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Decorin and biglycan are two small leucine-rich proteoglycans (SLRPs) that regulate collagen fibrillogenesis and extracellular matrix assembly in tendon. The objective of this study was to determine the individual roles of these molecules in maintaining the structural and mechanical properties of tendon during homeostasis in mature mice. We hypothesized that knockdown of decorin in mature tendons would result in detrimental changes to tendon structure and mechanics while knockdown of biglycan would have a minor effect on these parameters. To achieve this objective, we created tamoxifen-inducible mouse knockdown models targeting decorin or biglycan inactivation. This enables the evaluation of the roles of these SLRPs in mature tendon without the abnormal tendon development caused by conventional knockout models. Contrary to our hypothesis, knockdown of decorin resulted in minor alterations to tendon structure and no changes to mechanics while knockdown of biglycan resulted in broad changes to tendon structure and mechanics. Specifically, knockdown of biglycan resulted in reduced insertion modulus, maximum stress, dynamic modulus, stress relaxation, and increased collagen fiber realignment during loading. Knockdown of decorin and biglycan produced similar changes to tendon microstructure by increasing the collagen fibril diameter relative to wild-type controls. Biglycan knockdown also decreased the cell nuclear aspect ratio, indicating a more spindle-like nuclear shape. Overall, the extensive changes to tendon structure and mechanics after knockdown of biglycan, but not decorin, provides evidence that biglycan plays a major role in the maintenance of tendon structure and mechanics in mature mice during homeostasis.

During osteoarthritis, chondrocytes change their spatial arrangement from single to double strings, then to small and big clusters. This change in pattern has recently been established as an image-based biomarker for osteoarthritis. The pericellular matrix (PCM) appears to degrade together alongside cellular reorganization. The aim of this study was to characterize this PCM-degradation based on different cellular patterns. We additionally wanted to identify the earliest time point of PCM-breakdown in this physiopathological model. To this end, cartilage samples were selected according to their predominant cellular pattern. Qualitative analysis of PCM degradation was performed immunohistochemically by analysing five main PCM components: collagen type VI, perlecan, collagen type III, biglycan, and fibrillin-1 (n = 6 patients). Their protein content was quantified by enzyme-linked immunosorbent assay (127 patients). Accompanying spatial cellular rearrangement, the PCM is progressively destroyed, with a pericellular signal loss in fluorescence microscopy for collagen type VI, perlecan, and biglycan. This loss in protein signal is accompanied by a reduction in total protein content from single strings to big clusters (P < .001 for collagen type VI, P = .003 for perlecan, and P < .001 for biglycan). As a result of an increase in the number of cells from single strings to big clusters, the amount of protein available per cell also decreases for collagen type III and fibrillin-1, where total protein levels remain constant. Biochemical changes of the PCM and cellular rearrangement are thus highly interconnected hallmarks of osteoarthritis. Interestingly, the earliest point in time for a relevant PCM impairment appears to be at the transition to small clusters.

Tendons have complex mechanical properties that depend on their structure and composition. Some studies have assessed the role of small leucine-rich proteoglycans (SLRPs) in the mechanical response of tendon, but the relationships between sophisticated mechanics, assembly of collagen and SLRPs have not been well characterized. In this study, biglycan gene expression was varied in a dose dependent manner using biglycan null, biglycan heterozygote and wild type mice. Measures of mechanical (tension and compression), compositional and structural changes of the mouse patellar tendon were evaluated. Viscoelastic, tensile dynamic modulus was found to be increased in the biglycan heterozygous and biglycan null tendons compared to wild type. Gene expression analyses revealed biglycan gene expression was closely associated in a dose-dependent allelic manner. No differences were seen between genotypes in elastic or compressive properties or quantitative measures of collagen structure. These results suggest that biglycan, a member of the SLRP family, plays a role in tendon viscoelasticity that cannot be completely explained by its role in collagen fibrillogenesis.

We hypothesized that BMP-2 might induce non-tenocyte differentiation and increase production of proteoglycans of tendon-derived stem cells (TDSCs). This study investigated the effects of BMP-2 on the differentiation and production of proteoglycans in TDSCs in vitro. Rat patellar TDSCs were treated without or with BMP-2. The osteogenic, adipogenic, chondrogenic, and tenogenic differentiation of TDSCs were assessed by (1) Alizarin red-S staining assay; (2) Oil Red-O staining assay; (3) haematoxylin-eosin staining, Safranin-O staining, immunohistochemical staining of Sox9, and collagen type II; and (4) qRT-PCR analysis of lineage-specific markers. The production of glycoaminoglycans (GAG) in the BMP-2-treated TDSCs was assessed by alcian blue staining. The mRNA expression of aggrecan (Acan), decorin (Dcn), biglycan (Bgn), and fibromodulin (Fmod) in TDSCs after BMP-2 treatment was assessed by qRT-PCR. BMP-2 promoted the osteogenic, adipogenic, and chondrogenic differentiation but inhibited tenogenic marker expression of TDSCs. GAG production and Acan increased while Dcn, Bgn, and Fmod decreased in TDSCs after BMP-2 stimulation. In conclusion, BMP-2 promoted GAG deposition, aggrecan expression, and enhanced non-tenocyte differentiation of TDSCs in vitro. The effect of BMP-2 on TDSCs might provide insights into the histopathological changes of tendinopathy.

Cardiovascular risk factors are known to be associated with intervertebral disc degeneration, but the underlying mechanism is still unclear. The ApoE knockout (KO) mouse is a well-established model for atheroscelorosis. We hypothesized that ApoE is involved in maintaining disc health and that ApoE KO mice will develop early disc degeneration. Discs of ApoE KO and wild-type (WT) mice were characterized with histological/immunological, biochemical, and real-time RT-PCR assays. A comparison of the extracellular matrix production was also performed in disc cells. We demonstrated that ApoE was highly expressed in the endplates of WT discs, and ectopic bone formed in the endplates of ApoE KO discs. Glycosaminoglycan content was decreased in both ApoE KO annulus fibrosus (AF) and nucleus pulposus (NP) cells. Collagen levels were increased in AF and decreased in NP cells. Matrix metalloproteinase-3, -9, and -13 expressions were increased, which may partially explain the impaired matrix production. We also found collagen I, II, aggrecan, and biglycan mRNA expressions were increased in AF cells but decreased in NP cells. Apoptosis was increased in the ApoE KO NP tissue. These results suggest early disc degeneration changes in the ApoE KO mice. ApoE may play a critical role in disc integrity and function.

During neonatal development, tendons undergo a well-orchestrated process whereby extensive structural and compositional changes occur in synchrony to produce a normal tissue. Conversely, during the repair response to injury, structural and compositional changes occur, but a mechanically inferior tendon is produced. As a result, developmental processes have been postulated as a potential paradigm through which improved adult tissue healing may occur. By examining injury at distinctly different stages of development, vital information can be obtained into the structure-function relationships in tendon. The mouse is an intriguing developmental model due to the availability of assays and genetically altered animals. However, it has not previously been used for mechanical analysis of healing tendon due to the small size and fragile nature of neonatal tendons. The objective of this study was to evaluate the differential healing response in tendon at two distinct stages of development through mechanical, compositional, and structural properties. To accomplish this, a new in vivo surgical model and mechanical analysis method for the neonatal mouse Achilles tendons were developed. We demonstrated that injury during early development has an accelerated healing response when compared to injury during late development. This accelerated healing model can be used in future mechanistic studies to elucidate the method for improved adult tendon healing.

The acquisition of chondro-osteogenic phenotypes and erroneous matrix deposition may account for poor tissue quality after acute tendon injury. We investigated the presence of chondrocyte phenotype, ossification, and the changes in the expression of major collagens and proteoglycans in the window wound in a rat patellar tendon window injury model using histology, von Kossa staining and immunohistochemistry of Sox 9, major collagens, and proteoglycans. Our results showed that the repair tissue did not restore to normal after acute injury. Ectopic chondrogenesis was observed in 33% of samples inside wound at week 4 while ectopic ossification surrounded by chondrocyte-like cells were observed in the window wound in 50% of samples at week 12. There was sustained expression of biglycan and reduced expression of aggrecan and decorin in the tendon matrix in the repair tissue. The erroneous deposition of extracellular matrix and ectopic chondro-ossification in the repair tissue, both might influence each other, might account for the poor tissue quality after acute injury. Higher expression of biglycan and aggrecan were observed in the ectopic chondro-ossification sites in the repair tissue, suggesting that they might have roles in ectopic chondro-osteogenesis. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:37–46, 2012

Item does not contain fulltext Supraspinatus tendon overuse injuries lead to significant pain and disability in athletes and workers. Despite the prevalence and high social cost of these injuries, the early pathological events are not well known. We analyzed the potential relation between glycosaminoglycan (GAG) composition and phenotypic cellular alteration using a rat model of rotator cuff overuse. Total sulfated GAGs increased after 4 weeks of overuse and remained elevated up to 16 weeks. GAG accumulation was preceded by up-regulation of decorin, versican, and aggrecan proteoglycans (PGs) mRNAs and proteins and biglycan PG mRNA after 2 weeks. At 2 weeks, collagen 1 transcript decreased whereas mRNAs for collagen 2, collagen 3, collagen 6, and the transcription factor Sox9 were increased. Protein levels of heparin affine regulatory peptide (HARP)/pleiotrophin, a cytokine known to regulate developmental chondrocyte formation, were enhanced especially at 4 weeks, without up-regulation of HARP/pleiotrophin mRNA. Further results suggest that the increased GAGs present in early lesions may sequester HARP/pleiotrophin, which could contribute to a loss of tenocyte's phenotype. All these modifications are characteristic of a shift towards the chondrocyte phenotype. Identification of these early changes in the extra-cellular matrix may help to prevent the progression of the pathology to more disabling, degenerative alterations. 01 januari 2012

To evaluate changes in matrix molecules of the joint capsule, the right knees of 24 skeletally mature female NZW rabbits were immobilized while the contralateral limb served as an unoperated control. The immobilization was discontinued at 8 weeks and the rabbits were divided among four groups (n = 6) based on the number of weeks the right knees were remobilized: 0, 8, 16, or 32. Three rabbits (six knees) that did not have operations provided normal control joint capsules. The mRNA levels for collagen types I, II, and III, and MMP-1 and -13 were significantly increased in the joint capsules of the contracture knees in all groups when compared to normal and contralateral limb joint capsules. In contrast, the mRNA levels for TIMP-1, -2, and -3 were decreased in the joint capsules of the contracture knees in all groups when compared to normal and contralateral limb joint capsules. The mRNA levels for lumican and decorin were increased in the joint capsules of the contracture knees in all groups when compared to normal capsules. Many of the changes observed in this animal model are similar to those observed in human joint capsules from posttraumatic elbow contractures, supporting the value of this rabbit model.

Growth/differentiation factors (GDFs) play a significant role in numerous skeletal tissues and processes. Previous work using the brachypod mouse has suggested that GDF-5 affects Achilles tendon composition, ultrastructure, and material behavior, as well as tendon repair. The aim of the present study was to examine the role of a related GDF family member, GDF-7 (BMP-12), in intact tendon by studying the Achilles tendon of genetically engineered knockout mice. Achilles tendons from 16-week-old GDF-7 -/- mice contained 14% less GAG/DNA than did wild type littermates (p = 0.0481), although collagen content was comparable to controls. Quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) results show that GDF-5 was upregulated two-threefold in response to the absence of GDF-7 protein. GDF-6 was also upregulated in knockouts, but to a lesser extent (twofold, p = 0.0013). On an ultrastructural level, GDF-7 deficient Achilles tendons exhibited a shift towards smaller diameter fibrils which resulted in a small but significant reduction in mean fibril diameter (-8%, p = 0.05). GDF-7 deficiency did not noticeably affect the expression of fibrillar collagens (I, III, V) or tendon proteoglycans (decorin, fibromodulin, lumican, biglycan, versican, aggrecan). Differences in tendon composition and ultrastructure were not biologically significant enough to have a noticeable effect on the structural or material behavior of the tendons. These results demonstrate that GDF-7 deficiency has a subtle effect on the composition and ultrastructure of murine Achilles tendon. The small magnitude of the observed differences may be due to overcompensation by related GDF family members.

The unilateral canine model is the most commonly used model of experimental osteoarthritis (OA). In this model, the anterior cruciate ligament (ACL) of one knee is transected and the contralateral joint is usually used as a control. However, dogs, similar to humans, can develop OA spontaneously with old age. Additionally, certain breeds of dogs are genetically predisposed to OA and can develop symptoms at a young age. The goal of this study was to compare the pathological changes of proteoglycans in OA cartilage from dogs that developed OA spontaneously to those that underwent ACL transection. For this reason, biglycan, decorin and fibromodulin levels and degradation patterns were compared by Western blot hybridization, and aggrecan contents were quantified by dimethylmethylene blue assay. The changes in proteoglycan levels in the cartilage of dogs with spontaneous OA, regardless of their age, were very similar to those published for human OA cartilage. However, when OA developed as a result of ACL-surgery, the changes in proteoglycans were different from those of slowly developing spontaneous OA. Therefore, these differences should be taken into consideration when the ACL-transection model is used.

Osteoarthritis (OA) is the most common form of arthritis and patients with meniscal and ligament injuries of the knee are at high risk to develop the disease. The purpose of this study was to evaluate molecular and structural changes occurring in four articular cartilage (AC) regions from the knees of anterior cruciate ligament (ACL)-transected rabbits at 3 and 8 weeks post-surgery. Rabbit AC from the lateral and medial femoral condyles (LFC and MFC) as well as from the medial and lateral tibial plateau (MTP and LTP) were processed for histology and for semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for a subset of relevant molecules (collagen II, aggrecan. biglycan, decorin, fibromodulin, MMP-1, -3, -13, and TIMP-1). While the most severe histological changes were observed in the MTP starting as early as 3 weeks post-ACL transection based on Mankin scores, histological examination demonstrated a progression of osteoarthritic changes in the MFC from 3 to 8 weeks post-surgery. In contrast, very few changes were observed within both the LFC and LTP, and these changes did not worsen with increasing time after surgery. The water content increased significantly in the MFC at 8 weeks post-ACL transection and at both 3 and 8 weeks post-ACL transection in the MTP. Significant decreases in DNA content were observed for the MFC, LTP and MTP at 8 weeks post-ACL transection. Total RNA yields from the MFC and MTP were significantly elevated at 8 weeks post-ACL transection, while in the lateral compartment total RNA was unchanged following ACL transection. Analysis of mRNA levels for a subset of matrix molecules, proteinases and proteinase inhibitors, by RT-PCR demonstrated significant region-specific changes at the mRNA level following ACL transection. These results show that following ACL transection, complex molecular, as well as structural changes occur early in cartilage and that the observed changes are both region-specific and time-dependent.

To assess the mRNA expression of extracellular matrix genes which might correlate with or contribute to mechanically weaker medial collateral ligament (MCL) scars in the ACL-deficient rabbit knee joint compared to those in anterior cruciate ligament (ACL) intact knee joints, a bilateral MCL injury was induced in 10 skeletally mature female NZW rabbits. As part of the same surgical procedure, the ACL was transected in one of the knees while the contralateral knee had a sham procedure. The side having the combined MCL and ACL injury was randomly assigned. After six weeks, the rabbits were euthanized. Histological assessments were performed on samples of the MCL scars from each operated knee (n = 3 animals) and mRNA levels for collagen type I, III, V, decorin, biglycan, lumican, fibromodulin, TGF-beta, IL-1, TNF-alpha, MMP-1, MMP-13, and a housekeeping gene (GAPDH) were assessed using semiquantitative RT-PCR on RNA isolated from the MCL scar tissue of the remaining animals (n = 7 animals). Levels of mRNA for each gene were normalized using the corresponding GAPDH value. Results showed that the total RNA yield (per mg wet weight) in the MCL scar of the ACL-deficient knee was significantly greater than that in the MCL scar from the ACL-intact knee. Collagen type I mRNA levels were significantly lower and mRNA levels for TNF-alpha were significantly greater in the scars of ACL-deficient knees compared to scars from ACL-intact joints. There were no significant differences between ACL-deficient and ACL-intact knees with respect to MCL scar mRNA levels for the remaining genes assessed. Histologically, the "flaw" area, which has been shown to correlate with mechanical properties in previous studies, was significantly greater in MCL scars from ACL-deficient knees than in the ACL-intact MCL scars. The mean number of cells/mm2 in MCL scars from ACL-deficient knees was significantly greater than in MCL scars from ACL-intact knees. The present study suggests that MCL scar cell metabolism is differentially influenced by the combined injury environment.

To test the hypothesis that loading conditions can be used to engineer early ligament scar behaviors, we used an in vitro system to examine the effect that cyclic hydrostatic compression and cyclic tension applied to 6-week rabbit medial collateral ligament scars had on mRNA levels for matrix molecules, collagenase, and the proto-oncogenes c-fos and c-jun. Our specific hypothesis was that tensile stress would promote more normal mRNA expression in ligament whereas compression would lead to higher levels of mRNA for cartilage-like molecules. Femur (injured medial collateral ligament)-tibia complexes were subjected to a hydrostatic pressure of 1 MPa or a tensile stress of 1 MPa of 0.5 Hz for 1 minute followed by 14 minutes of rest. On the basis of a preliminary optimization experiment, this 15-minute testing cycle was repeated for 4 hours. Semiquantitative reverse transcription-polymerase chain reaction analysis was performed for mechanically treated medial collateral ligament scars with use of rabbit specific primer sets for types I, II, and III collagen, decorin, biglycan, fibromodulin, versican, aggrecan, collagenase, c-fos, c-jun, and a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. Cyclic hydrostatic compression resulted in a statistically significant increase in mRNA levels of type-II collagen (171% of nonloaded values) and aggrecan (313% of nonloaded values) but statistically significant decreases in collagenase mRNA levels (35% of nonloaded values). Cyclic tension also resulted in a statistically significant decrease in collagenase mRNA levels (66% of nonloaded values) and an increase in aggrecan mRNA levels (458% of nonloaded values) but no significant change in the mRNA levels for the other molecules. The results show that it is possible to alter mRNA levels for a subset of genes in scar tissue by supplying unique mechanical stimuli in vitro and thus that further investigation of scar engineering for potential reimplantation appears feasible.

A region of fibrocartilage develops in bovine deep flexor tendon where the tissue wraps around bone and is subjected to compressive and shear forces in addition to tension. There is no fibrocartilage at this location in fetal tendon or in adjacent adult tendon that is subjected to tensional load only. We investigated the development of fibrocartilage in tendon using in situ hybridization to localize cells that express collagen and proteoglycan genes typical of either tendon or cartilage. The signal for type I collagen and decorin was high in cells throughout fetal and newborn tendon, as is expected in a growing tissue composed predominantly of type I collagen. No signal for aggrecan was seen in either fetal or newborn tendon. No hybridization with any of the probes for collagen or proteoglycan was detected in cells in the tensional region of adult tendon, indicating that the cells in this tissue are normally quiescent. However, the cells in the fibrocartilage of adult tendon displayed a high level of expression for types I and II collagen, decorin, biglycan, and aggrecan. This suggests that the fibrocartilage in adult tendon is a dynamic tissue. Expression of type IIA collagen is considered a marker of prechondrocytes. Type IIA collagen gene expression was present throughout both the tensional and compressed regions of fetal and newborn tendon but was absent in cartilage and adult tendon. This suggests that cells located throughout fetal tendon may have the capacity to develop as chondrocytes. Fibrocartilage signal was detected for type I collagen in 75% of the cells and for type II collagen in 50% of the cells at one location in adult tendon, suggesting that some cells in this tissue could have expressed mRNA for both type I and type II collagen.

A human chondrosarcoma cell line has been established from an aggressive chondrosarcoma. The cells grow in a monolayer culture (doubling time: 2 days) and form aggregates. The aggregates consist of a rim of cells surrounding a hollow core. The cell line exhibits a unique pattern of mRNA expression with several molecules characteristic of the chondrocyte phenotype. Consistent with the chondrocyte phenotype, mRNAs encoding types IX and XI collagens were present along with an abundant expression of mRNAs encoding the core protein of the cartilage proteoglycans biglycan and aggrecan. No expression of mRNAs encoding types I or II fibrillar collagens or the proteoglycan decorin was observed. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of [35S]sulfate-radiolabeled material confirmed the translation of proteoglycans containing glycosaminoglycan chains. The expression of molecules that contribute to cartilage development and tumorigenesis was examined. The cell line produces abundant mRNA that encodes transforming growth factor-beta1, a member of a family of cartilage and bone inductive proteins. The expression of mRNA encoding two proteins associated specifically with chondrogenesis was detected: Cart-1, a homeobox protein involved in cartilage differentiation, and CD-RAP, a secreted molecule restricted under normal conditions to differentiating chondrocytes and cartilage. Overexpression of p53, a tumor-suppressor gene, was detected. DNA analysis revealed a loss of heterozygosity at the chromosomal locus encoding p53, with the deletion of one p53 allele and the mutation of the remaining allele in both the parent tumor and the cell line. The malignant chondrosarcoma phenotype may be related to the unique gene expression pattern that is characteristic in many ways of differentiating chondroblasts, as well as to the inactivation of the p53 function that could contribute to the proliferative capacity of the cell line. This cell line may serve as a biological model for further investigation of the etiology of human chondrosarcomas and for the synthesis and regulation of cartilage-specific genes.

The study was designed to determine the contents and distributions of the proteoglycans decorin and biglycan in adult human femoral condylar cartilage and whether these may change in osteoarthritis. New radioimmunoassays were established using peptides representing the amino-terminal 21 amino acid sequence of each proteoglycan (to which a tyrosine was added for radioiodination) and antibodies in a rabbit antiserum raised to both these molecules. Cartilage was extracted with 4 M guanidine HCl to determine total content, and extracts were analyzed by chromatography to determine molecular sizes. Frozen sections were cut parallel to the articular surface and were extracted to determine distribution within the tissue. Gel chromatography on Sepharose CL-2B under dissociative conditions revealed molecules with a partition coefficient of 0.7-0.75 in both normal and osteoarthritic cartilage. In normal adult cartilage, the mean contents of the core proteins of biglycan and decorin were calculated to be approximately 0.34 and 0.48 mg per gram wet weight, respectively. These represented molar contents similar to that of aggrecan. In osteoarthritic cartilage, there were no overall significant changes in the content and distribution of these molecules. There was, however, considerable individual variation in both distribution and content. Analyses indicated that there was a trend in osteoarthritic cartilage toward a loss of biglycan and decorin from the more superficial layers of intact cartilage, where both these molecules are normally more concentrated. This was accompanied by maintenance of proteoglycan content deeper in the cartilage, regardless of the degree of degeneration.

Rotator cuff and biceps tendons that appeared grossly normal were procured from adult cadavers without a history of shoulder problems. These tendons were analyzed for the amount and type of glycosaminoglycan, type of proteoglycan, and histology. When compared with the distal/tensional region of biceps tendon, the glycosaminoglycan content of supraspinatus, infraspinatus, and subscapularis tendons was 2.5-fold higher and the glycosaminoglycan content of the proximal/compressed region of biceps tendon was 3-fold higher. The ratio of hyaluronic acid to chondroitin sulfate/dermatan sulfate in all three cuff tendons was approximately 1. Rotator cuff tendons contained large proteoglycan similar to aggrecan, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel migration elution from Sepharose CL-4B, and content of both chondroitin sulfate and keratan sulfate chains. Both decorin and biglycan were also present, as demonstrated by migration in sodium dodecyl sulfate-polyacrylamide gels and core protein immunoreactivity. In contrast decorin was the only proteoglycan prominent in distal/tensional regions of biceps tendon. Histological analysis showed layers of loosely organized alcian blue-stained material running between the longitudinal collagen fiber bundles. The proteoglycan content of rotator cuff tendons was similar to fibrocartilage in tendons that have been subjected to compressive loads in situ. This suggests that cells of normal adult rotator cuff tendons have adapted to loads distinct from pure tension. However, the histological organization did not resemble mature fibrocartilage. The increased amount of proteoglycan in rotator cuff tendons may serve to separate and lubricate collagen bundles as they move relative to each other during normal shoulder motion.

The appearance of noncollagenous proteins and proteoglycans during induction of cartilage and bone by implanted demineralized bone powder was studied by immunohistochemistry with polyclonal antibodies. Three bone proteins (osteopontin, sialoprotein, and a 62 kDa protein) were present in the bone powder grains before implantation. They appeared to be lost slowly from the granulation tissue but reappeared when bone formation started. The raw powder also contained a cartilage protein, biglycan (S1), chondrocalcin, cartilage oligomeric matrix protein, and the large proteoglycan aggrecan. The amounts of these molecules, however, increased significantly both within and outside the grains on cartilage formation. Cartilage matrix protein (148 kDa protein) appeared sparsely. The 58 kDa protein and fibromodulin (59 kDa protein), particularly the latter, were prevalent in fibrillar bundles. Antibodies against the laminin-staining vessel basement membranes showed an abundant occurrence of capillaries within the matrix grains in the granulation tissue and in the precartilaginous tissue. Bone powder made noninductive by 4 M guanidine HCl did not induce cartilage and did not stain for antibodies against bone proteins or for molecules restricted to cartilage.

Proteoglycan content and tissue morphology were examined in tendons and ligaments from 24 cadavers, ranging in age at the time of death from 1.5 months to 83 years. The region of the human tibialis posterior tendon that passes under the medial malleolus was characterized by cells having a rounded shape, positive staining with alcian blue, and higher glycosaminoglycanuronic acid content than in the more proximal region of the same tendon. Analysis of proteoglycans by sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicated that the predominant small proteoglycan of the proximal/tensional region was decorin, whereas two types of small proteoglycans (decorin and biglycan) and large proteoglycans were present in the region passing under the medial malleolus and presumably subjected to compressive and shear forces in addition to tension. The pattern of proteoglycan accumulation in the compressed region of tendon was basically similar for all individuals and showed no distinctive trends related to age after puberty. In terms of type and amount of proteoglycan, the patellar tendon was like the tensional region of the tibialis posterior. Glycosaminoglycan content in the lateral collateral ligament and anterior cruciate ligament, however, was twofold higher than in the tendons. The ligaments contained large as well as small proteoglycans, just as in the compressed region of tendon.

Osteoarthritis is characterized by a loss of extracellular matrix that leads to cartilage degradation and joint space narrowing. Specific proteases, including the aggrecanases ADAMTS‐4 and matrix metalloproteinase 3, are important in initiating and promoting cartilage degradation in osteoarthritis. This study investigated protease‐specific and disease‐specific cleavage patterns of particular extracellular matrix proteins by comparing new peptide fragments, neopeptides, in specific exogenous protease‐driven digestion of a crude cartilage proteoglycan extract and an in‐vitro model of early osteoarthritis. Additionally, equine cartilage explants were treated with interleukin‐1 and the media collected. Proteolytic cleavage products following trypsin digestion were then identified using tandem mass spectrometry. Complete sequences of proteolytically cleaved neopeptides were determined for the major cartilage proteoglycans aggrecan, biglycan, decorin, fibromodulin plus cartilage oligomeric matrix protein. The generation of neopeptides varied with enzyme specificity; however, some peptides were common to all samples. Previous known and novel cleavage sites were identifies. The identification of novel peptide fragments provides a platform for the development of antibodies that could assist in the identification of biomarkers for osteoarthritis (OA), as well as the identification of basic biochemical processes underlying OA. © 2015 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 34:106–120, 2016.