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.

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.

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

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.

Immunoglobulin fold and tandem repeat structures in proteoglycan N-terminal domains and link protein.

Detailed primary sequence and secondary structure analyses are reported for the hyaluronate binding region (G1 domain) and link protein of proteoglycan aggregates. These are based on six full or partial sequences from the chicken, pig, human, rat and bovine proteins. Determinations of a full pig and a partial human link protein sequence are reported in the Appendix. Five sequences at the N terminus in both proteins were compared with the structures of 11 variable immunoglobulin (Ig) fold domains for which crystal structures are available. Despite only modest sequence homology, a clear alignment could be proposed. Analysis of this shows that the equivalents of the first and second hypervariable segments are now significantly longer, and both proteins have N-terminal extensions that are up to 23 residues in length. Secondary structure predictions showed that these sequences could be identified with available crystal structures for the variable Ig fold. However the hydrophobic residues involved in interactions between the light and heavy chains in Igs are replaced by hydrophilic charged groups in both proteins. These results imply that both proteins are members of the Ig superfamily, but exhibit structural differences distinct from other members of this superfamily for which crystal structures are known. The proteoglycan tandem repeat (PTR) is a repeat of 99 residues that is found twice in the amino acid sequence of link protein and the proteoglycan G1 domain adjacent to the Ig fold, and also twice in the proteoglycan G2 domain. A total of 16 PTRs was available for analysis. Compositional analyses show that these are positively charged if these originate from link protein, and negatively charged if from the G1 or G2 domains. The 16 Robson secondary structure predictions for the PTRs were averaged to improve the statistics of the prediction, and checked by comparison with Chou-Fasman calculations. A strong alpha-helix prediction was found at residues 13 to 25, and several beta-strands were predicted. The overall content is 18% alpha-helix and 28% beta-sheet, with 44% of the remaining sequence being predicted as turns. These analyses show that both the proteoglycan G1 domain and link protein are constructed from two distinct globular components, which may provide the two functional roles of these proteins in proteoglycan aggregation.

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.

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.

Expression of collagen types IX and XI and other major cartilage matrix components by human fetal chondrocytes in vivo.

Coordinate differentiation of the chondrocytes plays a crucial role during skeletal development. In the cascade of endochondral bone formation, mature chondrocytes of the fetal growth plate represent metabolically highly active cells. They show high expression levels of the major cartilage matrix genes, collagen types II, IX, and XI, the major cartilage proteoglycan aggrecan, and proteoglycan link protein. The strongest signals are found in areas of maximal growth, the proliferative and upper hypertrophic zones. The major cartilage matrix components are co-expressed by the chondrocytes of the resting and proliferative zones. Type X collagen is restricted to lower hypertrophic chondrocytes. Interestingly, in the lower hypertrophic zone type IX collagen, but not type II and XI collagen, mRNA expression is downregulated, indicating a discoordinate expression of these collagen types in hypertrophic chondrocytes. The results of this study confirm the strict zonal differentiation pattern of chondrocytes in the developing fetal growth plate, which can be monitored by the expression patterns of its major expression products, the collagen subtypes and aggrecan and proteoglycan link protein.

Complete primary sequence of equine cartilage link protein deduced from complementary DNA.

Investigation of the structure of the equine articular cartilage link protein (LP) from individuals ranging in age from 1 to 15 years identified 3 distinct isoforms having molecular weights of 46,000, 43,000 and 41,000. The relative amounts of each of the 3 isoforms altered with age. The largest form did not change with age; however, amounts of the Mr 43,000 and 41,000 forms increased with increasing age. The results suggested that an accumulation, in the extracellular matrix of cartilage, of these 2 smaller products may have arisen from proteolytic cleavage. The complete amino acid sequence of the protein core was determined from complementary DNA products prepared by polymerase chain reaction amplification of cartilage LP mRNA. The sequence had 96% similarity with human LP and with that of other species for which the primary structure has been determined. This high degree of sequence conservation and the isoform data indicate that extracellular processing of LP occurs by similar mechanisms in various species. At the transcription level, equine chondrocytes were found to express LP as 2 abundant mRNA of 5.0 and 3.0 kb, and a smaller mRNA of 1.5 kb. Processing of the LP m RNA in horses, thus, appears to be similar to that found in other species investigated, an although multiple transcripts are present, the coding region remains unaltered and only 1 protein product is made.

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.

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.

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.

Prolonged expression of differentiated phenotype by chondrocytes cultured at low density on a composite substrate of collagen and agarose that restricts cell spreading.

The dedifferentiation of chondrocytes in culture is frequently associated with transition from a rounded to a spread morphology. A number of culture methods which prevent cell spreading have been described; however, all have disadvantages that limit their widespread use. In this paper we describe a new technique which allows prolonged cultivation of attached chondrocytes at low density while inhibiting spreading: the cells are grown on a composite substrate of agarose and collagen. By varying the ratio of agarose to collagen in the gel, the degree of spreading can be varied. The cultures are suitable for ultrastructural and immunofluorescence analysis and for studies of the synthesis and secretion of macromolecules. In order to determine whether the differentiated phenotype was maintained on composite gels, we compared the levels of messenger RNAs for cartilage-specific proteoglycan, link protein, alpha 1 (II) and alpha 1 (I) collagens in chondrocytes grown at low density on composite gels or at high or low density on tissue culture plastic for up to 21 days. The rate of decline in the level of mRNAs encoding the cartilage-specific products and the rate of increase in the level of alpha 1 (I) collagen mRNA were slower in the composite cultures than in the cultures on plastic. This culture technique may, therefore, prolong expression of the differentiated phenotype of chondrocytes relative to cultivation on plastic and will be useful for further studies on the role of cell shape in regulating differentiated gene expression.

The structure, function and turnover of aggrecan, the large aggregating proteoglycan from cartilage.

Aggrecan, the large aggregating proteoglycan from cartilage contains chondroitin sulphate and keratan sulphate attached to a multidomain protein core. It aggregates by binding to hyaluronan and this is further stabilised by a separate globular link protein. There are two structurally related N-terminal globular domains, G1 and G2, of which only G1 and not G2 is involved in aggregation. The interglobular domain joining G1 and G2 contains proteinase sensitive sequences which appear to be the key site for cleavage during aggrecan turnover. Much of the keratan sulphate and all of the chondroitin sulphate is attached to the long extended glycosaminoglycan attachment region. The function of the C-terminal G3 domain is unknown. It contains a mammalian type C lectin and complement regulatory protein motifs. These may have interactive properties that contribute to matrix organisation. There is also an alternatively spliced form with an epidermal growth factor-like motif. The carbohydrate composition of aggrecan varies with cartilage source, development and age and is heterogeneous in each sample. There is evidence of a close control of chondroitin sulphate synthesis that determines chain length and disaccharide composition and which change during development and in pathology. Monoclonal antibodies that recognise specific sequences within chondroitin sulphate chains enable some of these changes in fine structure to be detected. Progressive digestion of chains with chondroitinase AC II has provided evidence of a pattern of sulphation, with 6-sulphated disaccharides more abundant towards the protein core, although the disaccharide next to the linkage region is predominantly non-sulphated.

Human link protein gene: structure and transcription pattern in chondrocytes.

We have examined the genomic organization and the transcription unit for the human link protein gene from genomic clones and RNA prepared from human cartilage over a wide age range. Five exons cover the gene which is greater than 60 kbp. Primer extension and S1 nuclease protection analysis revealed transcription initiation to be 315 bases upstream from the translation initiation codon in RNA derived from cartilage samples ranging from fetal to 53 years of age. The first exon size therefore is 289 bp and examination of the 5' flanking sequence indicated a lack of a TATA box in close proximity to the transcription start, although a TATAA-like motif (TCTAA) was present at -75 bp. Such a sequence at a similar distance can serve as a promoter in the chicken link protein gene. The large first exon of 289 bp is similar to that of the chicken but contrasts with that described previously for human (96 bp) and rat (62 bp). We also analysed human link protein mRNA by PCR for the presence of an alternatively spliced exon that is present in rat mRNA in low abundance, but could not detect such transcripts. Equine and porcine mRNA contained this spliced form but the results suggested that this was expressed as a rare transcript.

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.

Quantification of aggrecan and link-protein mRNA in human articular cartilage of different ages by competitive reverse transcriptase-PCR.

A competitive reverse transcriptase-PCR (RT-PCR) assay has been developed for the quantification of particular mRNA species in human articular cartilage. Competitor RNA species were synthesized that differed from the amplified target sequence only by the central insertion of an EcoRI restriction site. By using known amounts of synthetic target and competitor RNA, it was shown that competitor RNA molecules designed in this way are reverse-transcribed and amplified with equal efficiency to the target of interest. Furthermore quantification could be performed during the plateau phase of the PCR, which was necessary when using ethidium bromide fluorescence as a detection system. The inhibition of aggrecan and link-protein mRNA expression by interleukin 1 or tumour necrosis factor in monolayers of human articular chondrocytes quantified by this competitive RT-PCR method compared favourably with Northern hybridization studies. The main advantage of this technique is that it can be used to quantify levels of mRNA with RNA extracted directly from 100 mg wet weight of human articular cartilage. Age-related changes in aggrecan and link-protein mRNA were therefore quantified in human articular cartilage directly after dissection from the joint. The concentration of link-protein mRNA was higher in immature cartilage than in mature cartilage when expressed relative to the amount of glyceraldehyde-3-phosphate dehydrogenase mRNA, but no age-related changes were observed in aggrecan mRNA expression. The ratio of aggrecan to link-protein mRNA was higher in mature cartilage than in immature tissue. These age-related differences in the molecular stoichiometry of aggrecan and link-protein mRNA might have implications with respect to the regulation of the formation and the stability of the proteoglycan aggregates in cartilage.