Mos, M. de

Tendon degeneration is not mediated by regulation of Toll-like receptors 2 and 4 in human tenocytes (Article)

2009-08-01T00:00:00Z

We hypothesized that expression of Toll-like receptors (TLRs) 2 and 4 by tenocytes is involved in the catabolic processes of tendon degeneration. We investigated TLR2 and TLR4 expression by tenocytes in healthy and tendinotic Achilles tendons. We also investigated whether TLR2 and TLR4 could be upregulated in tendon explants using proinflammatory cytokines interleukin (IL)-1beta and tumor necrosis factor alphpa (TNFalpha). Peroperatively harvested healthy (n = 5) and tendinotic (n = 13) Achilles tendon samples were examined by real-time RT-PCR and immunohistochemical staining for TLR2 and TLR4. In addition, the catabolic process in tendinopathy was analyzed by real-time RT-PCR for matrix metalloproteinases MMP1, MMP3, MMP9, and MMP13. Furthermore, healthy tendon explants were cultured in the presence of 20 ng/ml IL-1beta (n = 10) or 10 ng/mL TNFalpha (n = 8) for 4, 24, 48, and 72 h before analysis of TLR and MMP expression levels. Although mRNA levels for both TLR2 and TLR4 were detected in healthy and tendinotic Achilles tendons, we could not confirm expression of these receptors by immunohistochemical staining in either healthy or tendinotic tendon samples. Both receptors did not show significant transcriptional regulation in tendinopathy, although MMP3 was downregulated and MMP9 was upregulated in tendinopathy. In tendon explant cultures TLR2 mRNA was upregulated by TNFalpha (p < 0.05) and IL-1beta (not significant). TLR4 gene expression was not altered by addition of IL-1beta or TNFalpha. Tendon tissue can be stimulated to increase TLR2 gene expression by addition of catabolic factors TNFalpha or IL-1beta. However, the catabolic processes in Achilles tendinopathy cannot be attributed to regulation of TLR2 and TLR4 by tenocytes.

In vitro model to study chondrogenic differentiation in tendinopathy (Article)

2009-06-01T00:00:00Z

BACKGROUND: Treatment of midportion Achilles tendinopathy is hampered by limited knowledge of the pathophysiology. HYPOTHESIS: Chondrogenic differentiation of tendon cells might take place in midportion Achilles tendinopathy and could be used as a target for drug treatment. An in vitro model for chondrogenic differentiation would be useful to evaluate existing and future treatment opportunities. Study: A controlled laboratory study. METHODS: Perioperatively harvested tissue from human midportion Achilles tendinotic lesions and healthy Achilles tendons was analyzed by microscopy and real-time reverse transcription polymerase chain reaction. In vitro chondrogenic differentiation of tendon explants was induced using transforming-growth-factor beta. This model was modulated by removing the chondrogenic stimulus or adding triamcinolone or platelet-rich plasma. RESULTS: Midportion Achilles tendinotic lesions had increased glycosaminoglycan staining and more rounded cell nuclei. Chondrogenic markers (sex-determining region Y)-box9, aggrecan, collagen 2, and RUNT-related transcription factor 2 were upregulated, but collagen 10 was not. Nondegenerative tendon explants cultured on chondrogenic medium had higher expression of aggrecan, collagen 2, and collagen 10 but not (sex-determining region Y)-box9 and RUNT-related transcription factor 2. Removing the chondrogenic stimulus decreased expression of aggrecan, collagen 2, and collagen 10. Both triamcinolone and platelet-rich plasma influenced the chondrogenic gene expression pattern in the in vitro model. CONCLUSION: Chondrogenic differentiation is present in midportion Achilles tendinopathy. An in vitro model to study this chondrogenic differentiation was developed. CLINICAL RELEVANCE: This model can be used to investigate chondrogenic differentiation as a possible target for drug treatment, contributing to the development of more successful mechanism-based treatment opportunities.

The effect of focused extracorporeal shock wave therapy on collagen matrix and gene expression in normal tendons and ligaments (Article)

2009-04-01T00:00:00Z

REASONS FOR PERFORMING STUDY: Extracorporeal shock wave therapy (ESWT) is frequently used in equine practice, but little is known about its biological action. OBJECTIVES: To study the effects of ESWT on matrix structure and gene expression levels in normal, physiologically loaded tendinous structures in ponies. METHODS: Six Shetland ponies, free of lameness and with ultrasonographically normal flexor and extensor tendons and suspensory ligaments (SL), were used. ESWT was applied at the origin of the suspensory ligament and the mid-metacarpal region of the superficial digital flexor tendon (SDFT) 6 weeks prior to sample taking, and at the mid-metacarpal region (ET) and the insertion on the extensor process of the distal phalanx (EP) of the common digital extensor tendon 3 h prior to tendon sampling. In all animals one forelimb was treated and the other limb was used as control. After euthanasia, tendon tissue was harvested for real-time PCR to determine gene expression levels and additional samples were taken for histological evaluation and biochemical analyses RESULTS: Histologically a disorganisation of the normal collagen structure was observed 3 h after ESWT, remnants of which were still visible after 6 weeks. While degraded collagen levels showed an increase at 3 h post treatment (P= 0.012) they were reduced at 6 weeks post ESWT (P = 0.039). Gene expression for both COL1 (P = 0.004) and MMP14 (P = 0.020) was upregulated at 6 weeks after treatment. CONCLUSIONS: Exposure of normal tendinous tissue to ESWT is not uneventful; it leads to a disorganisation of matrix structure and changes in degraded collagen levels. The upregulation of COL1 expression 6 weeks after ESWT may be indicative for repair. POTENTIAL RELEVANCE: The observed disorganisation of the collagen network warrants caution when using ESWT. Exposing noninjured tissue to ESWT should be avoided and it may be advisable to restrict exercise in recently treated patients. However, the induced tissue disorganisation might also be a trigger for repair in chronic tendinopathies.

Tendon Cell Behavior and Matrix Remodeling in Degenerative Tendinopathy (Doctoral Thesis)

2009-01-07T00:00:00Z

Tendon injuries are common in human athletes [1-4]. Furthermore, such injuries are also prevalent in the ageing sedentary population [5-7]. In recent decades, the incidence of tendon injuries has risen due to both an increase in an elderly population and a rise in participation in recreational and competitive sporting activities. In the general population the lifetime cumulative incidence of Achilles tendinopathy is 5.9 % among sedentary people and 50 % among elite endurance athletes [2]. Despite the high frequency, there are still many unsolved questions and differences of opinion concerning pathology, etiology, and even terminology. Until several years ago the most often used word for tendon disease in the clinical practice of orthopaedic and sports medicine was ’tendonitis/ tendinitis’, literally meaning tendon inflammation, reflecting the general idea that overuse tendinopathies were due to inflammation. However, this common wisdom was challenged by that time, as the histopathological feature usually described in tendinopathies was a degenerative process and inflammation was not typically seen [8-11]. Therefore Nicola Maffulli suggested to use the term ‘tendinopathy’ as a general descriptor of the clinical conditions in and around tendons arising from overuse [12, 13]. In addition the term ‘tendinosis’, literally meaning tendon degeneration, should be used after histopathological examination. This nomenclature is gradually being integrated now in research communication and clinicial practice. The clinical presentation of tendinopathy is characterized by a combination of pain, swelling, and impaired performance. A variety of tendons in humans may be affected including the supraspinatus tendon in the shoulder, the forearm extensor and flexor muscle tendons in the elbow, and the Achilles tendon and the patellar tendon in the lower limb. The respons of tendinopathy to the currently available treatment options is often unsatisfactory requiring lengthy periods of rehabilitation or even surgical intervention [14, 15].

Cutaneous tactile allodynia associated with microvascular dysfunction in muscle (Article)

2008-10-28T00:00:00Z

Background: Cutaneous tactile allodynia, or painful hypersensitivity to mechanical stimulation of the skin, is typically associated with neuropathic pain, although also present in chronic pain patients who do not have evidence of nerve injury. We examine whether deep tissue microvascular dysfunction, a feature common in chronic non-neuropathic pain, contributes to allodynia. Results: Persistent cutaneous allodynia is produced in rats following a hind paw ischemia-reperfusion injury that induces microvascular dysfunction, including arterial vasospasms and capillary slow flow/ no-reflow, in muscle. Microvascular dysfunction leads to persistent muscle ischemia, a reduction of intraepidermal nerve fibers, and allodynia correlated with muscle ischemia, but not with skin nerve loss. The affected hind paw muscle shows lipid peroxidation, an upregulation of nuclear factor kappa B, and enhanced pro-inflammatory cytokines, while allodynia is relieved by agents that inhibit these alterations. Allodynia is increased, along with hind paw muscle lactate, when these rats exercise, and is reduced by an acid sensing ion channel antagonist. Conclusion: Our results demonstrate how microvascular dysfunction and ischemia in muscle can play a critical role in the development of cutaneous allodynia, and encourage the study of how these mechanisms contribute to chronic pain. We anticipate that focus on the pain mechanisms associated with microvascular dysfunction in muscle will provide new effective treatments for chronic pain patients with cutaneous tactile allodynia.

Medical history and the onset of complex regional pain syndrome (CRPS) (Article)

2008-10-15T00:00:00Z

Knowledge concerning the medical history prior to the onset of complex regional pain syndrome (CRPS) might provide insight into its risk factors and potential underlying disease mechanisms. To evaluate prior to CRPS medical conditions, a case-control study was conducted in the Integrated Primary Care Information (IPCI) project, a general practice (GP) database in the Netherlands. CRPS patients were identified from the records and validated through examination by the investigator (IASP criteria) or through specialist confirmation. Cases were matched to controls on age, gender and injury type. All diagnoses prior to the index date were assessed by manual review of the medical records. Some pre-specified medical conditions were studied for their association with CRPS, whereas all other diagnoses, grouped by pathogenesis, were tested in a hypothesis-generating approach. Of the identified 259 CRPS patients, 186 cases (697 controls) were included, based on validation by the investigator during a visit (102 of 134 visited patients) or on specialist confirmation (84 of 125 unvisited patients). A medical history of migraine (OR: 2.43, 95% CI: 1.18-5.02) and osteoporosis (OR: 2.44, 95% CI: 1.17-5.14) was associated with CRPS. In a recent history (1-year before CRPS), cases had more menstrual cycle-related problems (OR: 2.60, 95% CI: 1.16-5.83) and neuropathies (OR: 5.7; 95% CI: 1.8-18.7). In a sensitivity analysis, including only visited cases, asthma (OR: 3.0; 95% CI: 1.3-6.9) and CRPS were related. Psychological factors were not associated with CRPS onset. Because of the hypothesis-generating character of this study, the findings should be confirmed by other studies.

Can platelet-rich plasma enhance tendon repair? A cell culture study (Article)

2008-06-01T00:00:00Z

BACKGROUND: Autologous platelet-rich plasma (PRP) application appears to improve tendon healing in traumatic tendon injuries, but basic knowledge of how PRP promotes tendon repair is needed. HYPOTHESIS: Platelet-rich plasma has a positive effect on cell proliferation and collagen production and induces the production of matrix-degrading enzymes and endogenous growth factors by human tenocytes. STUDY DESIGN: Controlled laboratory study. METHODS: Human tenocytes were cultured 14 days in 2% fetal calf serum medium complemented with 0%, 10%, or 20% vol/vol platelet-rich clot releasate ([PRCR] the active releasate of PRP) or platelet-poor clot releasate (PPCR). At day 4, 7, and 14, cell amount, total collagen, and gene expression of collagen I alpha 1 (COL1) and III alpha 1 (COL3), matrix metalloproteinases ([MMPs] MMP1, MMP3, and MMP13), vascular endothelial-derived growth factor (VEGF)-A, and transforming growth factor (TGF)-beta1 were analyzed. RESULTS: Platelet numbers in PRP increased to 2.55 times baseline. Growth-factor concentrations of VEGF and platelet-derived growth factor (PDGF)-BB were higher in PRCR than PPCR. Both PRCR and PPCR increased cell number and total collagen, whereas they decreased gene expression of COL1 and COL3 without affecting the COL3/COL1 ratio. PRCR, but not PPCR, showed upregulation of MMP1 and MMP3 expression. Matrix metalloproteinase 13 expression was not altered by either treatment. PRCR increased VEGF-A expression at all time points and TGF-beta1 expression at day 4. CONCLUSION: In human tenocyte cultures, PRCR, but also PPCR, stimulates cell proliferation and total collagen production. PRCR, but not PPCR, slightly increases the expression of matrix-degrading enzymes and endogenous growth factors. CLINICAL RELEVANCE: In vivo use of PRP, but also of PPP to a certain extent, in tendon injuries might accelerate the catabolic demarcation of traumatically injured tendon matrices and promote angiogenesis and formation of a fibrovascular callus. Whether this will also be beneficial for degenerative tendinopathies remains to be elucidated.

Achilles tendinosis: changes in biochemical composition and collagen turnover rate (Article)

2007-09-01T00:00:00Z

BACKGROUND: Understanding biochemical and structural changes of the extracellular matrix in Achilles tendinosis might be important for developing mechanism-based therapies. HYPOTHESIS: In Achilles tendinosis, changes occur in biochemical composition and collagen turnover rate. STUDY DESIGN: Descriptive laboratory study. METHODS: From 10 patients undergoing surgery for Achilles tendinopathy, 1 tendinosis biopsy specimen and 1 biopsy specimen of macroscopically healthy tendon tissue adjacent to the lesion were collected. Furthermore, biopsy samples were collected from 3 donors with asymptomatic Achilles tendons. Water content, collagen content, percentage of denatured collagen, amount of lysine hydroxylation, number of enzymatic and nonenzymatic crosslinks, matrix metalloproteinase activity, and matrix metalloproteinase and collagen gene-expression levels were analyzed. RESULTS: In tendinotic lesions, the water content was highest, and collagen content was subnormal with higher amounts of denatured/damaged collagen. Low pentosidine levels in tendinotic tissue indicated the presence of relatively young collagenous matrix. More hydroxylated lysine residues were present in tendinotic samples, but enzymatic crosslinks revealed no differences between tendinotic, adjacent, and healthy samples. In tendinotic specimens, matrix metalloproteinase activity was higher, matrix metalloproteinase gene-expression profile was altered, and collagen type I and III gene expression were upregulated. CONCLUSION: In Achilles tendinosis, the collagen turnover rate is increased, and the natural biochemical composition of the collagenous matrix is compromised. CLINICAL RELEVANCE: Although tendon tissue directly adjacent to an Achilles tendinosis lesion looks macroscopically healthy, histological and biochemical degenerative changes in adjacent tissue are evident, which may have implications for surgical interventions.

Intrinsic differentiation potential of adolescent human tendon tissue: an in-vitro cell differentiation study (Article)

2007-02-23T00:00:00Z

Tendinosis lesions show an increase of glycosaminoglycan amount, calcifications, and lipid accumulation. Therefore, altered cellular differentiation might play a role in the etiology of tendinosis. This study investigates whether adolescent human tendon tissue contains a population of cells with intrinsic differentiation potential. METHODS: Cells derived from adolescent non-degenerative hamstring tendons were characterized by immunohistochemistry and FACS-analysis. Cells were cultured for 21 days in osteogenic, adipogenic, and chondrogenic medium and phenotypical evaluation was carried out by immunohistochemical and qPCR analysis. The results were compared with the results of similar experiments on adult bone marrow-derived stromal cells (BMSCs). RESULTS: Tendon-derived cells stained D7-FIB (fibroblast-marker) positive, but alpha-SMA (marker for smooth muscle cells and pericytes) negative. Tendon-derived cells were 99% negative for CD34 (endothelial cell marker), and 73% positive for CD105 (mesenchymal progenitor-cell marker). In adipogenic medium, intracellular lipid vacuoles were visible and tendon-derived fibroblasts showed upregulation of adipogenic markers FABP4 (fatty-acid binding protein 4) and PPARG (peroxisome proliferative activated receptor gamma). In chondrogenic medium, some cells stained positive for collagen 2 and tendon-derived fibroblasts showed upregulation of collagen 2 and collagen 10. In osteogenic medium Von Kossa staining showed calcium deposition although osteogenic markers remained unaltered. Tendon-derived cells and BMCSs behaved largely comparable, although some distinct differences were present between the two cell populations. CONCLUSION: This study suggests that our population of explanted human tendon cells has an intrinsic differentiation potential. These results support the hypothesis that there might be a role for altered tendon-cell differentiation in the pathophysiology of tendinosis.