Influence of commonly used pharmaceutical agents on equine bone marrow-derived mesenchymal stem cell viability.

REASON FOR PERFORMING STUDY: To provide evidence to support recommendations regarding the co-administration of drugs with mesenchymal stem cell (MSC) therapy. OBJECTIVES: To determine the influence of sedatives, local anaesthetic and corticosteroids on MSC viability and proliferation, in comparison to somatic cells derived from tendon (TDCs). STUDY DESIGN: In vitro cell culture. MATERIALS AND METHODS: MSCs (n = 3) and TDCs (n = 2) were cultured in media containing a clinically relevant dose range of xylazine, romifidine, detomidine and butorphanol, mepivacaine, methylprednisolone, or triamcinolone acetonide. Cell viability in suspension culture was assessed at intervals up to 4 h using the trypan blue dye assay. MSCs in monolayer culture were exposed to the highest concentrations of drug and proliferation was measured using the alamarBlue fluorescence assay. RESULTS: Exposure to romifidine or mepivacaine did not significantly affect viability or proliferation rate of MSCs or TDCs at any of the dosages tested. At the highest concentration of detomidine and butorphanol, MSC viability was significantly reduced compared to controls. Although xylazine exposure caused a significant (P < 0.001), dose-dependent reduction in MSC viability compared to controls, overall population viability remained good. Conversely, both methylprednisolone and triamcinolone resulted in the rapid death of significant numbers of MSCs (P < 0.001). CONCLUSIONS: Clinicians can sedate horses and administer nerve blocks to assist in intratendinous or intrathecal injection of MSCs with confidence that these drugs will not impact the viability of implanted cells. However, the concomitant use of corticosteroids is likely to have a severely detrimental effect on cell viability and should not be performed. Similarly, steroid administration into the sheath of a damaged tendon is not recommended.

Expression of Sulf1 and Sulf2 in cartilage, bone and endochondral fracture healing.

SULF1/SULF2 enzymes regulate cell signalling that impacts the growth and differentiation of many tissues. To determine their possible role in cartilage and bone growth or repair, their expression was examined during development and bone fracture healing using RT-PCR and immunochemical analyses. Examination of epiphyseal growth plates revealed differential, inverse patterns of SULF1 and SULF2 expressions, with the former enriched in quiescent and the latter in hypertrophic chondrocyte zones. Markedly higher levels of both SULFs, however, were expressed in osteoblasts actively forming bone when compared with proliferating pre-osteoblasts in the periosteum or the entombed osteocytes which express the lowest levels. The increased expression of Sulf1 and Sulf2 in differentiating osteoblasts was further confirmed by RT-PCR analysis of mRNA levels in rat calvarial osteoblast cultures. SULF1 and SULF2 were expressed in most foetal articular chondrocytes but down-regulated in a larger subset of cells in the post-natal articular cartilage. Unlike adult articular chondrocytes, SULF1/SULF2 expression varied markedly in post-natal hypertrophic chondrocytes in the growth plate, with very high SULF2 expression compared with SULF1 apparent during neonatal growth in both primary and secondary centres of ossification. Similarly, hypertrophic chondrocytes expressed greatly higher levels of SULF2 but not SULF1 during bone fracture healing. SULF2 expression unlike SULF1 also spread to the calcifying matrix around the hypertrophic chondrocytes indicating its possible ligand inhibiting role through HSPG desulphation. Higher levels of SULF2 in both developing and healing bone closely correlated with parallel increases in hedgehog signalling analysed by ptc1 receptor expression.

Circulating concentrations of a marker of type I collagen metabolism are associated with hypertrophic cardiomyopathy mutation status in ragdoll cats.

OBJECTIVES: Human carriers of hypertrophic cardiomyopathy associated sarcomeric mutations have abnormal collagen metabolism before overt left ventricular hypertrophy is detectable. This study investigated whether differences in collagen biomarkers were present in blood samples of ragdoll cats positive for the MYBPC3:R820W mutation compared with negative controls. MATERIALS AND METHODS: Cats were recruited for hypertrophic cardiomyopathy screening using echocardiography and genotyping. Circulating markers of collagen turnover (C-terminal telopeptide of type I collagen [CITP; type I collagen degradation] and N-terminal propeptide of type III procollagen [type III collagen synthesis]) and cardiac biomarkers (N-terminal B-type natriuretic peptide and cardiac troponin I) were measured. Correlation between concentrations of collagen biomarkers and echocardiographic variables was analysed, and collagen biomarker concentrations were compared between MYBPC3 mutation positive and negative cats, without left ventricular hypertrophy. RESULTS: Linear regression analyses showed that genotype was independently associated with CITP concentration. CITP was higher in mutation carriers (25 · 4 µg/L, interquartile range 16 · 0-29 · 2 µg/L) than non-carriers (14 · 6 µg/L, interquartile range 9 · 38-19 · 2 µg/L; P = 0 · 024). CLINICAL SIGNIFICANCE: Circulating CITP was higher in MYBPC3-positive ragdoll cats than negative controls and may indicate altered collagen metabolism. Further studies are necessary to determine whether alterations in circulating collagen biomarker concentration relate to an early stage of hypertrophic cardiomyopathy.

Implantation of bone marrow-derived mesenchymal stem cells demonstrates improved outcome in horses with overstrain injury of the superficial digital flexor tendon.

REASONS FOR PERFORMING STUDY: Mesenchymal stem (progenitor; stromal) cell (MSC) therapy has gained popularity for the treatment of equine tendon injuries but without reports of long-term follow-up. OBJECTIVES: To evaluate the safety and reinjury rate of racehorses after intralesional MSC injection in a large study of naturally occurring superficial digital flexor tendinopathy and to compare these data with those published for other treatments. METHODS: Safety was assessed clinically, ultrasonographically, scintigraphically and histologically in a cohort of treated cases: 141 client-owned treated racehorses followed-up for a minimum of 2 years after return to full work. Reinjury percentages were compared to 2 published studies of other treatments with similar selection criteria and follow-up. The number of race starts, discipline, age, number of MSCs injected and interval between injury and treatment were analysed. RESULTS: There were no adverse effects of the treatment with no aberrant tissue on histological examination. The reinjury percentage of all racehorses with follow-up (n = 113) undergoing MSC treatment was 27.4%, with the rate for flat (n = 8) and National Hunt (n = 105) racehorses being 50 and 25.7%, respectively. This was significantly less than published for National Hunt racehorses treated in other ways. No relationship between outcome and age, discipline, number of MSCs injected or injury to implantation interval was found. CONCLUSIONS: Whilst recognising the limitations of historical controls, this study has shown that MPC implantation is safe and appears to reduce the reinjury rate after superficial digital flexor tendinopathy, especially in National Hunt racehorses. POTENTIAL RELEVANCE: This study has provided evidence for the long-term efficacy of MSC treatment for tendinopathy in racehorses and provides support for translation to human tendon injuries.

Cell-based therapies for tendon and ligament injuries.

Tendon and ligament injuries have proved difficult to treat effectively. Cell-based therapies offer the potential to harness the complex protein synthetic machinery of the cell to induce a regenerative response rather than fibrous scarring. This article reviews the current state of play with respect to the clinically used cell preparations for the treatment of tendon and ligaments overstrain injuries.

The relationship between in vivo limb and in vitro tendon mechanics after injury: a potential novel clinical tool for monitoring tendon repair.

REASONS FOR PERFORMING THE STUDY: Highly prevalent superficial digital flexor tendon (SDFT) injury results in compromised tendon function through fibrosis and high frequency of re-injury due to altered biomechanical function. This study investigated the consequences of SDF tendinopathy on limb mechanics in relation to the mechanical properties of injured tendon. OBJECTIVES: To develop and validate a noninvasive in vivo assessment of tendon mechanics to investigate the effect of recent SDFT injury on limb stiffness index, providing an objective method to assess quality of healing. HYPOTHESES: Limb stiffness index would reduce as a consequence of SDFT injury and progressively increase during tendon healing and correlate with in vitro mechanical properties of the respective SDFTs. METHODS: Kinematic analysis was performed at walk in 10 horses that had sustained career-ending SDFT injury. Stiffness index was derived from limb force recorded via a series of force plates and measurement of change in metacarpophalangeal joint angle using 3D motion analysis software. Horses were subjected to euthanasia 7 months after injury, the SDFTs removed and subjected to nondestructive in vitro mechanical testing. RESULTS: Limb stiffness index was reduced following SDFT injury in comparison with the contralateral limb and increased during the convalescent period, approximating that of the contralateral limb by 7 months post injury. There was a significant positive correlation between in vivo limb stiffness index and in vitro SDFT stiffness. CLINICAL RELEVANCE: The ability to assess and monitor SDFT mechanical competence through limb stiffness measurement techniques in horses recovering from SDFT injury and the possibility of corroborating this with functional tendon healing may permit a more objective and accurate assessment of optimal tendon repair in the horse. This technique may be a useful method for assessing the efficacy of treatment regimens for tendinopathy and could be utilised to predict time to safe return to performance or re-injury.

Age-related differences in prostaglandin E2 synthesis by equine cartilage explants and synoviocytes.

Time- and concentration-related actions of lipopolysaccharide (LPS) on the synthesis of prostaglandin E(2) (PGE(2)) were investigated in cartilage explants and synoviocytes harvested from 3 age groups of horses, all with clinically normal joint function: group A <10 years; group B 11-20 years and group C >20 years. Cartilage explants from group A horses were least and those from group C were most sensitive to LPS. Significant increases in PGE(2) concentration (P

Aggrecan, aging and assembly in articular cartilage.

The primary function of articular cartilage to act as a self-renewing, low frictional material that can distribute load efficiently at joints is critically dependent upon the composition and organisation of the extracellular matrix. Aggrecan is a major component of the extracellular matrix, forming high molecular weight aggregates necessary for the hydration of cartilage and to meet its weight-bearing mechanical demands. Aggregate assembly is a highly ordered process requiring the formation of a ternary complex between aggrecan, link protein and hyaluronan. There is extensive age-associated heterogeneity in the structure and molecular stoichiometry of these components in adult human articular cartilage, resulting in diverse populations of complexes with a range of stabilities that have implications for cartilage mechanobiology and integrity. Recent findings have demonstrated that aggrecan can form ligands with other matrix proteins. These findings provide new insights into mechanisms for aggregate assembly and functional protein networks in different cartilage compartments with maturation and aging.

Enhanced concentration of COMP (cartilage oligomeric matrix protein) in osteochondral fractures from racing Thoroughbreds.

The aim of the present study was to correlate the levels of COMP and aggrecan as indicators of tissue damage, in synovial fluid (sf) from carpal joints of acutely lame racehorses, with macroscopical lesions of articular cartilage (OA), osteochondral fractures and ligament tears found at arthroscopy. Sixty-three lame horses [49 Standardbred trotters (STB) and 14 Thoroughbreds (TB)] in conventional training and racing that underwent arthroscopy of their middle carpal or radiocarpal joints were included in the study. Intact as well as fragmented COMP and aggrecan released into the synovial fluid were quantified by western blot analyses and ELISA. The expression of COMP in tissues was estimated by mRNA in situ hybridisation and protein immunolocalisation in cartilage and osteochondral fractures. The concentration of sf-COMP was higher in TB with an osteochondral fracture than in STB with osteochondral fractures and TB and STB with OA. The chondrocytes in middle and deep zones of the articular cartilage of the osteochondral fragments (from a TB) expressed COMP mRNA, in contrast to the cartilage on the opposite side of the fracture where no expression was detected. In the synovial fluid from a joint (TB) with osteochondral fractures only intact COMP was present, whereas, fragmented COMP was more prominent in synovial fluid from a joint with OA. The concentration of sf-aggrecan did not differ between the two breeds, or between different lesions. The increased concentration of sf-COMP in TB with osteochondral fractures, but not in synovial fluid from equine joints with OA, is a novel finding. The results from this study indicate that elevated sf-COMP concentration in the joints of Thoroughbreds may be a useful marker for carpal joint osteochondral fragments.

Metalloproteinase and tissue inhibitor of metalloproteinase expression in the murine STR/ort model of osteoarthritis.

OBJECTIVE: To study the temporal expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in the STR/ort mouse model of osteoarthritis, using in situ hybridization with oligonucleotide probes and specific antisera for each protein. METHODS: In situ hybridization and immunolocalization experiments were performed on serial cryosections of knee joints from STR/ort and control CBA mice. The mRNA was localized using digoxygenin-labeled probes. RESULTS: MMP2, MMP3, MMP7, MMP9, MMP13, MT1-MMP and TIMP2 mRNA was detected in the tibial articular chondrocytes of STR/ort mice at all ages (12, 18, 24, 30 and 35 weeks). Levels were always higher than in age-matched CBA mice. Neither MMP8 nor TIMP1 mRNA was detected in murine cartilage. The location and distribution of each of the MMP mRNA transcripts varied within the tibial plateau. Immunolocalization consistently detected MMP3 and MT1-MMP in articular cartilage and MMP13 in calcified cartilage. Other proteases and their inhibitors were not detected in either of these cartilages but MMP2 and MMP9 were immunolocalized in bone marrow cells and growth cartilage respectively. CONCLUSION: Expression of all the detected MMPs and TIMP-2 is up-regulated in STR/ort mice at the mRNA level. However, failure to detect protein expression for MMPs 2, 7, 9, 13 and TIMPs 1 and 2 in murine chondrocytes by immunohistochemistry indicates that the changes in mRNA levels in STR/ort mice must be interpreted with caution.

Platelet-derived growth factor stimulates the formation of versican-hyaluronan aggregates and pericellular matrix expansion in arterial smooth muscle cells.

Hyaluronan and versican-rich pericellular matrices form around arterial smooth muscle cells (ASMC) preferentially during the detachment phase of proliferation and migration. PDGF is a potent mitogen and chemotactic agent for ASMC and also stimulates the production of extracellular matrix molecules which may regulate the proliferative and migratory capacity of the cells. We have examined the effect of PDGF on the formation of hyaluronan-dependent pericellular matrices, and on the synthesis and interaction of several major pericellular coat constituents. As demonstrated using a particle exclusion assay, PDGF stimulated the formation of pericellular matrices and was seen both in an increased proportion of cells with a coat and a greater coat size. This increase was accompanied by a transient increase in hyaluronan synthase 2 (HAS2) expression and an increase in hyaluronan synthesis and polymer length. PDGF also increased the synthesis of versican and link protein as measured at the mRNA and protein levels. The amount of native versican-hyaluronan aggregates and link-stabilized aggregate was also increased following PDGF treatment. Time lapse imaging showed that pericellular matrix formation occurred around trailing cell processes prior to their detachment. These data suggest that PDGF modulates the synthesis and organization of ASMC pericellular coat-forming molecules such as versican, hyaluronan, and link protein, which leads to extracellular matrix expansion and alterations in ASMC phenotype.

Age-related changes in the synthesis and mRNA expression of decorin and aggrecan in human meniscus and articular cartilage.

OBJECTIVE: To determine if the biosynthesis of aggrecan and decorin in the human meniscus and the potential of the cells to express these macromolecules (mRNA), is affected by the age of the individual and that if any changes are observed are they different to those measured in articular cartilage obtained from the same joint. DESIGN: Radiolabelling of tissue explants, anion-exchange chromatography and agarose-polyacrylamide gel electrophoresis were used to analyze newly synthesized proteoglycans. A quantitative, competitive reverse-transcriptase polymerase chain reaction was developed and applied to the tissue to measure the expression of decorin and aggrecan mRNA. RESULTS: Proteoglycan synthesis in the meniscus was higher in young donors (1-5 mmoles sulfate incorporated/h/mgDNA, under 20 years of age) than in adult tissues (0.5-1 mmoles incorporated/h/mgDNA, 20-62 years of age) and decorin was the major proteoglycan synthesized at this time. An age-related increase in the proportion of aggrecan synthesis in the meniscus was also observed using agarose-polyacrylamide gel electrophoresis. Both decorin (five-fold) and aggrecan (eight-fold) mRNA expression increased with age in meniscus whereas levels were relatively constant in articular cartilage. In addition, the synthesis of decorin and aggrecan and the expression of their mRNA was different in meniscus and articular cartilage from the same knee joint. CONCLUSION: The synthesis and turnover of aggrecan and decorin in the human meniscus is influenced by the age of the individual and is not the same as that observed for articular cartilage.

Up-regulation and differential expression of the hyaluronan-binding protein TSG-6 in cartilage and synovium in rheumatoid arthritis and osteoarthritis.

OBJECTIVE: TSG-6 [the product of tumor necrosis factor (TNF)-stimulated gene-6] is a hyaluronan-binding protein that is present in the synovial fluids of arthritis patients and is secreted by cells of articular joints (e.g. chondrocytes and synoviocytes). This study examines the pattern of TSG-6 expression in normal and diseased cartilage and synovium using immunohistochemical techniques. DESIGN: A polyclonal antibody was raised against recombinant Link module from human TSG-6 and used to detect the protein in tissue sections taken from osteoarthritis (OA) and rheumatoid arthritis (RA) patients and controls. RESULTS: There was no TSG-6 detected in normal tissues. In all OA synovium there was intense TSG-6 expression throughout the intimal layer, whereas in RA staining in this region was generally less pronounced and was absent at the synovial surface in tissues exhibiting significant inflammation. In RA TSG-6 was also expressed by infiltrating leukocytes and by cells at the cartilage-synovium pannus junction. TSG-6 immunoreactivity was present in the tunica intima of blood vessels in OA subintima, being particularly noticeable in the thickened smooth muscle of inflamed vessel walls, but was mostly confined to the lumen of the vessel in RA. In cartilage the majority of chondrocytes expressed TSG-6 in both OA and RA, usually with extensive staining in the surrounding matrix. CONCLUSION: TSG-6 is present within synovium and cartilage of arthritic joints, but not normal controls, and is synthesized by the resident cells. The pattern of TSG-6 expression is consistent with its proposed roles in extracellular matrix (ECM) remodeling and cellular proliferation.

Age-related effects of TGF-beta on proteoglycan synthesis in equine articular cartilage.

The synthesis of proteoglycans was measured in normal equine articular cartilage of ages 9 months to 20 years and the effect of TGF-beta1 on this activity was investigated. The rate of incorporation of [(35)S]Na(2)SO(4) decreased with age as did the responsiveness of the tissue to the growth factor. The enhanced synthesis of proteoglycan induced at all ages by TGF-beta1 was down-regulated by IL-1 beta and retinoic acid. The expression of mRNA for TGF-beta1, 2, and 3 was also measured, and although the level of TGF-beta1 was highest at all ages, the expression of each growth factor decreased with age.

The organization of aggrecan in human articular cartilage. Evidence for age-related changes in the rate of aggregation of newly synthesized molecules.

The effect of age on the incorporation of newly synthesized aggrecan into the extracellular matrix of human articular cartilage was investigated. This property was measured in a pulse-chase explant culture system by determining the distribution of radiolabeled molecules ([(35)S]sulfate-labeled) between a nondissociating extract (phosphate-buffered saline), which extracts mainly nonaggregated macromolecules, and a dissociating extract (4 M GnHCl) containing mainly aggrecan that was complexed in situ with hyaluronan. The rate of incorporation of aggrecan into aggregates was much slower in mature cartilage than in tissue obtained from younger individuals. Furthermore, autoradiography showed that in mature cartilage, newly synthesized aggrecan is not transported from the pericellular environment within the first 18 h of chase culture, whereas in immature cartilage, it moves into the intercellular space during the same period, i.e. aggrecan is processed in the extracellular space very differently in young and adult articular cartilage. Experiments were also performed to show that the interaction of link protein with newly synthesized aggrecan depends on the maturity of the G(1) domain of aggrecan. This investigation has shown that the extracellular aggregation of aggrecan in adult human articular cartilage involves a number of intermediate structures. These have not been identified in the very young cartilage obtained from laboratory animals or in porcine and bovine articular cartilage obtained from the abattoir.

Age-related changes in the synthesis of link protein and aggrecan in human articular cartilage: implications for aggregate stability.

The rates of incorporation of radiolabelled leucine into aggrecan and link protein have been measured in human articular cartilage of different ages. Aggrecan and link protein were purified in the A1 fraction of CsCl gradients as a result of their ability to form high-buoyant-density proteoglycan aggregates with hyaluronic acid. Separation of the aggrecan from the link protein was achieved by Mono Q anion-exchange chromatography. The rates of synthesis of both aggrecan and link protein decreased with age. The age-related decrease in synthesis of aggrecan was paralleled by a decrease in the rate of sulphate incorporation into glycosaminoglycan chains. The synthesis of link protein decreased with age to a greater extent than that of aggrecan such that the ratio of the rates of link protein to aggrecan synthesis decreased from 1 in immature cartilage to 0.2 in mature cartilage. The age-related decrease in link protein synthesis is controlled at least in part by transcriptional or post-transcriptional mechanisms, as shown by the accompanying age-related decrease in link-protein mRNA. The absence of any age-related decrease in aggrecan mRNA suggests that the decrease in synthesis of aggrecan core protein is controlled by a translational mechanism. Measurement of the total tissue content of aggrecan and link protein by radioimmunoassay revealed an age-related increase in the accumulation of these matrix proteins, even though their de novo synthesis was decreasing. This illustrates the importance that the regulation of extracellular post-translational modification also has in controlling the overall turnover of the cartilage matrix.

Phenotypic diversity of neoplastic chondrocytes and extracellular matrix gene expression in cartilaginous neoplasms.

Chondrocyte differentiation is characterized by distinct cellular phenotypes, which can be identified by specific extracellular matrix gene expression profiles. By applying in situ analysis on the mRNA and protein level in a series of benign and malignant human chondrogenic neoplasms, we were able to identify for the first time different phenotypes of neoplastic chondrocytes in vivo: 1) mature chondrocytes, which synthesized the characteristic cartilaginous extracellular tumor matrix, 2) cells resembling hypertrophic chondrocytes of the fetal growth plate, 3) cells resembling so-called dedifferentiated chondrocytes, and 4) well differentiated chondrocytic cells, which expressed type I collagen, indicating the presence of post-hypertrophic differentiated neoplastic chondrocytes. Chondrocytes exhibiting a range of phenotypes were found to be present in the same neoplasm. The different observed phenotypes, including the dedifferentiated phenotype, were in contrast to the anaplastic cells of high-grade chondrosarcomas. Comparison of expression data with tumor morphology revealed a relationship between the cellular phenotypes, the tumor matrix composition, and the matrix and cell morphology within the neoplasms. The distinctly different phenotypes of neoplastic chondrocytes are the basis of the characteristic high biochemical and morphological heterogeneity of chondroid neoplasms and shed light on their biological and clinical behavior.