Wen-Chung Tsai

Background: Diabetes mellitus is associated with tendinopathy or tendon injuries. However, the mechanism underlying diabetic tendinopathy is unclear. The purpose of this study was to examine the effects of high glucose concentrations on the activity and expression of matrix metalloproteinases, type I collagen, and type III collagen in tendon cells. Methods: Tendon cells from rat Achilles tendons were treated with 6 mM, 12 mM, and 25 mM glucose, and then cell proliferation was evaluated by the 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Messenger RNA (mRNA) expression of MMP-2, MMP-8, MMP-9, and MMP-13 and type I and type III collagen was assessed by quantitative real-time polymerase chain reaction (PCR). The enzymatic activity of MMP-2 and MMP-9 was measured by gelatin zymography. Results: The MTT assay results showed that the glucose concentration did not affect tendon cell proliferation. The results of the real-time PCR assay revealed that the mRNA expression of MMP-9 and MMP-13 was up-regulated by treatment with 25 mM glucose, whereas the mRNA expression of type I and III collagen was not affected. Gelatin zymography showed that 25 mM glucose increased the enzymatic activity of MMP-9. Conclusions: High glucose concentration up-regulates the expression of MMP-9 and MMP-13 in tendon cells, which may account for the molecular mechanisms underlying diabetic tendinopathy.

Background: Most tendon pathology is associated with degeneration, which is thought to involve cyclic loading and cumulative age-related changes in tissue architecture. However, the association between aging and degeneration of the extracellular matrix (ECM) in tendons has not been investigated extensively. Methods: We examined tenocytes from Achilles tendons taken from rats of three different ages (2, 12, and 24 months). Tenocyte viability was assessed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Quantitative real-time polymerase chain reaction (PCR) was used to determine the levels of mRNAs that encode type-I collagen, matrix metalloproteinase (MMP)-2 and −9, tissue inhibitor of metalloproteinase (TIMP)-1 and −2 and transforming growth factor (TGF)-β1. Gelatin zymography was used to evaluate the enzymatic activities of MMP-2 and −9. Furthermore, the concentration of TGF-β1 in conditioned medium was evaluated using enzyme-linked immunosorbent assay (ELISA). Results: The results of the MTT assay showed that the number of viable tenocytes decreased with age. No differences were observed in the levels of mRNAs that encode type-I collagen and TGF-β1 among the three age groups, and the TGF-β1 concentration did not change with age. However, mRNAs that encode MMP-2 and −9 were significantly more abundant in tenocytes from the aging group, and gelatin zymography revealed that the enzymatic activities of MMP-2 and −9 also increased significantly with age. Furthermore, as compared with young group, mRNAs that encode TIMP-1 and −2 were significantly decreased in tenocytes from the aging group. Conclusions: Activities of MMP-2 and MMP-9 in tenocytes increase with age. This might provide a mechanistic explanation of how aging contributes to tendinopathy or tendon rupture with age.

Quinolone‐induced tendinopathy or tendon rupture tends to be age‐related. However, the synergistic effects of quinolone and aging on tenocytes remained to be explored. Tenocytes intrinsic to rat Achilles tendon from two age groups (young: 2 months; and near senescent (old): 24 months) were treated with ciprofloxacin. Tenocyte migration and proliferation were assessed by transwell filter migration assay and MTT (3‐[4,5‐Dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide) assay, respectively. Messenger RNA and protein expressions of types I and III collagen were determined by reverse transcription‐polymerase chain reaction (RT/PCR) and Western blot analysis, respectively. Transwell filter migration assay revealed that ciprofloxacin inhibited tenocytes migration, which became more significant in old tenocytes (p < 0.05). The results of MTT assay revealed that tenocytes proliferation decreased after ciprofloxacin treatment (p < 0.05), which also became more significant in old tenocytes. The results of RT‐PCR and Western blot analysis revealed that mRNA and protein expressions of type I collagen remained unchanged in either young or old tenocytes with ciprofloxacin treatment, whereas the expressions of type III collagen were down‐regulated by ciprofloxacin, which was more significant in old tenocytes. In conclusion, aging potentiated the ciprofloxacin‐mediated inhibition of migration, proliferation, and expression of type III collagen of tenocytes. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:764–768, 2012

Ultrasound is a therapeutic agent commonly used to treat sports-related tendinopathy. Tendon healing requires tendon cells migration to the repair site, followed by the proliferation and synthesis of extracellular matrix. This study was designed to determine the effect of ultrasound on migration of tendon cells intrinsic to rat Achilles tendon. Furthermore, the existence of a correlation between this effect and the expression of the contractile actin isoform, α-smooth muscle (SM) actin, which is associated with cell mobility, was also examined. Cell migration was evaluated by transwell filter migration assay. The mRNA expressions of α-SM actin were determined by reverse transcription-polymerase chain reaction. Dose-dependent ultrasound enhancement of tendon cells migration through the transwell filter was demonstrated. Using immunofluorescence stain for α-SM actin, the percentages of α-SM actin-positive cells of total cells, nonmigrated cells, and migrated cells on the filter were calculated. Ultrasound-treated cells which had migrated to the bottom side of the filter were more likely to express α-SM actin than migrated control cells and nonmigrated cells. However, there was no change of mRNA and protein expression of α-SM actin as well as expression of FAK and p-FAK. In conclusion, ultrasound stimulates tendon cell migration in association with increased expression of α-SM actin of tendon cells.

Sports-related tendinopathy is commonly treated with nonsteroidal anti-inflammatory drugs including cyclooxygenase-2 inhibitor. Tendon healing requires migration of tendon cells to the repair site, followed by proliferation and synthesis of collagen. This study was designed to determine the effects of COX-2 inhibitor (celecoxib) on the migration, proliferation, and types I and III collagen expression of tendon cells intrinsic to rat Achilles tendon. Using cultured tendon cells, cell migration and proliferation were evaluated by transwell filter migration assay and by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, respectively. The mRNA expression of alpha1(I) procollagen and alpha1(III) procollagen were determined by reverse transcription-polymerase chain reaction (RT-PCR). The protein expression of types I and III collagen were determined by immunocytochemistry. Dose-dependent celecoxib inhibition was demonstrated for migration of tendon cells through the transwell filter migration assay (p=0.002). Dose-dependent celecoxib inhibition of tendon cell proliferation also was demonstrated by MTT assay (p=0.004). However, both RT-PCR and immunocytochemical staining revealed that mRNA and protein expression of types I and III collagen remained constant after celecoxib treatment. In conclusion, celecoxib inhibits tendon cell migration and proliferation. However, the expression of types I and III collagen remained unchanged.

Traumatic tendon injuries are commonly treated with ultrasound. However, previous research has not examined the molecular mechanism of this therapeutic effect on collagen synthesis of tendon cells. This study was designed to determine the effect of ultrasound on the expression of type I and type III collagen of tendon cells intrinsic to rat Achilles tendon. Whether a correlation exits between this effect and the expression of transforming growth factor β (TGF‐β), which enhances collagen synthesis, was also investigated. Tendon cells after ultrasound treatment and protein expression of types I and III collagen were determined by immunocytochemistry. The mRNA expressions of α1(I) procollagen, α1(III) procollagen, and TGF‐β were determined by reverse transcription‐polymerase chain reaction (RT‐PCR). Furthermore, the concentration of TGF‐β in conditioned medium was evaluated by enzyme‐linked immunosorbent assay (ELISA). Immunocytochemical staining revealed that ultrasound‐treated tendon cells were stained more strongly for types I and III collagen than were control cells. Upregulation of procollagen α1(I) gene, procollagen α1(III) gene, and TGF‐β at the mRNA level was confirmed by RT‐PCR. A dose‐dependent increase in the concentration of TGF‐β in conditioned medium obtained from cells treated with ultrasound was demonstrated by ELISA assay (p = 0.043). In conclusion, ultrasound stimulates the expression of type I and type III collagen in a process that is likely mediated by the upregulation of TGF‐β. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 24:1310–1316, 2006