Tom Samiric

Changes in the composition of the extracellular matrix in patellar tendinopathy.

OBJECTIVE To compare the chemical levels and mRNA expression of proteoglycan and collagen in normal human patellar tendons and tendons exhibiting chronic overuse tendinopathy. METHODS Sulfated glycosaminoglycan and hydroxyproline content were investigated by spectrophotometric measurement using papain-digested samples. Deglycosylated proteoglycan core proteins were analysed by Western blot using specific antibodies. Total mRNA isolated from samples of frozen tendons was assayed by relative quantitative RT-PCR for decorin, biglycan, fibromodulin, versican, aggrecan, and collagens Type I, II and III and normalised to glyceraldehyde-3-phosphate dehydrogenase. RESULTS There was a significant increase in sulfated glycosaminoglycan content in pathologic tendons compared to normal. This was attributed to an increased deposition of the large aggregating proteoglycans versican and aggrecan and the small proteoglycans biglycan and fibromodulin, but not decorin. Aggrecan and versican were extensively degraded in both normal and pathologic tendons, biglycan was more fragmented in the pathologic tendons while predominantly intact fibromodulin and decorin were present in normal and pathologic tendons. There was a greater range in total collagen content but no change in the level of total collagen in pathologic tendons. There were no significant differences between the pathologic and normal tendon for all genes, however p values close to 0.05 indicated a trend in downregulation of Type I collagen and fibromodulin, and upregulation in versican and Type III genes in pathologic tissue. CONCLUSION The changes in proteoglycan and collagen levels observed in patellar tendinopathy appear to be primarily due to changes in the metabolic turnover of these macromolecules. Changes in the expression of these macromolecules may not play a major role in this process.

The Pain of Tendinopathy: Physiological or Pathophysiological?

Tendon pain remains an enigma. Many clinical features are consistent with tissue disruption—the pain is localised, persistent and specifically associated with tendon loading, whereas others are not—investigations do not always match symptoms and painless tendons can be catastrophically degenerated. As such, the question ‘what causes a tendon to be painful?’ remains unanswered. Without a proper understanding of the mechanism behind tendon pain, it is no surprise that treatments are often ineffective. Tendon pain certainly serves to protect the area—this is a defining characteristic of pain—and there is often a plausible nociceptive contributor. However, the problem of tendon pain is that the relation between pain and evidence of tissue disruption is variable. The investigation into mechanisms for tendon pain should extend beyond local tissue changes and include peripheral and central mechanisms of nociception modulation. This review integrates recent discoveries in diverse fields such as histology, physiology and neuroscience with clinical insight to present a current state of the art in tendon pain. New hypotheses for this condition are proposed, which focus on the potential role of tenocytes, mechanosensitive and chemosensitive receptors, the role of ion channels in nociception and pain and central mechanisms associated with load and threat monitoring.

Change in proteoglycan metabolism is a characteristic of human patellar tendinopathy

OBJECTIVE To determine differences in the metabolism of proteoglycans and the gene expression of proteinases and their inhibitors between patellar tendons exhibiting chronic overuse tendinopathy and normal patellar tendons in humans. METHODS Rates of loss and synthesis of proteoglycans were determined. Radiolabeled and total proteoglycans retained in and lost from the tissue were analyzed by fluorography and Western blotting. Levels of messenger RNA for matrix metalloproteinase 1 (MMP-1), MMP-2, MMP-3, MMP-9, MMP-13, ADAMTS-1, ADAMTS-4, ADAMTS-5, tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, TIMP-3, and TIMP-4 were determined in fresh tissue. RESULTS The rate of loss of (35)S-labeled proteoglycans was greater in abnormal tendons, as was the rate of synthesis of proteoglycans. Fluorography and Western blotting revealed the presence of greater amounts of large proteoglycans (aggrecan and versican) in abnormal tendons, and these proteoglycans were rapidly lost from the matrix of abnormal tendons. There was no significant difference in the expression of ADAMTS-1, ADAMTS-4, ADAMTS-5, MMP-1, MMP-2, MMP-3, MMP-13, TIMP-2, TIMP-3, or TIMP-4. There was a significant increase in the expression of MMP-9 and TIMP-1 in abnormal tendons. CONCLUSION Our findings suggest that a change in the proteoglycan content of the extracellular matrix in abnormal tendons results from the altered metabolism of the cells, reflected in the enhanced synthesis of the large proteoglycans aggrecan and versican, and does not appear to result from changes at the level of gene expression.

Relationship between compressive loading and ECM changes in tendons.

Tendons are designed to absorb and transfer large amounts of tensile load. The well organised, strong yet flexible, extracellular matrix allows for this function. Many tendons are also subject to compressive loads, such as at the entheses, as the tendon wraps around bony protuberances or from internal compression during tensile loading or twisting. Tendinopathy, the clinical syndrome of pain and dysfunction in a tendon is usually the result of overload. However, it is not only the tensile overload that should be considered, as it has been shown that compressive loads change tendon structure and that combination loads can induce tendon pathology. This review summarises how load is detected by the tenocytes, how they respond to compressive load and the resulting extracellular matrix changes that occur. Understanding the effect of compression on tendon structure and function may provide directions for future matrix based interventions.

Structure, metabolism, and tissue roles of chondroitin sulfate proteoglycans

Publisher Summary Proteoglycans (PGs) are a complex group of glycoproteins that are characterized by the presence of one or more sulfated glycosaminoglycan (GAG) chains and various oligosaccharides that are covalently attached to a core protein. The majority of chondroitin sulfate proteoglycans (CSPGs) belongs to the group of large aggregating PGs (hyalectins) and small leucine-rich PGs. The sulfated GAG chains, oligosaccharides, and domains within the core proteins of these macromolecules allow PGs to interact with a large number of macromolecules. As a result, PGs have wide‐ranging roles in tissues where they are involved in the organization and function of extracellular matrices, matrix cell interactions, and the regulation of cellular processes. The cellular and tissue location of PGs is largely dependent on the interactions of specific domains in the core proteins and the GAG chains with other molecules present in the cell membrane or extracellular structures. Proteoglycans are multifunctional macromolecules that have wide-ranging functions within the body. Not only are they involved in the organization of extracellular functions, they also have cell regulatory functions. These attributes of PGs arise from structural domains that are present on both the core proteins and the GAG chains of this group of macromolecules.

Short- and long-term exposure of articular cartilage to curcumin or quercetin inhibits aggrecan loss ☆

The aim of this study was to determine if curcumin and quercetin inhibit induced aggrecan loss from bovine articular cartilage explants given that these polyphenols have been shown to suppress the expression of matrix-degrading enzymes. The kinetics of loss of ³⁵S-aggrecan and the loss of total aggrecan in cartilage explants maintained in catabolic medium containing either 1 μM retinoic acid or 50 ng/ml interleukin (IL)-1α were studied in the presence of either 1-25 μM curcumin or 10-50 μM quercetin. The reversibility of catabolism of ³⁵S-aggrecan was also studied in catabolically stimulated cultures treated with 25 μM curcumin or 50 μM quercetin for the initial 4-5 days of culture followed by 10-15 days of culture in catabolic medium in the absence of either polyphenol. Curcumin and quercetin suppressed ³⁵S-aggrecan and total aggrecan loss from the explants in a dose-dependent manner. When the exposure of explants to curcumin or quercetin was limited to the first 4-5 days of culture, the suppression of ³⁵S-aggrecan loss was maintained in the extended culture period when the tissue was stimulated with either retinoic acid or IL-1α. Quercetin suppressed IL-1α-stimulated expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4. Curcumin suppressed retinoic acid stimulated expression of ADAMTS-5, and both polyphenols suppressed basal expression of ADAMTS-5. The ability of curcumin and quercetin to protect cartilage from stimulated aggrecan loss and to maintain this protection posttreatment may, at least in part, be due to the suppression of gene expression of ADAMTS-4 and -5.

Extracellular matrix content of ruptured anterior cruciate ligament tissue

Anterior cruciate ligaments (ACLs) can rupture with simple movements, suggesting that structural changes in the ligament may reduce the loading capacity of the ligament. We aimed to investigate if proteoglycan and collagen levels were different between ruptured and non-ruptured ACLs. We also compared changes in ruptured tissue over time. During arthroscopic knee reconstruction surgery 24 ruptured ACLs were collected from participants (10 females; 14 males; mean age 24 years). Four non-ruptured ACLs were obtained from participants undergoing total knee replacement surgery (one female, three males; mean age 66 years). Western blot analysis was used to characterise core proteins of aggrecan, versican, decorin and biglycan and glycosaminoglycan assays were also conducted. Collagen levels were measured by hydroxyproline (OHPr) assays. Significantly lower levels of collagen, were found in ruptured ACL compared to non-ruptured ACL (p=0.004). Lower levels of both small and large proteoglycans were found in ruptured than non-ruptured ACLs. No correlation was found between time since rupture and proteoglycan or collagen levels. Ruptured ACLs had less collagen and proteoglycans than non-ruptured ACLs. These changes indicate either extracellular matrix protein levels were reduced prior to rupture or levels decreased immediately after rupture. It is possible that the composition and structure of ACLs that rupture are different to normal ACLs, potentially reducing the tissues ability to withstand loading. An enhanced understanding of the aetiology of ACL injury could help identify individuals who may be predisposed to rupture.

The effectiveness of separating theory and practicum as a conduit to learning physiology

Many conventional science courses contain subjects embedded with laboratory-based activities. However, research on the benefits of positioning the practicals within the theory subject or developing them distinctly from the theory is largely absent. This report compared results in a physiology theory subject among three different cohorts of students: those taking the theory subject alone, those taking it concurrent with a physiology practicum subject, and those who previously took the subject when it had practicums embedded within the one subject. The path model shows that students taking both physiology theory and physiology practicum attained a significantly higher result in online tests compared with those who took the theory subject alone (P < 0.05) and that this translated to a significantly higher result in the end-of-semester examination. Similarly, students taking both physiology theory and the physiology practicum attained a significantly higher end-examination result compared with those who took the physiology subject in previous years when the practicums were embedded within the theory subject (P < 0.05). In both cases, this increase was largely attained in components that tested critical thinking and deep learning (short theory application questions and extended written questions). We conclude that students undertaking both physiology theory and the physiology practicum likely performed better in the theory subject due to better problem-solving skills and a more developed understanding of theoretical content. We suggest that consideration be given in all science curricula to the separation of theory and practicum by developing two subjects with clearly defined different learning outcomes.

Sulfated polysaccharides inhibit the catabolism and loss of both large and small proteoglycans in explant cultures of tendon

This study investigated the effects of two highly sulfated polysaccharides, calcium pentosan polysulfate and heparin, on the loss of newly synthesized proteoglycans from the matrix of explant cultures of bovine tendon. The tensional region of deep flexor tendon was incubated with [35S]sulfate for 6 h and then placed in culture for up to 15 days. The amount of radiolabel associated with proteoglycans lost to the medium and retained in the matrix was determined for each day in culture. It was shown that both sulfated polysaccharides at concentrations of 1000 µg·mL−1 inhibited the loss of 35S‐labeled large and small proteoglycans from the matrix and concomitant with this was a retention of chemical levels of proteoglycans in the explant cultures. In other explant cultures that were maintained in culture in the presence of both agents for more than 5 days after incubation with [35S]sulfate, inhibition of the intracellular catabolic pathway was evident, indicating that these highly sulfated polysaccharides also interfered with the intracellular uptake of small proteoglycans by tendon cells.

Promoter methylation status of the TIMP2 and ADAMTS4 genes and patellar tendinopathy

OBJECTIVES Patellar tendinopathy (PT) is a debilitating and prevalent condition that tends to affect those who are physically active or engaged in jumping sports. Although tendinopathies are known to have a genetic basis, the role of DNA methylation as an epigenetic factor and risk determinant for human PT has never been described. We sought to determine whether differences existed between the methylation profiles of both the TIMP2 and ADAMTS4 gene promoter sequences in a cohort of males having undergone surgery for patellar tendinopathy compared to controls. DESIGN Case-control epigenetic study using DNA from 10 males with PT and 10 males with healthy tendons. METHODS We used PCR and targeted pyrosequencing to interrogate the methylation profiles of CpG sites upstream of both the TIMP2 (4 sites) and ADAMTS4 (6 sites) genes. We compared methylation differences between the two groups using t-tests. RESULTS We report no significant (p>0.05) methylation differences within the TIMP2 gene promoter between the PT group and controls across the 4 CpG sites investigated. In contrast, we detected a significant (p=0.016) difference in the methylation status of 1 CpG site, approximately 3kb upstream of the ADAMTS4 gene between the PT group and controls. CONCLUSIONS To our knowledge, this is the first study to investigate how DNA methylation impacts on the risk of human tendinopathy. Our data indicate that the methylation status of the ADAMTS4 gene is altered in patellar tendinopathy and we speculate on how this change might modify the patellar tendon extra-cellular matrix environment.