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.

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.

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.

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.

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.

Genome-wide analysis identifies differential promoter methylation of Leprel2, Foxf1, Mmp25, Igfbp6, and Peg12 in murine tendinopathy.

We have used a murine Achilles tendinopathy model to investigate whether tissue changes (such as collagen disorganization, chondroid metaplasia, and loss of tensile properties) which are broadly characteristic of human tendinopathies, are accompanied by changes in the expression of chromatin-modifying enzymes and the methylation status of promoter regions of tendon cell DNA. Tendinopathy was induced by two intra-tendinous TGF-ß1 injections followed by cage activity or treadmill running for up to 28 days. Activation of DNA methyltransferases occurred at 3 days after the TGF-ß1 injections and also at 14 days, but only with treadmill activity. Genome-wide Methyl Mini-Seq™ analysis identified 19 genes with differentially methylated promoters, five of which perform functions with an apparent direct relevance to tendinopathy (Leprel2, Foxf1, Mmp25, Igfbp6, and Peg12). The functions of the genes identified included collagen fiber assembly and pericellular interactions, therefore their perturbation could play a role in the characteristic disorganization of fibers in affected tendons. We postulate that a study of the functional genomics of these genes in animal and human tendon could further delineate the pathogenesis of this multi-factorial complex disease. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:947-955, 2017.

Recent Scientific Advances Towards the Development of Tendon Healing Strategies.

There exists a range of surgical and non-surgical approaches to the treatment of both acute and chronic tendon injuries. Despite surgical advances in the management of acute tears and increasing treatment options for tendinopathies, strategies frequently are unsuccessful, due to impaired mechanical properties of the treated tendon and/or a deficiency in progenitor cell activities. Hence, there is an urgent need for effective therapeutic strategies to augment intrinsic and/or surgical repair. Such approaches can benefit both tendinopathies and tendon tears which, due to their severity, appear to be irreversible or irreparable. Biologic therapies include the utilization of scaffolds as well as gene, growth factor, and cell delivery. These treatment modalities aim to provide mechanical durability or augment the biologic healing potential of the repaired tissue. Here, we review the emerging concepts and scientific evidence which provide a rationale for tissue engineering and regeneration strategies as well as discuss the clinical translation of recent innovations.

ADAMTS5 is required for biomechanically-stimulated healing of murine tendinopathy.

A recently developed murine model of tendinopathy, induced by TGF-ß1 injection, has been used to examine the reparative capacity of tendinopathic Achilles in Adamts5(-/-) mice. After TGF-ß1 injection and 2 weeks of treadmill exercise, the Achilles from Adamts5(-/-) mice exhibited a reduction in maximum tensile stress of approximately 60%. However, in contrast to wild type mice previously characterized by this model, Adamts5(-/-) mice subjected to further treadmill exercise were unable to reverse this biomechanical deficit. This nonreparative phenotype was accompanied by a major deficiency, relative to wild-type, in expression of Col1a1 and Col3a1 and an abnormally elevated expression of a wide range of integrins. In addition, the tendinopathic Adamts5(-/-) mice showed a persistent accumulation of chondrogenic cells in the tendon body and an aggrecan-rich fibrocartilaginous matrix within disorganized collagen fiber bundles. Moreover, consistent with the compromised biomechanical properties of the Achilles in the Adamts5(-/-) mice, in vivo gait analysis revealed a strong trend (p = 0.07) towards increased swing time of the injected limb in Adamts5(-/-) relative to wild-type mice. These findings demonstrate that a deficiency in ADAMTS5 promotes a chondrogenic response to TGF-ß1 injection that is not reversed by treadmill exercise. Hence, repair of biomechanically compromised tendons exhibiting midsubstance chondroid accumulation requires ADAMTS5.

Controlled treadmill exercise eliminates chondroid deposits and restores tensile properties in a new murine tendinopathy model.

Tendinopathy is a widespread and disabling condition characterized by collagen fiber disruption and accumulation of a glycosaminoglycan-rich chondroid matrix. Recent clinical reports have illustrated the potential of mechanical loading (exercise) therapies to successfully treat chronic tendinopathies. We have developed a new murine tendinopathy model which requires a single injection of TGF-ß1 into the Achilles tendon midsubstance followed by normal cage activity for 2 weeks. At this time, tendon maximum stress showed a dramatic (66%) reduction relative to that of normal controls and this persisted at four weeks. Loss of material properties was accompanied by abundant chondroid cells within the tendon (closely resembling the changes observed in human samples obtained intra-operatively) and increased expression of Acan, Col1a1, Col2a1, Col3a1, Fn1 and Mmp3. Mice subjected to two weeks of daily treadmill exercise following TGF-ß1 injection showed a similar reduction in tendon material properties as the caged group. However, in mice subjected to 4 weeks of treadmill exercise, tendon maximum stress values were similar to those of naive controls. Tendons from the mice exercised for 4 weeks showed essentially no chondroid cells and the expression of Acan, Col1a1, Col2a1, Col3a1, and Mmp3 was significantly reduced relative to the 4-week cage group. This technically simple murine tendinopathy model is highly amenable to detailed mechanistic and translational studies of the biomechanical and cell biological pathways, that could be targeted to enhance healing of tendinopathy.

Hyaluronan injection in murine osteoarthritis prevents TGFbeta 1-induced synovial neovascularization and fibrosis and maintains articular cartilage integrity by a CD44-dependent mechanism.

INTRODUCTION: The mechanism by which intra-articular injection of hyaluronan (HA) ameliorates joint pathology is unknown. Animal studies have shown that HA can reduce synovial activation, periarticular fibrosis and cartilage erosion; however, its specific effects on the different cell types involved remain unclear. We have used the TTR (TGFbeta1 injection and Treadmill Running) model of murine osteoarthritis (OA), which exhibits many OA-like changes, including synovial activation, to examine in vivo tissue-specific effects of intra-articular HA. METHODS: The kinetics of clearance of fluorotagged HA from joints was examined with whole-body imaging. Naïve and treated knee joints were examined macroscopically for cartilage erosion, meniscal damage and fibrosis. Quantitative histopathology was done with Safranin O for cartilage and with Hematoxylin & Eosin for synovium. Gene expression in joint tissues for Acan, Col1a1, Col2a1, Col3a1, Col5a1, Col10a1, Adamts5 and Mmp13 was done by quantitative PCR. The abundance and distribution of aggrecan, collagen types I, II, III, V and X, ADAMTS5 and MMP13 were examined by immunohistochemistry. RESULTS: Injected HA showed a half-life of less than 2 h in the murine knee joint. At the tissue level, HA protected against neovascularization and fibrosis of the meniscus/synovium and maintained articular cartilage integrity in wild-type but not in Cd44 knockout mice. HA injection enhanced the expression of chondrogenic genes and proteins and blocked that of fibrogenic/degradative genes and proteins in cartilage/subchondral bone, whereas it blocked activation of both groups in meniscus/synovium. In all locations it reduced the expression/protein for Mmp13 and blocked Adamts5 expression but not its protein abundance in the synovial lining. CONCLUSIONS: The injection of HA, 24 h after TGFbeta1 injection, inhibited the cascade of OA-like joint changes seen after treadmill use in the TTR model of OA. In terms of mechanism, tissue protection by HA injection was abrogated by Cd44 ablation, suggesting that interaction of the injected HA with CD44 is central to its protective effects on joint tissue remodeling and degeneration in OA progression.

Murine tendon function is adversely affected by aggrecan accumulation due to the knockout of ADAMTS5.

The present study examined the effect of ADAMTS5 (TS5) knockout on the properties of murine flexor digitorum longus (FDL) and Achilles tendons. FDL and Achilles tendons were analyzed using biomechanical testing, histology, and immunohistochemistry; further characterization of FDL tendons was conducted using transmission electron microscopy (collagen fibril ultrastructure), SDS-PAGE (collagen content and type), fluorescence-assisted carbohydrate electrophoresis for chondroitin sulfate and hyaluronan, and Western blotting for aggrecan, versican, and decorin abundance and distribution. FDL tendons of TS5(-/-) mice showed a 33% larger cross-sectional area, increased collagen fibril area fraction, and decreased material properties relative to those of wild type mice. In TS5(-/-) mice, aggrecan accumulated in the pericellular matrix of tendon fibroblasts. In Achilles tendons, cross-sectional area, stress relaxation, and structural properties were similar in TS5(-/-) and wild type mice; however, the TS5(-/-) tendons exhibited a higher tensile modulus and a weakened enthesis. These results demonstrate that TS5 deficiency disturbs normal tendon collagen organization and alters biomechanical properties. Hence, the role of ADAMTS5 in tendon is to remove pericellular and interfibrillar aggrecan to maintain the molecular architecture responsible for normal tissue function.

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 hyaluronan prevents cartilage erosion, periarticular fibrosis and mechanical allodynia and normalizes stance time in murine knee osteoarthritis.

INTRODUCTION: Intraarticular hyaluronan (HA) is used clinically for symptomatic relief in patients with knee osteoarthritis (OA); however, the mechanism of action is unclear. In this study, we examined the effects of a single injection of HA on joint tissue pathology, mechanical allodynia and gait changes (measured by stride times) in a murine model of OA. METHODS: OA was induced in the right knee joint (stifle) of 12-week-old male C57BL/6 mice by transforming growth factor ß1 (TGFß1) injection and treadmill running for 14 days. Gait parameters were quantified by using TreadScan, mechanical allodynia was evaluated with von Frey filaments, and joint pathology was evaluated by scoring of macroscopic images for both cartilage erosion and periarticular fibrosis. HA or saline control was injected 1 day after TGFß1 injection but before the start of treadmill running. RESULTS: OA development in this model was accompanied by significant (P < 0.01) enhancement of the stance and propulsion times of affected legs. HA injection (but not saline injection) blocked all gait changes and also protected joints from femoral cartilage erosion as well as tibial and femoral tissue fibrosis. Both HA injection and saline injection attenuated acute allodynia, but the HA effect was more pronounced and prolonged than the saline injection. CONCLUSIONS: We conclude that videographic gait analysis is an objective, sensitive and reproducible means of monitoring joint pathology in experimental murine OA, since stance time appears to correlate directly with OA severity. A single injection of HA prevents acute and prolonged gait changes and ameliorates the cartilage erosion and periarticular fibrosis normally seen in this model. We speculate that the capacity of HA to prevent cartilage erosion results from its normalization of joint biomechanics and its inhibitory effects on periarticular cells, which are involved in tissue hyperplasia and fibrosis. This effect of exogenous HA appears to mimic the protective effects of ablation of Adamts5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) on experimental murine OA, and we speculate that a common mechanism is involved.

Knockout of ADAMTS5 does not eliminate cartilage aggrecanase activity but abrogates joint fibrosis and promotes cartilage aggrecan deposition in murine osteoarthritis models.

To investigate the role of ADAMTS5 in murine osteoarthritis (OA), resulting from destabilization of the medial meniscus (DMM model) or from TGFb1 injection and enforced uphill treadmill running (TTR model). Wild-type (WT) and ADAMTS5-/- mice were subjected to either DMM or TTR and joints were evaluated for meniscal damage, cartilage changes, and fibrotic ingrowths from the joint margins. Cartilage lesions were quantified on an 8-point scoring system. Cartilage chondroitin sulfate (CS) content was evaluated by SafraninO staining and by quantitative electrophoresis (FACE). The abundance of aggrecan, versican, and specific aggrecanase-generated products was determined by Western analysis. Joint changes were similar for WT mice taken through either the DMM or the TTR model. ADAMTS5 ablation essentially eliminated cartilage erosion and fibrous overgrowth in both models. In the TTR model, ADAMTS5 ablation did not eliminate aggrecanase activity from the articular cartilage but blocked fibrosis and resulted in the accumulation of aggrecan in the articular cartilage. The cartilage protection provided by ADAMTS5 ablation in the mouse does not result from prevention of aggrecanase activity per se, but it appears to be due to a blockade of joint tissue fibrosis and a concomitant increase in cartilage aggrecan content.

Biochemical identification and immunolocalizaton of aggrecan, ADAMTS5 and inter-alpha-trypsin-inhibitor in equine degenerative suspensory ligament desmitis.

We describe analysis of suspensory ligaments from horses with advanced degenerative suspensory ligament desmitis (DSLD) to identify the major proteoglycans (PGs), ADAMTS-aggrecanases and inter-alpha-trypsin inhibitor (IαI) components associated with ligament degeneration. Specific anatomical regions of suspensory ligaments from two normal horses and four diagnosed with DSLD were analyzed by Western blot and immunohistochemistry for the following: aggrecan, aggrecan fragments, decorin, ADAMTS4, ADAMTS5, and IαI components. When compared to normal, DSLD ligaments showed about a 15-fold increase (P < 0.0014) in aggrecan levels and markedly enhanced staining with Safranin O. The aggrecan was composed of two distinct high molecular weight core protein species. The largest species was found only in DSLD samples and it co-migrated with aggrecan synthesized by equine mesenchymal stem cells (MSC). Many of the DSLD samples also contained abnormally high concentrations of ADAMTS4, ADAMTS5, and IαI. Notably, the ADAMTS5 in DSLD samples, but not normals, was present largely as a high molecular weight complex. We conclude that ligament degeneration in DSLD is associated with matrix changes characteristic of an inflammatory nonhealing wound, specifically containing chondrogenic progenitor cells. Since aggrecan accumulation is a major feature of incomplete healing in tendon and skin of the ADAMTS5 knockout mouse, we propose that ligament failure in DSLD results from a process involving tissue inflammation and the complexation of ADAMTS5.

Age-related differences in human skin proteoglycans.

Previous work has shown that versican, decorin and a catabolic fragment of decorin, termed decorunt, are the most abundant proteoglycans in human skin. Further analysis of versican indicates that four major core protein species are present in human skin at all ages examined from fetal to adult. Two of these are identified as the V0 and V1 isoforms, with the latter predominating. The other two species are catabolic fragments of V0 and V1, which have the amino acid sequence DPEAAE as their carboxyl terminus. Although the core proteins of human skin versican show no major age-related differences, the glycosaminoglycans (GAGs) of adult skin versican are smaller in size and show differences in their sulfation pattern relative to those in fetal skin versican. In contrast to human skin versican, human skin decorin shows minimal age-related differences in its sulfation pattern, although, like versican, the GAGs of adult skin decorin are smaller than those of fetal skin decorin. Analysis of the catabolic fragments of decorin from adult skin reveals the presence of other fragments in addition to decorunt, although the core proteins of these additional decorin catabolic fragments have not been identified. Thus, versican and decorin of human skin show age-related differences, versican primarily in the size and the sulfation pattern of its GAGs and decorin in the size of its GAGs. The catabolic fragments of versican are detected at all ages examined, but appear to be in lower abundance in adult skin compared with fetal skin. In contrast, the catabolic fragments of decorin are present in adult skin, but are virtually absent from fetal skin. Taken together, these data suggest that there are age-related differences in the catabolism of proteoglycans in human skin. These age-related differences in proteoglycan patterns and catabolism may play a role in the age-related changes in the physical properties and injury response of human skin.

Controlled delivery of transforming growth factor ß1 by self-assembling peptide hydrogels induces chondrogenesis of bone marrow stromal cells and modulates Smad2/3 signaling.

Self-assembling peptide hydrogels were modified to deliver transforming growth factor ß1 (TGF-ß1) to encapsulated bone-marrow-derived stromal cells (BMSCs) for cartilage tissue engineering applications using two different approaches: (i) biotin-streptavidin tethering; (ii) adsorption to the peptide scaffold. Initial studies to determine the duration of TGF-ß1 medium supplementation necessary to stimulate chondrogenesis showed that 4 days of transient soluble TGF-ß1 to newborn bovine BMSCs resulted in 10-fold higher proteoglycan accumulation than TGF-ß1-free culture after 3 weeks. Subsequently, BMSC-seeded peptide hydrogels with either tethered TGF-ß1 (Teth-TGF) or adsorbed TGF-ß1 (Ads-TGF) were cultured in the TGF-ß1-free medium, and chondrogenesis was compared to that for BMSCs encapsulated in unmodified peptide hydrogels, both with and without soluble TGF-ß1 medium supplementation. Ads-TGF peptide hydrogels stimulated chondrogenesis of BMSCs as demonstrated by cell proliferation and cartilage-like extracellular matrix accumulation, whereas Teth-TGF did not stimulate chondrogenesis. In parallel experiments, TGF-ß1 adsorbed to agarose hydrogels stimulated comparable chondrogenesis. Full-length aggrecan was produced by BMSCs in response to Ads-TGF in both peptide and agarose hydrogels, whereas medium-delivered TGF-ß1 stimulated catabolic aggrecan cleavage product formation in agarose but not peptide scaffolds. Smad2/3 was transiently phosphorylated in response to Ads-TGF but not Teth-TGF, whereas medium-delivered TGF-ß1 produced sustained signaling, suggesting that dose and signal duration are potentially important for minimizing aggrecan cleavage product formation. Robustness of this technology for use in multiple species and ages was demonstrated by effective chondrogenic stimulation of adult equine BMSCs, an important translational model used before the initiation of human clinical studies.

Interleukin-1alpha treatment of meniscal explants stimulates the production and release of aggrecanase-generated, GAG-substituted aggrecan products and also the release of pre-formed, aggrecanase-generated G1 and m-calpain-generated G1-G2.

Pro-inflammatory cytokines induce meniscal matrix degradation and inhibition of endogenous repair mechanisms, but the pathogenic mechanisms behind this are mostly unknown. Therefore, we investigated details of interleukin-1 (IL-1alpha)-induced aggrecan turnover in mature meniscal tissue explants. Fibro-cartilagenous disks (3 mm diameter x 1 mm thickness) were isolated from the central, weight-bearing region of menisci from 2-year-old cattle. After 3 or 6 days of IL-1alpha-treatment, GAG loss (DMMB assay), biosynthetic activity ([(35)SO(4)]-sulfate and [(3)H]-proline incorporation), gene expression (quantitative RT-PCR) and the abundance (zymography, Western blot) of matrix-degrading enzymes and specific aggrecan products were determined. Meniscal fibrocartilage had a 4-fold lower GAG content (per wet weight) than adjacent articular cartilage, and expressed MMPs-1, -2, -3 and ADAMTS4 constitutively, whereas ADAMTS5 m-RNA was essentially undetectable. Significant IL-1 effects were a decrease in biosynthetic activity, an increase in GAG release and in the expression/abundance of MMP-2, MMP-3 and ADAMTS4. Fresh tissue contained aggrecan core protein products similar to those previously described for bovine articular cartilage of this age. IL-1 induced the release of aggrecanase-generated CS-substituted products including both high (>250 kDa) and low molecular weight (about 75 kDa) species. TIMP-3 (but not TIMP-1 and -2 or a broad spectrum MMP inhibitor) inhibited IL-1-dependent GAG loss. In addition, IL-1 induced the release of preformed pools of three known G1-bearing products. We conclude that aggrecanases are responsible for IL-1-stimulated GAG release from meniscal explants, and that IL-1 also stimulates release of G1-bearing products, by a process possibly involving hyaluronan fragmentation.

Aggrecanolysis and in vitro matrix degradation in the immature bovine meniscus: mechanisms and functional implications.

INTRODUCTION: Little is known about endogenous or cytokine-stimulated aggrecan catabolism in the meniscal fibrocartilage of the knee. The objectives of this study were to characterize the structure, distribution, and processing of aggrecan in menisci from immature bovines, and to identify mechanisms of extracellular matrix degradation that lead to changes in the mechanical properties of meniscal fibrocartilage. METHODS: Aggrecanase activity in the native immature bovine meniscus was examined by immunolocalization of the aggrecan NITEGE neoepitope. To investigate mechanisms of cytokine-induced aggrecan catabolism in this tissue, explants were treated with interleukin-1alpha (IL-1) in the absence or presence of selective or broad spectrum metalloproteinase inhibitors. The sulfated glycosaminoglycan (sGAG) and collagen contents of explants and culture media were quantified by biochemical methods, and aggrecan catabolism was examined by Western analysis of aggrecan fragments. The mechanical properties of explants were determined by dynamic compression and shear tests. RESULTS: The aggrecanase-generated NITEGE neoepitope was preferentially localized in the middle and outer regions of freshly isolated immature bovine menisci, where sGAG density was lowest and blood vessels were present. In vitro treatment of explants with IL-1 triggered the accumulation of NITEGE in the inner and middle regions. Middle region explants stimulated with IL-1 exhibited substantial decreases in sGAG content, collagen content, and mechanical properties. A broad spectrum metalloproteinase inhibitor significantly reduced sGAG loss, abrogated collagen degradation, and preserved tissue mechanical properties. In contrast, an inhibitor selective for ADAMTS-4 and ADAMTS-5 was least effective at blocking IL-1-induced matrix catabolism and loss of mechanical properties. CONCLUSIONS: Aggrecanase-mediated aggrecanolysis, typical of degenerative articular cartilage, may play a physiologic role in the development of the immature bovine meniscus. IL-1-induced release of sGAG and loss of mechanical properties can be ascribed primarily to the activity of MMPs or aggrecanases other than ADAMTS-4 and ADAMTS-5. These results may have implications for the clinical management of osteoarthritis.

Cell death-associated ADAMTS4 and versican degradation in vascular tissue.

High blood flow through baboon polytetrafluorethylene aorto-iliac grafts increases neointimal vascular smooth muscle cell (SMC) death, neointimal atrophy, and cleavage of versican to generate the DPEAAE neoepitope, a marker of ADAMTS-mediated proteolysis. In this study, we have determined the effect of high blood flow on transcript abundance in the neointima for ADAMTS1, -4, -5, -8, -9, -15, and -20. We found that after 24 hr of flow, the mRNA for ADAMTS4 was significantly increased, whereas that for the other family members was unchanged. Because vascular SMC death is markedly increased in the graft after 24 hr of high flow, we next examined the possibility that the ADAMTS4 induction and the cell death are causally related. The addition of Fas ligand to SMC cultures increased both ADAMTS4 mRNA and cell death approximately 5-fold, consistent with the idea that ADAMTS4-dependent cleavage of versican may be partly responsible for cell death and tissue atrophy under these conditions.