation, whereas cells from Adamts5(-/-) mice phosphorylate Smad1/5/8. The altered TGFß1 response in the Adamts5(-/-) cells is dependent on the presence of aggrecan and expression of CD44, because Cd44(-/-)/Adamts5(-/-) cells respond like WT cells. We propose that ADAMTS5 deficiency in fibrous tissues results in a poor repair response due to the accumulation of aggrecan in the pericellular matrix of fibroblast progenitor cells, which prevents their transition to mature fibroblasts. Thus, the capacity of ADAMTS5 to modulate critical tissue repair signaling events suggests a unique role for this enzyme, which sets it apart from other members of the ADAMTS family of proteases.

Intraarticular injection of heparin-binding insulin-like growth factor 1 sustains delivery of insulin-like growth factor 1 to cartilage through binding to chondroitin sulfate.

OBJECTIVE: Insulin-like growth factor 1 (IGF-1) stimulates cartilage repair but is not a practical therapy due to its short half-life. We have previously modified IGF-1 by adding a heparin-binding domain and have shown that this fusion protein (HB-IGF-1) stimulates sustained proteoglycan synthesis in cartilage. This study was undertaken to examine the mechanism by which HB-IGF-1 is retained in cartilage and to test whether HB-IGF-1 provides sustained growth factor delivery to cartilage in vivo and to human cartilage explants. METHODS: Retention of HB-IGF-1 and IGF-1 was analyzed by Western blotting. The necessity of heparan sulfate (HS) or chondroitin sulfate (CS) glycosaminoglycans (GAGs) for binding was tested using enzymatic removal and cells with genetic deficiency of HS. Binding affinities of HB-IGF-1 and IGF-1 proteins for isolated GAGs were examined by surface plasmon resonance and enzyme-linked immunosorbent assay. RESULTS: In cartilage explants, chondroitinase treatment decreased binding of HB-IGF-1, whereas heparitinase had no effect. Furthermore, HS was not necessary for HB-IGF-1 retention on cell monolayers. Binding assays showed that HB-IGF-1 bound both CS and HS, whereas IGF-1 did not bind either. After intraarticular injection in rat knees, HB-IGF-1 was retained in articular and meniscal cartilage, but not in tendon, consistent with enhanced delivery to CS-rich cartilage. Finally, HB-IGF-1 was retained in human cartilage explants but IGF-1 was not. CONCLUSION: Our findings indicate that after intraarticular injection in rats, HB-IGF-1 is specifically retained in cartilage through its high abundance of CS. Modification of growth factors with heparin-binding domains may be a new strategy for sustained and specific local delivery to cartilage.

Disulfide-bonded multimers of proteoglycan 4 PRG4 are present in normal synovial fluids.

BACKGROUND: The proteoglycan 4 (PRG4) gene encodes for a mucin-like O-linked glycosylated protein with several names, including lubricin and superficial zone protein. The objective of this study was to analyze PRG4 in normal bovine calf and steer synovial fluids for evidence of native multimers formed by intermolecular disulfide bonds. METHODS: A combination of mucin biochemical techniques, with antibodies to both terminal domains and the mucin-like domain of PRG4, were used for analyses. RESULTS: Multimers were present in both calf and steer fluids, and reduction and alkylation converts the multimeric complex (likely dimeric) into monomeric subunits. Tandem mass spectrometry analyses supported the Western blot data and identified PRG4 in the reduced approximately 345 kDa monomeric form. Interestingly, approximately 70 kDa fragments released upon reduction contained peptides from both the N and C terminal regions, which most likely represent fragments of a sparsely glycosylated PRG4 population that are disulfide-linked to extensively glycosylated, intact monomers. CONCLUSIONS: The analyses described here have demonstrated the presence of native disulfide-bonded multimers of PRG4 in normal bovine synovial fluids. GENERAL SIGNIFICANCE: These structures are similar to those described for multimerization of mucins in general. Such multimerization and proteolytic cleavage of PRG4 may have functional significance in joint health and disease.

Modulation of hyaluronan production by CD44 positive glioma cells.

This study examines the functional relationship between glioma cell production of hyaluronan (HA), known to play a role in glioma invasion, expression of its CD44 receptor, and glioma cell viability. Production of HA by CD44 positive mouse G26 and human U373 glioma cell lines was evaluated and compared to that of a CD44 positive mouse fibroblast-like L929 cell line. We found that both G26 and U373 MG glioma cells, but not L929 fibroblast-like cells, synthesized HA. The synthesis of HA by glioma cells was found during the proliferative phase as well as post-confluency, as detected by fluorophore-assisted carbohydrate electrophoresis. Eighty to ninety percent of the HA synthesized was secreted into the medium and 10-20% remained associated with the cells. To examine a possible mechanistic link between the CD44-HA interaction and endogenous HA production, glioma cells were treated with either anti-CD44 antibodies (clones KM201 or IM7) or HA oligosaccharides (hexamer oligoHA-6 or decamer oligoHA-10). We found that oligoHA-10, which was previously shown to compete effectively with the CD44-HA interaction, enhanced glioma HA synthesis by approximately 1.5-fold, without affecting cell viability. IM7 treatment of human U373 glioma cells resulted in over 50% decrease of HA production, which was associated with changes in cell size and apoptosis. Taken together, these data show that CD44 specific ligands, such as the IM7 antibody or oligoHA-10 could down-regulate or up-regulate glioma HA production, respectively. Our results suggest that interference with CD44/HA may lead to the discovery and development of new treatment modalities for glioma.

Addition of High Molecular Weight Hyaluronic Acid to Fibroblast-Like Stromal Cells Modulates Endogenous Hyaluronic Acid Metabolism and Enhances Proteolytic Processing and Secretion of Versican.

We have examined the effect of exogenous linear chain high molecular weight hyaluronic acid (HMW HA) on endogenously synthesized hyaluronic acid (HA) and associated binding proteins in primary cultures of fibroblast-like stromal cells that were obtained by collagenase digestion of the murine peripatellar fat pad. The cultures were expanded in DMEM that was supplemented with fetal bovine serum and basic fibroblast growth factor (bFGF) then exposed to macrophage-colony-stimulating factor (MCSF) to induce macrophage properties, before activation of inflammatory pathways using E. coli lipopolysaccharide (LPS). Under all culture conditions, a significant amount of endogenously synthesized HA localized in LAMP1-positive lysosomal vesicles. However, this intracellular pool was depleted after the addition of exogenous HMW HA and was accompanied by enhanced proteolytic processing and secretion of de novo synthesized versican, much of which was associated with endosomal compartments. No changes were detected in synthesis, secretion, or proteolytic processing of aggrecan or lubricin (PRG4). The addition of HMW HA also modulated a range of LPS-affected genes in the TLR signaling and phagocytosis pathways, as well as endogenous HA metabolism genes, such as Has1, Hyal1, Hyal2, and Tmem2. However, there was no evidence for association of endogenous or exogenous HMW HA with cell surface CD44, TLR2 or TLR4 protein, suggesting that its physiochemical effects on pericelluar pH and/or ionic strength might be the primary modulators of signal transduction and vesicular trafficking by this cell type. We discuss the implications of these findings in terms of a potential in vivo effect of therapeutically applied HMW HA on the modification of osteoarthritis-related joint pathologies, such as pro-inflammatory and degradative responses of multipotent mesenchymal cells residing in the synovial membrane, the underlying adipose tissue, and the articular cartilage surface.

In-Vivo Efficacy of Recombinant Human Hyaluronidase (rHuPH20) Injection for Accelerated Healing of Murine Retrocalcaneal Bursitis and Tendinopathy.

The deposition of aggrecan/hyaluronan (HA)-rich matrix within the tendon body and surrounding peritenon impede tendon healing and result in compromised biomechanical properties. Hence, the development of novel strategies to achieve targeted removal of the aggrecan-HA pericellular matrix may be effective in treating tendinopathy. The current study examined the therapeutic potential of a recombinant human hyaluronidase, rHuPH20 (FDA approved for reducing HA accumulation in tumors) for treating murine Achilles tendinopathy. The 12-week-old C57Bl/6 male mice were injected with two doses of rHuTGF-ß1 into the retrocalcaneal bursa (RCB) to induce a combined bursitis and tendinopathy. Twenty-four hours following induction of injury, treatment groups were administered rHuPH20 Hyaluronidase (rHuPH20; Halozyme Therapeutics) into the RCB. At either 6 h (acute), 9 days, or 25 days following hyaluronidase treatment, Achilles tendons were analyzed for gene expression, histology and immunohistochemistry, fluorophore-assisted carbohydrate electrophoresis, and biomechanical properties. The rHuPH20 treatment was effective, particularly at the acute and 9-day time points, in (a) removing HA deposits from the Achilles tendon and surrounding tissues, (b) improving biomechanical properties of the healing tendon, and (c) eliciting targeted increases in expression of specific cell fate, extracellular matrix metabolism, and inflammatory genes. The potential of rHuPH20 to effectively clear the pro-inflammatory, HA-rich matrix within the RCB and tendon strongly supports the future refinement of injectable glycosidase preparations as potential treatments to protect or regenerate tendon tissue by reducing inflammation and scarring in the presence of bursitis or other inducers of damage such as mechanical overuse. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:59-69, 2020.

Intra-articular Hyaluronan Therapy for Symptomatic Knee Osteoarthritis.

Intra-articular hyaluronan therapy (IAHA) is a common but controversial nonsurgical treatment of symptomatic knee osteoarthritis. The overall treatment effect of IAHA for osteoarthritis pain is substantial, but the majority of this benefit is mediated by the placebo effect. Despite the large overall benefit of IAHA when the effect of the hyaluronan is combined with the placebo effect, there is only a small benefit of questionable clinical significance of IAHA compared with intra-articular placebo. Compared with intra-articular corticosteroids, however, IAHA most likely is comparable for long-term pain relief and possibly slightly inferior for short-term pain relief.

Regulation of proteoglycan production by varying glucose concentrations controls fiber formation in tissue engineered menisci.

Fibrillar collagens are highly prevalent in the extracellular matrix of all connective tissues and therefore commonly used as a biomaterial in tissue engineering applications. In the native environment, collagen fibers are arranged in a complex hierarchical structure that is often difficult to recreate in a tissue engineered construct. Small leucine rich proteoglycans as well as hyaluronan binding proteoglycans, aggrecan and versican, have been implicated in regulating fiber formation. In this study, we modified proteoglycan production in vitro by altering culture medium glucose concentrations (4500, 1000, 500, 250, and 125 mg/L), and evaluated its effect on the formation of collagen fibers inside tissue engineered meniscal constructs. Reduction of extracellular glucose resulted in a dose dependent decrease in total sulfated glycosaminoglycan (GAG) production, but minimal decreases of decorin and biglycan. However, fibromodulin doubled in production between 125 and 4500 mg/L glucose concentration. A peak in fiber formation was observed at 500 mg/L glucose concentration and corresponded with reductions in total GAG production. Fiber formation reduction at 125 and 250 mg/L glucose concentrations are likely due to changes in metabolic activity associated with a limited supply of glucose. These results point to proteoglycan production as a means to manipulate fiber architecture in tissue engineered constructs. STATEMENT OF SIGNIFICANCE: Fibrillar collagens are highly prevalent in the extracellular matrix of all connective tissues; however achieving appropriate assembly and organization of collagen fibers in engineered connective tissues is a persistent challenge. Proteoglycans have been implicated in regulating collagen fiber organization both in vivo and in vitro, however little is known about methods to control proteoglycan production and the subsequent fiber organization in tissue engineered menisci. Here, we show that media glucose content can be optimized to control proteoglycan production and collagen fiber assembly, with optimal collagen fiber assembly occurring at sub-physiologic levels of glucose.

Effect of intra-articular hyaluronan injection on inflammation and bone remodeling in the epiphyses and metaphyses of the knee in a murine model of joint injury.

The TTR (transforming growth factor ß1 (TGFß1) injection with treadmill running) model of murine joint injury was used to examine effects of intra-articular Hyaluronan (IA HA) on the metabolism of subchondral bone. HA was injected 24 h after TGFß1 injection and its effects on the mRNA of 80 genes in the Nfkb pathway, and bone remodeling genes, Acp5, Nos2 and Arg1, in femoral and tibial epiphyses/metaphyses of injected and contralateral legs was assessed. Structural bone parameters at those sites were determined by Micro-computed tomography (micro CT) and bone remodeling cells identified with histochemistry for tartrate-resistant acid phosphatase and immunohistochemistry for Nitric oxide synthase 2 (NOS2) and Arginase 1. Gene expression responses in femoral compartments were generally inhibitory and notably biphasic whereas the tibia was relatively non-responsive. Gene expression was also altered in the contralateral femoral compartment but were predominantly activated. IA TGFb did not alter bone structure in the injected leg, but resulted in a statistically significant reduction (25-40%) in trabecular bone of the contralateral limb. IA HA did not affect such changes. This bone loss was associated with an acute decrease in transcript abundance for Acp5, Nos2, Arg1 and this decrease persisted for Nos2 and Arg1. In conclusion, the data illustrate that in this model, IA TGFß1 injection results in marked biphasic changes in NfKb-regulated apoptosis, IL1 and IL12 pathways, which were transiently altered after IA HA therapy. The finding that all modulations are essentially restricted to the femoral compartment is consistent with the predominant localization and clearance of injected HA from this site.

Innate immune system activation in osteoarthritis: is osteoarthritis a chronic wound?

PURPOSE OF REVIEW: Synovial inflammation is increasingly recognized as an important pathophysiologic process in osteoarthritis, but the stimuli and downstream pathways activated are not well defined. Innate immune system activation, best documented in responses to pathogens, likely plays a role in induction of inflammatory mediators and the specific cellular infiltrate seen in osteoarthritis. Thus, the Toll-like receptors (TLRs) and their signaling pathways are of particular interest. These innate pattern-recognition receptors are activated not only by pathogens but by endogenous 'danger signals'. In this report, we review evidence that certain extracellular matrix components of joint tissues (hyaluronan and fibronectin) may act as TLR stimuli, and summarize recent literature implicating TLR activation in osteoarthritis. RECENT FINDINGS: Convincing evidence exists that hyaluronan/TLR interactions drive responses to tissue injury. Evidence of a similar role for fibronectin is growing. TLRs are expressed and functional in the joint, and many proteases and cytokines that promote cartilage catabolism are dependent on nuclear factor-kappaB, a TLR-activated transcription factor. SUMMARY: Activation of TLR pathways seems likely in osteoarthritis and may play a central role in disease development and progression. A model of osteoarthritis as a chronic wound, in which the innate immune response is triggered by molecular signals of tissue damage, is presented as a framework for future study of inflammation in this prevalent joint disease.

Knee joint injury and repair modeled by membrane systems.

Tissue reparative processes following tissue injury are modeled by a basic membrane system, dealing only with objects, non-active membranes, and non-deterministic evolution rules. At the biological level, tissue repair is regulated by multiple interactions between cells and macromolecules, the latter acting as signals. Such signals modify cell behavior including proliferation, migration, differentiation, and phagocytosis. The signaling components themselves are produced and removed by the resident cell population, and this set of events may provide additional stimuli for altering cell activities. In this paper we have focused on modeling the biology of events following an injury to the knee joint, and have used hyaluronan (a polymer produced by cartilage and synovial cells) as an example for a signaling component in the healing process. The intrinsic non-determinism of the model is a key feature, which allows a mathematical description of the repair responses as well as a possibility for either functional restoration or chronic degeneration, leading to arthritis.

Pirfenidone reduces subchondral bone loss and fibrosis after murine knee cartilage injury.

Pirfenidone is an anti-inflammatory and anti-fibrotic drug that has shown efficacy in lung and kidney fibrosis. Because inflammation and fibrosis have been linked to the progression of osteoarthritis, we investigated the effects of oral Pirfenidone in a mouse model of cartilage injury, which results in chronic inflammation and joint-wide fibrosis in mice that lack hyaluronan synthase 1 (Has1-/- ) in comparison to wild-type. Femoral cartilage was surgically injured in wild-type and Has1-/- mice, and Pirfenidone was administered in food starting after 3 days. At 4 weeks, Pirfenidone reduced the appearance, on micro-computed tomography, of pitting in subchondral bone at, and cortical bone surrounding, the site of cartilage injury. This corresponded with a reduction in fibrotic tissue deposits as observed with gross joint surface photography. Pirfenidone resulted in significant recovery of trabecular bone parameters affected by joint injury in Has1-/- mice, although the effect in wild-type was less pronounced. Pirfenidone also increased Safranin-O staining of growth plate cartilage after cartilage injury and sham operation in both genotypes. Taken together with the expression of selected extracellular matrix, inflammation, and fibrosis genes, these results indicate that Pirfenidone may confer chondrogenic and bone-protective effects, although the well-known anti-fibrotic effects of Pirfenidone may occur earlier in the wound-healing response than the time point examined in this study. Further investigations to identify the specific cell populations in the joint and signaling pathways that are responsive to Pirfenidone are warranted, as Pirfenidone and other anti-fibrotic drugs may encourage tissue repair and prevent progression of post-traumatic osteoarthritis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:365-376, 2018.

Knockout of hyaluronan synthase 1, but not 3, impairs formation of the retrocalcaneal bursa.

Hyaluronan (HA), a high molecular weight non-sulfated glycosaminoglycan, is an integral component of the extracellular matrix of developing and mature connective tissues including tendon. There are few published reports quantifying HA content during tendon growth and maturation, or detailing its effects on the mechanical properties of the tendon extracellular matrix. Therefore, the goal of the current study was to examine the role of HA synthesis during post-natal skeletal growth and maturation, and its influence on tendon structure and biomechanical function. For this purpose, the morphological, biochemical, and mechanical properties of Achilles tendons from wild type (WT) and hyaluronan synthase 1 and 3 deficient mouse strains (Has1-/- (Has1KO), Has3-/- (Has3KO), and Has1-/- 3-/- (Has1/3KO)) were determined at 4, 8, and 12 weeks of age. Overall, HAS-deficient mice did not show any marked differences from WT mice in Achilles tendon morphology or in the HA and chondroitin/dermatan sulfate (CS/DS) contents. However, HAS1-deficiency (in the single or Has1/3 double KO) impeded post-natal formation of the retrocalcaneal bursa, implicating HAS1 in regulating HA metabolism by cells lining the bursal cavity. Together, these data suggest that HA metabolism via HAS1 and HAS3 does not markedly influence the extracellular matrix structure or function of the tendon body, but plays a role in the formation/maintenance of peritendinous bursa. Additional studies are warranted to elucidate the relationship of HA and CS/DS metabolism to tendon healing and repair in vivo. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2622-2632, 2018.

Oral Ibuprofen Interferes with Cellular Healing Responses in a Murine Model of Achilles Tendinopathy.

BACKGROUND: The attempted healing of tendon after acute injury (overloading, partial tear or complete rupture) proceeds via the normal wound healing cascade involving hemostasis, inflammation, matrix synthesis and matrix remodeling. Depending on the degree of trauma and the nature of the post-injury milieu, a variable degree of healing and recovery of function occurs. Post-injury analgesia is often achieved with NSAIDs such as Ibuprofen, however there is increasing evidence that NSAID usage may interfere with the healing process. This study aimed to investigate the cellular mechanism by which IBU therapy might lead to a worsening of tendon pathology. METHODS: We have examined the effect of oral Ibuprofen, on Achilles tendon healing in a TGFb1-induced murine tendinopathy model. Dosing was started 3 days after initial injury (acute cellular response phase) and continued for 22 days or started at 9 days after injury (transition to matrix regeneration phase) and given for 16 days. Cellular changes in tendon and surrounding peritenon were assessed using Hematoxylin/Eosin, chondroid accumulation with Safranin O and anti-aggrecan immunohistochemistry, and neo-vessel formation with GSI Lectin histochemistry. Markers of inflammation included histochemical localization of hyaluronan, immunohistochemistry of heavy chain 1 and TNFα-stimulated glycoprotein-6 (TSG6). Cell responses were further examined by RT-qPCR of 84 NFκB target genes and 84 wound healing genes. Biomechanical properties of tendons were evaluated by tensile testing. RESULTS: At a clinically-relevant dosage, Ibuprofen prevented the process of remodeling/removal of the inflammatory matrix components, hyaluronan, HC1 and TSG6. Furthermore, the aberrant matrix remodeling was accompanied by activation at day 28 of genes (Col1a2, Col5a3, Plat, Ccl12, Itga4, Stat3, Vegfa, Mif, Col4a1, Rhoa, Relb, F8, Cxcl9, Lta, Ltb, Ccl12, Cdkn1a, Ccl22, Sele, Cd80), which were not activated at any time without the drug, and so appear most likely to be involved in the pathology. Of these, Vegfa, Col4a1, F8, Cxcl9 and Sele, have been shown to play a role in vascular remodeling, consistent with the appearance at 25 days of vasculogenic cell groups in the peritenon and fat pad stroma surrounding the Achilles of the drug-dosed mice. Tensile stiffness (p = 0.004) and elastic modulus (p = 0.012) were both decreased (relative to age-matched uninjured and non-dosed mice) in mice dosed with Ibuprofen from day 3 to day 25, whether injured or not. CONCLUSION: We conclude that the use of Ibuprofen for pain relief during inflammatory phases of tendinopathy, might interfere with the normal processes of extracellular matrix remodeling and cellular control of expression of inflammatory and wound healing genes. It is proposed that the known COX2-mediated anti-inflammatory effect of ibuprofen has detrimental effects on the turnover of a pro-inflammatory HA matrix produced in response to soft-tissue injury, thus preventing the switch to cellular responses associated with functional matrix remodeling and eventual healing.