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To fulfil international conservation commitments, governments have begun to recognise the need for more proactive marine planning policies, advocating sensitive engineering design that can deliver secondary benefits above and beyond the primary purpose of developments. In response, there is growing scientific interest in novel multi-functional coastal defence structures with built-in secondary ecological and/or socio-economic benefits. To ensure research efforts are invested effectively, it is first necessary to determine what secondary benefits can potentially be built-in to engineered coastal defence structures, and further, which of these benefits would be most desirable. It is unlikely that secondary benefits are perceived in the same way across different stakeholder groups. Further, their order of priority when evaluating different options is unlikely to be consistent, since each option will present a suite of compromises and trade-offs. The aim of this study was to investigate stakeholder attitudes towards multi-functional coastal defence developments across different sector groups. A preliminary questionnaire indicated unanimous support for implementing multi-functional structures in place of traditional single-purpose ones. This preliminary survey informed the design of a Delphi-like study, which revealed a more nuanced and caveated level of support from a panel of experts and practitioners. The study also elicited a degree of consensus that the most desirable secondary benefits that could be built-in to developments would be ecological ones – prioritised over social, economic and technical benefits. This paper synthesises these findings, discusses the perceived barriers that remain, and proposes a stepwise approach to effective implementation of multi-functional coastal defence developments.

Design, synthesis and structure–activity relationship of a series of biphenylsulfonamido-3-methylbutanoic acid based aggrecanase-1 inhibitors are described. In addition to robust aggrecanase-1 inhibition, these compounds also exhibit potent MMP-13 activity. In cell-based cartilage explants assay compound 48 produced 87% inhibition of proteoglycan degradation at 10 μg/mL. Good pharmacokinetic properties were demonstrated by 46 with a half-life of 6 h and bioavailability of 23%.

Summary The clinical implications of ventilatory alterations induced by left recurrent laryngeal neuropathy (LRLN) are reduced performance due to increased metabolic requirements for breathing and a reduction in peak aerobic power (VO2peak). To test this hypothesis, the metabolic cost of locomotion (MCL) and peak aerobic power (VO2peak) were measured in 4 non racing crossbred horses (low VO2peak) with reversibly induced LRLN using an incremental treadmill exercise test. Reversible LRLN was achieved with regional anaesthesia. These results were compared to previous measurements in 4 Thoroughbred racehorses (high VO2peak, Ehrlich et al. 1993). No significant difference in MCL (based on VO2 and venous lactate) was found in either group of horses at exercise intensities where VO2 < VO2peak (

Osteoarthritis (OA), the most common arthritic condition in humans, is characterized by the progressive degeneration of articular cartilage accompanied by chronic joint pain. Inflammatory mediators, such as cytokines and prostaglandin E 2 (PGE 2 ) that are elevated in OA joints, play important roles in the progression of cartilage degradation and pain-associated nociceptor sensitivity. We have found that the nuclear receptor family transcription factors L iver X R eceptors (LXRα and -β) are expressed in cartilage, with LXRβ being the predominant isoform. Here we show that genetic disruption of Lxrβ gene expression in mice results in significantly increased proteoglycan (aggrecan) degradation and PGE 2 production in articular cartilage treated with IL-1β, indicating a protective role of LXRβ in cartilage. Using human cartilage explants, we found that activation of LXRs by the synthetic ligand GW3965 significantly reduced cytokine-induced degradation and loss of aggrecan from the tissue. Furthermore, LXR activation dramatically inhibited cytokine-induced PGE 2 production by human osteoarthritic cartilage as well as by a synovial sarcoma cell line. These effects were achieved at least partly by repression of the expression of ADAMTS4, a physiological cartilage aggrecanase, and of cyclooxygenase-2 and microsomal prostaglandin E synthase-1, key enzymes in the PGE 2 synthesis pathway. Consistent with our in vitro observations, oral administration of GW3965 potently alleviated joint pain in a rat meniscal tear model of osteoarthritis.

Mass spectrometry-based proteomic analyses performed on cartilage tissue extracts identified the serine protease HtrA1/PRSS11 as a major protein component of human articular cartilage, with elevated levels occurring in association with osteoarthritis. Overexpression of a catalytically active form of HtrA1, but not an active site mutant (S328A), caused a marked reduction in proteoglycan content in chondrocyte-seeded alginate cultures. Aggrecan degradation fragments were detected in conditioned media from the alginate cultures overexpressing active HtrA1. Incubation of native or recombinant aggrecan with wild type HtrA1 resulted in distinct cleavage of these substrates. Cleavage of aggrecan by HtrA1 was strongly enhanced by HtrA1 agonists such as CPII, a C-terminal hexapeptide derived from the C-propeptide of procollagen IIα1 (i.e. chondrocalcin). A novel HtrA1-susceptible cleavage site within the interglobular domain (IGD) of aggrecan was identified, and an antibody that specifically recognizes the neoepitope sequence (VQTV356) generated at the HtrA1 cleavage site was developed. Western blot analysis demonstrated that HtrA1-generated aggrecan fragments containing the VQTV356 neoepitope were significantly more abundant in osteoarthritic cartilage compared with cartilage from healthy joints, implicating HtrA1 as a critical protease involved in proteoglycan turnover and cartilage degradation during degenerative joint disease.

Summary Objective Compare the expression and regulation of nuclear receptors (NRs) in osteoarthritic and normal human articular cartilage. Method The transcriptional levels of 48 NRs and additional related proteins were measured in mRNA from human articular cartilage from subjects with osteoarthritis (OA) and compared to samples from subjects without OA, using microarrays, individual quantitative reverse transcriptase polymerase chain reaction assays, and a custom human NR TaqMan ® Low Density Array (TLDA). The functional effect of liver X receptor (LXR) activity in cartilage was studied by measuring proteoglycan (PG) synthesis and degradation in articular cartilage explant cultures following treatment with the synthetic LXR agonist T0901317. Results Thirty-one of 48 NRs analyzed by TLDA were found to be measurably expressed in human articular cartilage; 23 of these 31 NRs showed significantly altered expression in OA vs unaffected cartilage. Among these, LXRα and LXRβ, and their heterodimeric partners retinoid X receptor (RXR)α and RXRβ were all expressed at significantly lower levels in OA cartilage, as were LXR target genes ABCG1 and apolipoproteins D and E. Addition of LXR agonist to human OA articular chondrocytes and to cartilage explant cultures resulted in activation of LXR-mediated transcription and significant reduction of both basal and interleukin (IL)-1-mediated PG degradation. Conclusions Articular cartilage expresses a substantial number of NRs, and a large proportion of the expressed NRs are dysregulated in OA. In particular, LXR signaling in OA articular cartilage is impaired, and stimulation of LXR transcriptional activity can counteract the catabolic effects of IL-1. We conclude that LXR agonism may be a possible therapeutic option for OA.

The prevention of aggrecan (a key component of cartilage) cleavage via the inhibition of aggrecanase-1 may provide a unique opportunity to stop the progression of cartilage degradation in osteoarthritis. The evaluation of a series of biphenylsulfonamides resulted in the identification of the ((4-keto)-phenoxy)methyl biphenyl-4-sulfonamides analogs (19-21 and 24) with improved Agg-1 inhibition and MMP-2, MMP-13 activity.

Objective Lubricin, also referred to as superficial zone protein and PRG4, is a synovial glycoprotein that supplies a friction-resistant, antiadhesive coating to the surfaces of articular cartilage, thereby protecting against arthritis-associated tissue wear and degradation. This study was undertaken to generate and characterize a novel recombinant lubricin protein construct, LUB:1, and to evaluate its therapeutic efficacy following intraarticular delivery in a rat model of osteoarthritis (OA). Methods Binding and localization of LUB:1 to cartilage surfaces was assessed by immunohistochemistry. The cartilage-lubricating properties of LUB:1 were determined using a custom friction testing apparatus. A cell-binding assay was performed to quantify the ability of LUB:1 to prevent cell adhesion. Efficacy studies were conducted in a rat meniscal tear model of OA. One week after the surgical induction of OA, LUB:1 or phosphate buffered saline vehicle was administered by intraarticular injection for 4 weeks, with dosing intervals of either once per week or 3 times per week. OA pathology scores were determined by histologic analysis. Results LUB:1 was shown to bind effectively to cartilage surfaces, and facilitated both cartilage boundary lubrication and inhibition of synovial cell adhesion. Treatment of rat knee joints with LUB:1 resulted in significant disease-modifying, chondroprotective effects during the progression of OA, by markedly reducing cartilage degeneration and structural damage. Conclusion Our findings demonstrate the potential use of recombinant lubricin molecules in novel biotherapeutic approaches to the treatment of OA and associated cartilage abnormalities.

In the treatment of osteoarthritis (OA), some of the therapeutic approaches require delivery of drug(s) to the diseased cartilage. Presence of adequate drug levels in the cartilage is one of the important criteria in selection and ranking of lead compounds. The purpose of this study was to investigate the correlation in cartilage compound levels between in vitro experiments and in vivo animal studies. Bovine cartilage samples were incubated with test compounds of various concentrations in a culture medium, in the absence or presence of 25 mg/ml of serum albumin which served as an artificial synovial fluid (SF). The test compounds were also dosed to rabbits, the animal model used for efficacy studies, over a six-week treatment period. Test article concentrations in plasma, SF, and cartilage were determined by LC/MS/MS analysis. A correlation in cartilage drug concentration was observed between in vitro and in vivo studies. Plasma protein binding and the test article’s affinity to cartilage were the major determining factors for drug delivery to cartilage in vivo.

Objective Protein kinase Czeta (PKCzeta), an atypical PKC, has been found to be transcriptionally up-regulated in human osteoarthritic (OA) articular cartilage. This study was undertaken to examine the role of PKCzeta in interleukin-1beta (IL-1beta)-induced NF-kappaB signaling in human OA chondrocytes, and ultimately to better understand its function in the regulation of downstream mediators of cartilage matrix degradation. Methods Pharmacologic inhibitors or genetic knockdown techniques were used to investigate the role of PKCzeta. Western blot analysis was used to evaluate phosphorylation of PKCzeta and NF-kappaB. Quantitative polymerase chain reaction (PCR) and activity assays were used to evaluate ADAMTS-4 expression and aggrecanase activity, respectively. Quantitative PCR, biochemical identification, and Western blot analysis were used to evaluate type 2 nitric oxide synthase (NOS2) and NO production. Results Phosphorylation of PKCzeta and NF-kappaB was induced by IL-1beta treatment in a time-dependent manner, and was specifically inhibited by inhibitors of atypical PKCs. Inhibition of PKCzeta suppressed IL-1beta-induced up-regulation of ADAMTS-4 messenger RNA (mRNA) and aggrecanase activity. Inhibitors of atypical PKCs also inhibited IL-1beta-induced NO production and NOS2 mRNA expression, demonstrating a novel link between PKCzeta and NO production. Furthermore, small interfering RNA- or short hairpin RNA-mediated knockdown of PKCzeta mRNA resulted in significant repression of both ADAMTS-4 and NOS2 mRNA expression. Conclusion Our results show that PKCzeta is involved in the regulation of IL-1beta-induced NF-kappaB signaling in human OA chondrocytes, which in turn regulates downstream expression of ADAMTS-4 and NOS2. Therefore, inhibition of PKCzeta could potentially regulate the production of matrix-degrading enzymes as well as NO production and have a profound effect on disease progression in OA.

5'-Phenyl-3'H-spiro[indoline-3,2'-[1,3,4]thiadiazol]-2-one inhibitors of ADAMTS-5 (Aggrecanase-2) have been prepared via commercially available starting materials. Selected compounds 23, 33-35 show sub-micromolar ADAMTS-5 potency and strong SAR trends with selectivity over the related metalloproteases ADAMTS-4 (Aggrecanase-1), MMP12, and MMP13. This series of compounds represents progress toward a selective ADAMTS-5 inhibitor as a disease modifying osteoarthritis drug.

Summary Objective To evaluate anterior cruciate ligament transection (ACLT) and destabilization of the medial meniscus (DMM) surgical instability models of osteoarthritis (OA) in the 129/SvEv mouse knee joint. Design Micro-surgical techniques were used to perform ACLT or DMM under direct visualization. Histological scoring was performed on multiple sections to assess cartilage damage across the entire joint. Results The ACLT model gave severe OA, chondrogenesis of the joint capsule and, in some cases, severe subchondral erosion of the posterior tibial plateau. Surgical DMM was less invasive than the ACLT procedure and resulted in lesions primarily on the central weight-bearing region of the medial tibial plateau and medial femoral condyles. Lesions in the DMM model progressed from mild-to-moderate OA at 4 weeks, to moderate-to-severe OA at 8 weeks post-surgery. Destruction of the subchondral bone was never observed in the DMM model. Conclusions ACLT is not recommended in the mouse due to the high surgical proficiency required and the development of severe OA that may involve subchondral bone erosion. The severity and location of lesions following DMM are consistent with lesions observed in aged spontaneous mouse models of OA. The DMM model has sufficient sensitivity to show disease modification, as observed with the ADAMTS-5 knock out (KO) mouse. The DMM model should be a first choice to challenge mice with gene deletions of potential targets in OA.

Lubricin is a secreted, cytoprotective glycoprotein that contributes to the essential boundary lubrication mechanisms necessary for maintaining low friction levels at articular cartilage surfaces. Diminishment of lubricin function is thereby implicated as an adverse contributing factor in degenerative joint diseases such as osteoarthritis. Lubricin occurs as a soluble component of synovial fluid, and is synthesized and localized in the superficial layer of articular cartilage (and thus has also been described as "superficial zone protein", or SZP); however, defined interactions responsible for lubricin retention at this site are not well characterized. In the current studies, we identified molecular determinants that enable lubricin to effectively bind to articular cartilage surfaces. Efficient and specific binding to the superficial zone was observed for synovial lubricin, as well as for recombinant full-length lubricin and a protein construct comprising the lubricin C-terminal (hemopexin-like) domain (LUB-C, encoded by exons 7-12). A construct representing the N-terminal region of lubricin (LUB-N, encoded by exons 2-5) exhibited no appreciable cartilage-binding ability, but displayed the capacity to dimerize, and thus potentially influence lubricin aggregation. Disulfide bond disruption significantly attenuated recombinant lubricin and LUB-C binding to cartilage surfaces, demonstrating a requirement for protein secondary structure in facilitating the appropriate localization of lubricin at relevant tissue interfaces. These findings help identify additional key attributes contributing to lubricin functionality, which would be expected to be instrumental in maintaining joint homeostasis.

Objective To identify potential molecular mediators and biomarkers for osteoarthritis (OA), through comparative proteomic analysis of articular cartilage tissue obtained from normal donors without OA (n = 7) and patients with OA (n = 7). Methods The proteomic analyses comprised extraction of soluble proteins from cartilage, separation of the protein mixtures by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by in-gel digestion, and subsequent nano-liquid chromatography–tandem mass spectrometry analysis in conjunction with a database search for protein identification and semiquantitation. Results A total of 814 distinct proteins were identified with high confidence from 14 samples; 420 of these proteins were detected with ≥3 unique peptides in at least 4 samples from the same group. Using stringent criteria, 59 proteins were found to be differentially expressed in OA cartilage. Gene Ontology and Ingenuity pathway analysis tools were used to characterize these proteins into functional categories. One of the up-regulated proteins, HtrA1, a serine protease, was detected at high levels in cartilage. Conclusion Altered protein expression in the disease state is associated with many aspects of the pathogenesis of OA, such as increased proteolysis, lipid metabolism, immune response, and decreased signal transduction. To our knowledge, this is the first time that a large portion of these proteins and their expression patterns were identified in cartilage, thus providing new insights for finding novel pathologic mediators and biomarkers of OA.

Objective To phenotypically characterize ADAMTS-4– and ADAMTS-5–double-knockout mice, and to determine the effect of deletion of ADAMTS-4 and ADAMTS-5 on the progression of osteoarthritis (OA) in mice. Methods Mice lacking the catalytic domain of ADAMTS-4 and ADAMTS-5 were crossed to generate ADAMTS-4/5–double-knockout animals. Twelve-week-old and 1-year-old male and female ADAMTS-4/5–double-knockout mice were compared with age- and sex-matched wild-type (WT) mice by evaluating terminal body weights, organ weights, clinical pathology parameters, PIXImus mouse densitometry findings, and macroscopic and microscopic observations. ADAMTS-4/5–double-knockout mice were challenged by surgical induction of joint instability to determine the importance of these genes in the progression of OA. Articular and nonarticular cartilage explants from WT and ADAMTS-4/5–double-knockout mice were treated with interleukin-1 (IL-1) plus retinoic acid ex vivo, to examine proteoglycan degradation. Results There were no genotype-related phenotype differences between ADAMTS-4/5–double-knockout and WT mice through 1 year of age, with the exception that female ADAMTS-4/5–double-knockout mice had a lower mean terminal body weight at the 12-week time point. Eight weeks after surgical induction of joint instability, OA was significantly less severe in ADAMTS-4/5–double-knockout mice compared with WT mice. Following stimulation of cartilage explants with IL-1 plus retinoic acid, aggrecanase-mediated degradation in ADAMTS-4/5–double-knockout mice was ablated, to a level comparable with that in ADAMTS-5–knockout mice. Conclusion Dual deletion of ADAMTS-4 and ADAMTS-5 generated mice that were phenotypically indistinguishable from WT mice. Deletion of ADAMTS-4/5 provided significant protection against proteoglycan degradation ex vivo and decreased the severity of murine OA. These effects in the ADAMTS-4/5–double-knockout mice were comparable with those observed with deletion of ADAMTS-5 alone.

Aggrecanases are ADAMTS (a disintegrin and metalloproteinase with thrombospondin type I motifs) proteases capable of primary (patho)physiological cleavage at specific Glu-Xaa bonds within the core protein of the hyaluronan-binding proteoglycan aggrecan. Accumulating evidence suggests that regulation of the activity of one such aggrecanase, ADAMTS-4 (or Aggrecanase-1), involves post-translational C-terminal processing (truncation) which modulates both glycosaminoglycan (GAG)-binding affinity and enzymatic activity. In the present study, we compared the effects of C-terminal truncation on the GAG-binding properties and aggrecanase activity of ADAMTS-5 (Aggrecanase-2) relative to three other ADAMTS family members, ADAMTS-9, ADAMTS-16 and ADAMTS-18. Full-length recombinant human ADAMTS-5 (M(r) approximately 85 kDa; ADAMTS-5p85) underwent autolytic cleavage during expression by CHO/A2 cells, and co-purified with C-terminally truncated (tr) isoforms of M(r) approximately 60 kDa (ADAMTS-5p60 and M(r) approximately 45 kDa (ADAMTS-5p45). All three ADAMTS-5 isoforms bound to sulfated GAGs (heparin and chondroitin sulfate (CS)). An ADAMTS-5p45 structural mimetic, terminating at Phe628 and comprising the catalytic domain, disintegrin-like domain and thrombospondin type I repeat (TSR)-1 domain (designated trADAMTS-5F628), also bound to heparin, and exhibited potent aggrecanase activity toward cleavage sites both in the aggrecan CS-2-attachment region (at Glu1771-Ala1772) and in the interglobular domain (at Glu373-Ala374). Further truncation (deletion of the TSR-1 domain) of ADAMTS-5 significantly reduced aggrecanase activity, although appreciable GAG (heparin)-binding affinity was maintained. Other TSR-1 domain-bearing truncated ADAMTS constructs demonstrating either positive GAG-binding ability (trADAMTS-9F649) or negligible GAG-affinity (trADAMTS-16F647 and trADAMTS-18F650) displayed comparably low aggrecanase activities. Thus, the presence of TSR-1 on truncated ADAMTSs appears to be necessary, but not sufficient, for effective aggrecanase-mediated catalysis of target Glu-Xaa bonds. Similarly, GAG-binding ability, irrespective of the presence of a TSR-1 domain, does not necessarily empower truncated ADAMTSs with proficient aggrecanase activity.

Chondrocytes are unique to cartilage and the study of these cells in vitro is important for advancing our understanding of the role of these cells in normal homeostasis and disease including osteoarthritis (OA). As there are limitations to the culture of primary chondrocytes, cell lines have been developed to overcome some of these obstacles. In this study, we developed a procedure to immortalize and characterize chondrocyte cell lines from mouse xiphisternum. The cells displayed a polygonal to fibroblastic morphology in monolayer culture. Gene expression studies using quantitative PCR showed that the cell lines responded to bone morphogenetic protein 2 (BMP-2) by increased expression of matrix molecules, aggrecan, and type II collagen together with transcriptional factor, Sox9. Stimulation by IL-1 results in the increased expression of catabolic effectors including MMP-13, nitric oxide synthase, ADAMTS4, and ADAMTS5. Cells cultured in alginate responded to BMP-2 by increased synthesis of proteoglycan (PG), a major matrix molecule of cartilage. IL-1 treatment of cells in alginate results in increased release of PG into the conditioned media. Further analysis of the media showed the presence of Aggrecanase-cleaved aggrecan fragments, a signature of matrix degradation. These results show that the xiphisternum chondrocyte cell lines preserve their chondrocyte phenotype cultured in either monolayer or 3-dimensional alginate bead culture systems. In summary, this study describes the establishment of chondrocyte cell lines from the mouse xiphisternum that may be useful as a surrogate model system to understand chondrocyte biology and to shed light on the underlying mechanism of pathogenesis in OA.

Aggrecanases are recently discovered enzymes that cleave aggrecan, a key component of cartilage. Aggrecanase inhibitors may provide a unique means to halt the progression of cartilage destruction in osteoarthritis. The synthesis and evaluation of biphenylsulfonamidocarboxylic acid inhibitors of aggrecanase-1 are reported. Compound 24 demonstrated 89% inhibition of proteoglycan degradation at 10 μg/mL and has an oral bioavailability in rat of 35%.

The structure-based design and synthesis of a series of novel biphenyl sulfonamide carboxylic acids as potent MMP-13 inhibitors with selectivity over MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-14, Aggrecanase 1, and TACE are described.

Members of the bone morphogenetic protein (BMP) group of the TGF-beta superfamily have been shown to enhance matrix synthesis and maintain cartilage phenotype in long-term culture. These proteins have also been shown to augment cartilage repair in vivo, and may be of potential therapeutic benefit in the treatment of damaged articular cartilage. The present study was undertaken to examine the effects of BMP-9 on the metabolism of juvenile and adult bovine cartilage in vitro, and to compare the effects to those produced by two previously characterized BMPs: BMP-2 and 13 (CDMP-2). BMP-9 lead to a 7-8-fold stimulation of proteoglycan synthesis at the highest concentration tested, and a 6.4-fold stimulation of collagen synthesis at a concentration of 50 ng/mL in juvenile cartilage. BMP-2 also lead to a 7-8-fold increase in proteoglycan synthesis at the highest concentration tested, and was able to induce collagen synthesis 6.4-fold, but at a concentration of 1000 ng/mL. Proteoglycans isolated from BMP-9 treated cartilage exhibited an increased hydrodynamic size possibly due to increased glycosaminoglycan substitution or decreased C-terminal proteolysis. Consistent with the idea of limited C-terminal proteolysis, BMP-9 treatment lead to a significant reduction in the turnover rate of proteoglycans in juvenile explants. Interestingly, all three BMPs were unable to induce a measurable anabolic response in adult cartilage explants.

Multipotential bone marrow stromal cells have the ability to differentiate along multiple connective tissue lineages including cartilage. In this study, we developed an efficient and reproducible procedure for the isolation of stromal cells from bone marrow aspirates of normal human donors based on the expression of endoglin, a type III receptor of the transforming growth factor-beta (TGF-β) receptor family. We demonstrate that these cells have the ability of multiple lineage differentiation. Stromal cells represented 2–3% of the total mononuclear cells of the marrow. The cells displayed a fibroblastic colony formation in monolayer culture and maintained similar morphology with passage. Expression of cell surface molecules by flow cytometry displayed a stable phenotype with culture expansion. When cocultured with hematopoietic CD34+ progenitor cells, stromal cells were able to maintain their ability to support hematopoiesis in vitro. Culture expanded stromal cells were placed in a 3-dimensional matrix of alginate beads and cultured in serum-free media in the presence of TGFβ-3 for chondrogenic lineage progression. Increased expression of type II collagen messenger RNA was observed in the TGFβ3 treated cultures. Immunohistochemistry performed on sections of alginate beads detected the presence of type II collagen protein. This isolation procedure for stromal cells and the establishment of the alginate culture system for chondrogenic progression will contribute to the understanding of chondrogenesis and cartilage repair. J. Cell. Physiol. 185:98–106, 2000. © 2000 Wiley-Liss, Inc.

OBJECTIVE To determine the effects of dimethylsulfoxide (DMSO) exposure on cartilage proteoglycan (PG) synthesis, PG degradation, chondrocyte viability, and matrix water content. STUDY DESIGN Using a cartilage explant culture system, PG synthesis, PG degradation, matrix water content, and chondrocyte viability were determined for cartilage exposed to DMSO daily for selected periods of time. ANIMALS OR SAMPLE POPULATION Juvenile bovine (calf) carpometacarpal joint cartilage explants. METHODS PG synthesis: Explants (n = 30/group) were separated into 10 groups based on the time of daily exposure to 10% DMSO. Exposure time was repeated daily for 3 days. The control group was incubated in basal medium alone for 3 days, with daily medium changes. Once all DMSO exposure times were complete for the third day, PG synthesis was determined by analysis of incorporation of radiolabelled sulfate. Cell viability: Explants (n = 3/group) were subjected to an identical DMSO exposure protocol, and examined histologically. The percentage of viable cells/high power field (hpf) was calculated for each group. PG degradation: Explants (n = 21/group) were preincubated with radiolabelled sulfate, then subjected to a similar DMSO exposure protocol. The medium was collected from all explants daily and assayed for PG content. After 3 days, the explants were digested and total labelled PG content determined. Percent of total explant labelled PG content released into the medium daily was determined for each group. Water content: Explants (n = 21/group) were separated into three treatment groups, one of which had no treatments performed, whereas the other two groups were incubated in basal medium for 72 hours, one with, and one without, 10% DMSO. Wet and dry weights were determined, and percent water calculated, for all three groups. Separate 1-way ANOVA were performed, with appropriate post hoc tests (P < .05). RESULTS PG synthesis was significantly lower than control for all time periods of DMSO exposure except for 1 and 3 hours, and decreased in a time-dependent manner after the 1-hour exposure time. The mean percentage of viable cells/hpf was significantly lower than control for the 1-, 3-, 9-, 12-, and 24-hour treatment groups. There was no significant difference in PG degradation for any group compared with control for the first 2 days of incubation. All groups except the 24-hour group had a significantly higher degradation compared with control for the third day of incubation. Cartilage exposed to DMSO for 72 hours had a significantly lower water content, and cartilage incubated in basal medium alone for 72 hours had a significantly higher water content than cartilage that received no DMSO and no incubation. CONCLUSIONS DMSO, in relatively low concentration, is detrimental to articular cartilage PG synthesis in a time-dependent manner. Dehydration of the cartilage and chondrocyte death also occur with increasing time of DMSO exposure. Significant PG degradation occurs on the third day of culture with daily DMSO exposure. CLINICAL RELEVANCE As a joint lavage solution, DMSO has potentially deleterious effects on the metabolism of chondrocytes.

Articular cartilage has a limited capacity for repair. We investigated the effect of rhBMP-2 (recombinant human bone morphogenetic protein-2) on the healing of full-thickness osteochondral defects in adult New Zealand White rabbits. A single defect, three millimeters wide by three millimeters deep, was created in the trochlear groove of the right femur in eighty-nine rabbits. The defect was either left empty, filled with a plain collagen sponge, or filled with a collagen sponge impregnated with five micrograms of rhBMP-2. The animals were killed at four, eight, or twenty-four weeks, and the repair tissue was examined histologically and evaluated with use of a grading scale. The defects also were examined immunohistochemically for the presence of type-II collagen at four and eight weeks. The rate of bone repair was evaluated with fluorescent labeling of bone at two and four weeks and with use of fluorescence microscopy at eight weeks. Treatment with rhBMP-2 greatly accelerated the formation of new subchondral bone and improved the histological appearance of the overlying articular surface. At twenty-four weeks, the thickness of the repair cartilage was 70 per cent that of the normal adjacent cartilage and a new tidemark usually had formed between the repair cartilage and the underlying subchondral bone. The average total scores on the histological grading scale were significantly better (p < 0.01) for the defects treated with rhBMP-2 than for the untreated defects (those left empty or filled with a plain collagen sponge) at all time-points. Immunostaining with an antibody against type-II collagen showed the diffuse presence of this cartilage-specific collagen throughout the repair cartilage in the treated defects. The untreated defects demonstrated minimum staining with this antibody. CLINICAL RELEVANCE: The operative removal of cartilage damaged as a result of trauma or focal osteoarthrosis is of little value because of the limited capability of articular cartilage to repair. If the damaged cartilage were to be removed and the tissue were induced to heal, the self-perpetuating process of osteoarthrosis might be prevented. We describe the capacity of rhBMP-2 to accelerate the healing of full-thickness defects of articular cartilage and to improve the histological appearance and biochemical characteristics of the repair cartilage. These improvements were evident as long as twenty-four weeks postoperatively in adult rabbits. Because of the technical simplicity of delivering a recombinant protein growth factor compared with transplanting cells, and because of the improvement in healing afforded by rhBMP-2, the use of this growth factor and related proteins to influence the healing of defects of articular cartilage should be investigated further.

Coastal defences are proliferating in response to anticipated climate change and there is increasing need for ecologically sensitive design in their construction. Typically, these structures support lower biodiversity than natural rocky shores. Although several studies have tested habitat enhancement interventions that incorporate novel water-retaining features into coastal defences, there remains a need for additional long-term, fully replicated trials to identify alternative cost-effective designs. We created artificial rock pools of two depths (12cm, 5cm) by drill-coring into a shore-parallel intertidal granite breakwater, to investigate their potential as an intervention for delivering ecological enhancement. After 18 months the artificial rock pools supported greater species richness than adjacent granite rock surfaces on the breakwater, and similar species richness to natural rock pools on nearby rocky shores. Community composition was, however, different between artificial and natural pools. The depth of artificial rock pools did not affect richness or community structure. Although the novel habitats did not support the same communities as natural rock pools, they clearly provided important habitat for several species that were otherwise absent at mid-shore height on the breakwater. These findings reveal the potential of drill-cored rock pools as an affordable and easily replicated means of enhancing biodiversity on a variety of coastal defence structures, both at the design stage and retrospectively.

Summary Objective To evaluate aggrecanase activity after traumatic knee injury in a rat model by measuring the level of aggrecanase-generated Ala-Arg-Gly-aggrecan (ARG-aggrecan) fragments in synovial fluid, and compare with ARG-aggrecan release into joint fluid following human knee injury. To evaluate the effect of small molecule inhibitors on induced aggrecanase activity in the rat model. Method An enzyme-linked immunosorbent assay (ELISA) was developed to measure ARG-aggrecan levels in animal and human joint fluids. A rat model of meniscal tear (MT)-induced joint instability was used to assess ARG-aggrecan release into joint fluid and the effects of aggrecanase inhibition. Synovial fluids were also obtained from patients with acute joint injury or osteoarthritis and assayed for ARG-aggrecan. Results Joint fluids from human patients after knee injury showed significantly enhanced levels of ARG-aggrecan compared to uninjured reference subjects. Similarly, synovial fluid ARG-aggrecan levels increased following surgically-induced joint instability in the rat MT model, which was significantly attenuated by orally dosing the animals with AGG-523, an aggrecanase specific inhibitor. Conclusions Aggrecanase-generated aggrecan fragments were rapidly released into human and rat joint fluids after injury to the knee and remained elevated over a prolonged period. Our findings in human and preclinical models strengthen the connection between aggrecanase activity in joints and knee injury and disease. The ability of a small molecule aggrecanase inhibitor to reduce the release of aggrecanase-generated aggrecan fragments into rat joints suggests that pharmacologic inhibition of aggrecanase activity in humans may be an effective treatment for slowing cartilage degradation following joint injury.

Potent 3,4-disubstituted benzofuran P1' MMP-13 inhibitors have been prepared. Selectivity over MMP-2 was achieved through a substituent at the C4 position of the benzofuran P1' moiety of the molecule. By replacing a backbone benzene with a pyridine and valine with threonine, compounds (e.g., 44) with greatly reduced plasma protein binding were also obtained.

N-((8-Hydroxy-5-substituted-quinolin-7-yl)(phenyl)methyl)-2-phenyloxy/amino-acetamide inhibitors of ADAMTS-5 (Aggrecanase-2) have been prepared. Selected compounds 10, 14, 25, and 53 show sub-μM ADAMTS-5 potency and good selectivity over the related metalloproteases ADAMTS-4 (Aggrecanase-1), MMP-13, and MMP-12. Compound 53 shows a good balance of potent ADAMTS-5 inhibition, moderate CYP3A4 inhibition and good rat liver microsome stability. This series of compounds represents progress towards selective ADAMTS-5 inhibitors as disease modifying osteoarthritis agents.

Insulin-like growth factor-I (IGF-I) is thought to play an important role in skeletal growth and development through its mitogenic and anabolic effects on epiphyseal growth plate chondrocytes. The bone morphogenetic proteins (BMPs) have been shown to promote endochondral osteogenesis, and some members of the BMP family, including BMP-2 and BMP-9, have anabolic effects on chondrocyte metabolism. We tested the hypothesis that BMP-2 and BMP-9 interact with IGF-I to modulate growth plate chondrocyte mitotic activity. IGF-I, but neither BMP-2 nor BMP-9, stimulated chondrocyte DNA synthesis. However, both BMP-2 and BMP-9 augmented the mitogenic action of IGF-I. BMP-2, but not BMP-9 increased IGF-I binding to growth plate chondrocytes in kinetic studies. In affinity labeling studies, 125I-IGF-I predominantly labeled an Mr approximately 135-kDa moiety, consistent with the alpha subunit of the type 1 IGF receptor and an Mr approximately 250-kDa moiety consistent with the type 2 IGF receptor. 125I-IGF-I labeling also appeared at Mr approximately 43 kDa, consistent with 125I-IGF-I binding to insulin-like growth binding protein-3. Treatment of chondrocytes with BMP-2, but not with BMP-9, increased the intensity of the Mr approximately 135-kDa band and decreased the intensity of the Mr approximately 43-kDa band. Taken together, these data suggest that the BMPs may modulate the action of IGF-I via the type 1 IGF receptor and/or IGF binding proteins.

Protein kinase Czeta (PKCzeta) is an intracellular serine/threonine protein kinase that has been implicated in the signaling pathways for certain inflammatory cytokines, including interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha), in some cell types. A study of gene expression in articular chondrocytes from osteoarthritis (OA) patients revealed that PKCzeta is transcriptionally up-regulated in human OA articular cartilage clinical samples. This finding led to the hypothesis that PKCzeta may be an important signaling component of cytokine-mediated cartilage matrix destruction in articular chondrocytes, believed to be an underlying factor in the pathophysiology of OA. IL-1 treatment of chondrocytes in culture resulted in rapidly increased phosphorylation of PKCzeta, implicating PKCzeta activation in the signaling pathway. Chondrocyte cell-based assays were used to evaluate the contribution of PKCzeta activity in NF-kappaB activation and extracellular matrix degradation mediated by IL-1, TNF, or sphingomyelinase. In primary chondrocytes, IL-1 and TNF-alpha caused an increase in NF-kappaB activity resulting in induction of aggrecanase-1 and aggrecanase-2 expression, with consequent increased proteoglycan degradation. This effect was blocked by the pan-specific PKC inhibitors RO 31-8220 and bisindolylmaleimide I, partially blocked by Go 6976, and was unaffected by the PKCzeta-sparing inhibitor calphostin C. A cell-permeable PKCzeta pseudosubstrate peptide inhibitor was capable of blocking TNFand IL-1-mediated NF-kappaB activation and proteoglycan degradation in chondrocyte pellet cultures. In addition, overexpression of a dominant negative PKCzeta protein effectively prevented cytokine-mediated NF-kappaB activation in primary chondrocytes. These data implicate PKCzeta as a necessary component of the IL-1 and TNF signaling pathways in chondrocytes that result in catabolic destruction of extracellular matrix proteins in osteoarthritic cartilage.

Human osteoarthritis is a progressive disease of the joints characterized by degradation of articular cartilage. Although disease initiation may be multifactorial, the cartilage destruction appears to be a result of uncontrolled proteolytic extracellular matrix destruction. A major component of the cartilage extracellular matrix is aggrecan, a proteoglycan that imparts compressive resistance to the tissue. Aggrecan is cleaved at a specific 'aggrecanase' site in human osteoarthritic cartilage; this cleavage can be performed by several members of ADAMTS family of metalloproteases. The relative contribution of individual ADAMTS proteases to cartilage destruction during osteoarthritis has not been resolved. Here we describe experiments with a genetically modified mouse in which the catalytic domain of ADAMTS5 (aggrecanase-2) was deleted. After surgically induced joint instability, there was significant reduction in the severity of cartilage destruction in the ADAMTS5 knockout mice compared with wild-type mice. This is the first report of a single gene deletion capable of abrogating the course of cartilage destruction in an animal model of osteoarthritis. These results demonstrate that ADAMTS5 is the primary 'aggrecanase' responsible for aggrecan degradation in a murine model of osteoarthritis, and suggest rational strategies for therapeutic intervention in osteoarthritis.

Objective To determine whether aggrecanase (ADAMTS) activities in articular cartilage can directly lead to the release of hyaluronan (HA) and hyaladherins (aggrecan G1 domain and link proteins), as may occur ex vivo during stimulation of cartilage explants with interleukin-1 (IL-1) or retinoic acid or in vivo in synovial joints during aging and joint pathology. Methods Bovine articular cartilage discs (live or freeze-killed) were cultured in the presence of IL-1 or were incubated in digestion buffer containing recombinant human ADAMTS-4 (rHuADAMTS-4; aggrecanase 1) or rHuADAMTS-5 (aggrecanase 2). Culture media, digestion supernatants, and tissue extracts were assayed for sulfated glycosaminoglycan (sGAG) content and analyzed by Western blotting to detect aggrecanase-generated G1 domain (using neoepitope monoclonal antibody AGG-C1/anti-NITEGE373) and link proteins (using monoclonal antibody 8-A-4), as well as by quantitative enzyme-linked immunosorbent assays to detect aggrecanase-generated G1 domain (G1-NITEGE373) and HA. Results IL-1 treatment of live cartilage explants induced a time-dependent release of sGAG, aggrecanase-generated G1 domain (G1-NITEGE373), and HA into the culture media. Exposure of live or freeze-killed articular cartilage discs to rHuADAMTS-4 or rHuADAMTS-5 resulted in a dose- and time-dependent release of sGAG and hyaluronan from the tissue, accompanied by a concomitant release of functionally intact hyaladherins (aggrecan G1-NITEGE373 and link proteins). Conclusion Coincident with aggrecanolysis, aggrecanase activities in articular cartilage may actuate the release of HA and associated hyaladherins, thereby further compromising the integrity of the cartilage matrix during degenerative joint diseases such as osteoarthritis.

Objective To determine the importance of the enzymatic activity of ADAMTS-4 in normal growth and development and to evaluate the role of ADAMTS-4 in the progression of osteoarthritis (OA). Methods We generated catalytic domain–deleted ADAMTS-4–transgenic mice and performed extensive gross and histologic analyses of various organs. The mice were challenged by surgical induction of joint instability leading to OA, to determine the importance of the enzymatic activity of ADAMTS-4 in the progression of the disease. The response of wild-type (WT) and ADAMTS-4–knockout (ADAMTS-4–KO) articular cartilage to interleukin-1 and retinoic acid challenge in vitro was also evaluated. Results ADAMTS-4–KO mice up to 1 year of age exhibited no gross or histologic abnormalities in 36 tissue sites examined. Despite evidence of ADAMTS-4 expression and activity in growth plates of WT mice, catalytic silencing of this proteinase caused no abnormalities in skeletal development, growth, or remodeling. There was no effect of ADAMTS-4 knockout on the progression or severity of OA 4 weeks or 8 weeks after surgical induction of joint instability. Enzymatic cleavage of aggrecan at the TEGE373–374ARGS site was clearly evident after exposure of articular cartilage from ADAMTS-4–KO mice to inflammatory cytokines. Conclusion Although expression of the ADAMTS-4 gene has been found in many tissues throughout the body, deletion of enzymatic activity did not appear to have any effect on normal growth and physiology. Our study provides evidence that ADAMTS-4 is the primary aggrecanase in murine growth plates; however, deletion of its enzymatic activity did not affect normal long bone remodeling. Our results also lead to the hypothesis that, in the mouse, ADAMTS-4 is not the primary enzyme responsible for aggrecan degradation at the TEGE373–374ARGS site. The elucidation of the relative importance of ADAMTS-4 in the pathologic process of human OA will require examination of human OA tissues and evidence of disease modification in patients following therapeutic intervention.

Introduction Articular cartilage matrix synthesis and degradation are dynamic processes that must be balanced for proper maintenance of the tissue. In osteoarthritis (OA), this balance is skewed toward degradation and ultimate loss of matrix. The transcriptional and/or activity levels of hundreds of genes are dysregulated in chondrocytes from osteoarthritic cartilage, and a subset of these genes may represent pivotal factors that could be modulated if their specific role in the disease process could be identified. Objective To investigate the role of ADAMTS-4 and ADAMTS-5 in cartilage matrix degradation by developing a chondrocyte pellet culture assay in combination with adenoviral gene expression, and to demonstrate the utility of this assay by assessing the specific functional contribution of these genes to cartilage matrix metabolism. Methods A full-length cDNA for bovine ADAMTS-4 (bADAMTS-4) was isolated, and used to evaluate the expression, regulation, and activity of this gene in bovine cartilage. Adenoviruses expressing bADAMTS-4, human ADAMTS-5 (hADAMTS-5) or human bone morphogenetic protein 2 (BMP-2) were used to infect primary chondrocytes, and their effect on extracellular matrix metabolism was assessed by monitoring the accumulation and release of glycosaminoglycans (GAG) in three-dimensional chondrocyte pellet cultures. Results Analysis of bADAMTS-4 transcriptional regulation in chondrocytes revealed that interleukin-1α (IL-1α) was the most potent inducer of bADAMTS-4 mRNA and subsequent aggrecan degradation in cartilage explant cultures of those cytokines tested. bADAMTS-4 mRNA induction by IL-1α was greater in nasal cartilage than in articular cartilage. Chondrocytes infected with adenovirus expressing either bADAMTS-4 or hADAMTS-5 genes showed increased aggrecan degradation in newly synthesized matrix by pellet cultures while chondrocytes overexpressing BMP-2 showed increased aggrecan synthesis. Conclusion Adenoviral delivery of genes to primary bovine chondrocytes, followed by culture in three-dimensional pellet format and evaluation of extracellular matrix protein metabolism, is a useful functional assay for assessing the role of genes on cartilage matrix synthesis and degradation.

Members of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family share common structural features including a disintegrin domain, a zinc metalloprotease domain, and at least one thrombospondin motif. Aberrant expression of several of these proteins has led to an understanding of their role in human disease; however, a link to function for many has not yet been made. One such uncharacterized family member, ADAMTS-8, shares significant protein sequence homology with a subgroup of ADAMTSs that includes ADAMTS-1, ADAMTS-4, ADAMTS-5, and ADAMTS-15. Each of these proteases has been shown to cleave ‘aggrecanase-susceptible’ site(s) within the extracellular matrix (ECM) proteoglycan aggrecan, and ADAMTS-4 and ADAMTS-5 have been postulated to play a role in the depletion of articular cartilage in osteoarthritic disease. Based on sequence relationships, in the present study we examined the ability of ADAMTS-8 to exhibit ‘aggrecanase’ activity. A neoepitope monoclonal antibody (MAb; AGG-C1; anti-NITEGE373) was developed and used to demonstrate the ability of ADAMTS-8 to cleave aggrecan at the aggrecanase-susceptible Glu373–Ala374 peptide bond. In addition, expression analyses demonstrated the presence of ADAMTS-8 mRNA transcripts in normal and osteoarthritic human cartilage.

Apoptosis may limit the utility of cell-based therapies for articular cartilage disorders. We tested the hypothesis that chondrocyte apoptosis can be reduced by optimizing the conditions employed to expand chondrocyte numbers in culture. Chondrocyte apoptosis was examined in monolayer and suspension culture, in the presence of fetal bovine serum (FBS) or autologous serum, and for culture periods of 2 or 4 days. The effect of these variables was assessed by measuring cell viability, Annexin V labeling and mitochondrial membrane potential. After 2 days of culture, the greatest increase in viable cell number (3.7-fold) occurred in monolayer cultures with autologous serum. After 4 days of culture, the greatest increase in cell number (9.0-fold) occurred in monolayer cultures supplemented with FBS. By Annexin V staining, the proportion of cells undergoing apoptosis after 2 days was not affected by the type of serum used or by culture in monolayer versus suspension. After 4 days of culture, the proportion of apoptotic cells was significantly reduced (35% to 13%, p

ADAMTS-4, also referred to as aggrecanase-1, is a glutamyl endopeptidase capable of generating catabolic fragments of aggrecan analogous to those released from articular cartilage during degenerative joint diseases such as osteoarthritis. Efficient aggrecanase activity requires the presence of sulfated glycosaminoglycans (GAGs) attached to the aggrecan core protein, implying the contribution of substrate recognition/binding site(s) to ADAMTS-4 activity. In the present study, we demonstrate that full-length ADAMTS-4 (M(r) approximately 68,000) undergoes autocatalytic C-terminal truncation to generate two discrete isoforms (M(r) approximately 53,000 and M(r) approximately 40,000), which exhibit a marked reduction in affinity of binding to sulfated GAGs. C-terminal sequencing and mass analyses revealed that the GAG-binding thrombospondin type I motif was retained following autocatalysis, indicating that sites present in the C-terminal cysteine (cys)-rich and/or spacer domains also effect binding of full-length ADAMTS-4 to sulfated GAGs. Binding-competition experiments conducted using native and deglycosylated aggrecan provided direct evidence for interaction of the ADAMTS-4 cysteine-rich/spacer domains with aggrecan GAGs. Furthermore, synthetic peptides mimicking putative (consensus) GAG-binding sequences located within the ADAMTS-4 cysteine-rich and spacer domains competitively blocked binding of sulfated GAGs to full-length ADAMTS-4, thereby identifying multiple GAG-binding sites, which may contribute to the regulation of ADAMTS-4 function.

Two major proteolytic cleavages, one at NITEGE(373)/A(374)RGSVI and the other at VDIPEN(341)/F(342)FGVGG, have been shown to occur in vivo within the interglobular domain of aggrecan. The Glu(373)-Ala(374) site is cleaved in vitro by aggrecanase-1 (ADAMTS4) and aggrecanase-2 (ADAMTS5), whereas the other site, at Asn(341)-Phe(342), is efficiently cleaved by matrix metalloproteinases (MMPs) and by cathepsin B at low pH. Accordingly, the presence of the cleavage products globular domain 1 (G1)-NITEGE(373) and G1-VDIPEN(341) in vivo has been widely interpreted as evidence for the specific involvement of ADAMTS enzymes and MMPs/cathepsin B, respectively, in aggrecan proteolysis in situ. We show here, in digests with native human aggrecan, that purified ADAMTS4 cleaves primarily at the Glu(373)-Ala(374) site, but also, albeit slowly and secondarily, at the Asn(341)-Phe(342) site. Cleavage at the Asn(341)-Phe(342) site in these incubations was due to bona fide ADAMTS4 activity (and not a contaminating MMP) because the cleavage was inhibited by TIMP-3 (a potent inhibitor of ADAMTS4), but not by TIMP-1 and TIMP-2, at concentrations that totally blocked MMP-3-mediated cleavage at this site. Digestion of recombinant human G1-G2 (wild-type and cleavage site mutants) confirmed the dual activity of ADAMTS4 and supported the idea that the enzyme cleaves primarily at the Glu(373)-Ala(374) site and secondarily generates G1-VDIPEN(341) by removal of the Phe(342)-Glu(373) peptide from G1-NITEGE(373). These results show that G1-VDIPEN(341) is a product of both MMP and ADAMTS4 activities and challenge the widely held assumption that this product represents a specific indicator of MMP- or cathepsin B-mediated aggrecan degradation.

Bone morphogenetic proteins play important roles in connective tissue morphogenesis. In this study, we used human multipotential mesenchymal cells as a target to analyze the effect of bone morphogenetic proteins on chondrogenesis. We also analyzed the effect of proinflammatory cytokine interleukin-1 on chondrogenic-differentiated cells and the interaction of IL-1β with bone morphogenetic proteins. Cells placed in a 3-dimensional matrix of alginate beads and cultured in a serum-free media with bone morphogenetic protein-2 and -9 induced expression of type II collagen (Col2A1) mRNA and increased expression of aggrecan and cartilage oligomeric matrix protein suggesting chondrogenic differentiation of the cells. The transcription factor Sox-9 that regulates both Col2A1 and aggrecan gene expression showed increased expression with BMP treatment. Chondrogenic differentiated cells treated with interleukin-1 decreased Sox-9, Col2A1 and aggrecan gene expression. Removal of interleukin-1 and further addition of bone morphogenetic proteins resulted in returned expression of chondrogenic markers. Chondrogenic differentiated cells cultured in the presence of different concentrations of bone morphogenetic proteins and interleukin-1 showed that bone morphogenetic proteins were able to partially block the suppressive effect of interleukin-1. This study shows that bone morphogenetic proteins play an important role in chondrogenesis and may prove to be potential therapeutics in cartilage repair. © 2001 Wiley-Liss, Inc.

Objective—To correlate substance P content of synovial fluid with prostaglandin E2content, radiographic evidence of joint abnormality, and anatomic location of the joint for normal and osteoarthritic joints of horses.Sample Population—Synovial fluid from 46 normal joints in 21 horses and 16 osteoarthritic joints in 10 horses.Procedure—Normal and osteoarthritic joints were identified by clinical and radiographic examination, by response to nerve blocks, during scintigraphy or surgery, or by clinicopathologic evaluation. Substance P and prostaglandin E2contents of synovial fluid were determined by radioimmunoassay. Radiographs of joints were assigned a numeric score reflecting severity of lesions. Joints were assigned a numeric score reflecting anatomic location.Results—Median concentrations of substance P and prostaglandin E2 were significantly increased in osteoarthritic joints, compared with normal joints. A significant correlation was found between concentrations of substance P and prostaglandin E2in synovial fluid, but a correlation was not detected between substance P concentration in synovial fluid and anatomic location of the joint or between radiographic scores of osteoarthritic joints and concentrations of substance P or prostaglandin E2.Conclusions and Clinical Relevance—A correlation existed between concentrations of substance P and prostaglandin E2in synovial fluid obtained from normal and osteoarthritic joints. However, content of substance P in synovial fluid cannot be predicted by the radiographic appearance of the joint or its anatomic location. Substance P and prostaglandin E2may share an important and related role in the etiopathogenesis of osteoarthritis, lending credence to the importance of neurogenic inflammation in horses. (Am J Vet Res2000;61: 714–718)

Background: Damaged articular cartilage has a limited ability to repair. Operative removal of damaged cartilage and penetration into the subchondral bone to allow population of the defect with progenitor cells can result in filling of the defect with repair tissue. However, this repair tissue often degenerates over time because of its inability to withstand the mechanical forces to which it is subjected. We previously reported that recombinant human bone morphogenetic protein-2 (rhBMP-2) improves the repair of full-thickness defects of cartilage as long as six months postoperatively. We have now extended that study to examine the quality of the repair tissue at one year. Methods: Full-thickness defects of cartilage were created in the trochlear groove of twenty-five adult New Zealand White rabbits. Eight defects were left empty, eight were filled with a collagen sponge, and nine were filled with a collagen sponge impregnated with five micrograms of rhBMP-2. The animals were killed at fifty-two weeks postoperatively, and the gross appearance of the healed defect was assessed. The repair tissue was examined histologically and was evaluated, according to a grading scale, by four individuals who were blinded with respect to the treatment. The tissue sections were immunostained with antibodies against type-I collagen, type-II collagen, aggrecan, and link protein. The residence time of the rhBMP-2 in the cartilage defect was evaluated in vivo with use of scintigraphic imaging of radiolabeled protein. Results: One year after a single implantation of a collagen sponge containing five micrograms of rhBMP-2, the defects had a significantly better histological appearance than the untreated defects (those left empty or filled with a collagen sponge). The histological features that showed improvement were integration at the margin, cellular morphology, architecture within the defect, and reformation of the tidemark. The total scores were also better for the defects treated with rhBMP-2 than for the untreated defects, but in no instance was the repair tissue identical to normal articular cartilage. The thickness of the cartilage in the defects treated with rhBMP-2 was 70 percent that of the normal cartilage, an observation that was identical to that at twenty-four weeks postoperatively. Immunostaining demonstrated significantly less type-I collagen in the defects treated with rhBMP-2 than in the untreated defects. Immunostaining for other matrix components showed no difference among the treatment groups. The mean residence time of rhBMP-2 in the cartilage defects was eight days with an elimination half-life of 5.6 days. Detectable amounts of rhBMP-2 were present as long as fourteen days after implantation. Conclusions: The problems associated with operative repair of cartilage include the formation of fibrocartilage rather than normal articular cartilage and the degeneration of that repair tissue over time. Our results demonstrate that the addition of rhBMP-2 to the operative site after creation of a full-thickness defect results in an improvement in the histological appearance and composition of the extracellular matrix at one year postoperatively. If these experimental results translate directly to the clinical situation, it is possible that the addition of rhBMP-2 to existing operative treatments for the repair of cartilage may improve the repair process and may help to maintain the integrity of the repair tissue.

Real–time ultrasonography was used to confirm diaphragmatic rupture in a horse that developed pleural effusion following a traumatic event. Pleural effusion, a cranially displaced crus, and a cranially positioned sacculated loop of bowel were seen radiographically. The presence of gas–filled intestine cranial and dorsal to the diaphragm was detected by ultrasound imaging. The use of ultrasound and radiography in the diagnosis of diaphragmatic rupture is discussed.

Bone morphogenetic proteins have been shown to increase matrix synthesis by articular chondrocytes in short-term cultures. Members of this family of proteins have also been shown to induce endochondral ossification in vivo. The present study was performed to determine if the addition of human recombinant bone morphogenetic protein-2 to a long-term monolayer articular chondrocyte cell culture system affected the ability of the chondrocytes to divide in vitro, whether the cytokine altered expression of the articular chondrocyte phenotype and synthesis of matrix proteoglycans, and whether the cytokine was capable of inducing differentiation to a hypertrophic chondrocyte. Human recombinant bone morphogenetic protein-2 did not alter cell proliferation. It caused 3.5-6.2 times more proteoglycan synthesis by articular chondrocytes during each of the time points tested after 4 days in culture. Total proteoglycan accumulation in the extracellular matrix after 28 days in culture was 6.7 times as great in the treated cultures as in the control. Treatment with human recombinant bone morphogenetic protein-2 maintained the articular chondrocyte phenotype of cells in culture as demonstrated by Northern blot analysis: the expression of type-I collagen genes was increased and that of type-II collagen and aggrecan mRNA was lost in untreated chondrocyte cultures after 14-21 days in culture. In contrast, exposure to 100 ng/ml human recombinant bone morphogenetic protein-2 maintained expression of type-II collagen and increased expression of aggrecan compared with controls during the 28-day culture period. Northern blot analysis of the expression of type-X collagen and osteocalcin by chondrocytes treated with human recombinant bone morphogenetic protein-2 showed a lack of expression of these genes, indicating no alteration in phenotype. These experiments demonstrated the ability of human recombinant bone morphogenetic protein-2 to promote the articular chondrocyte phenotype and matrix synthesis in long-term culture. Characteristics of cell growth were not affected, and the cytokine did not induce differentiation to a hypertrophic chondrocyte.