Miao-Sui Lin

Ibuprofen upregulates expressions of matrix metalloproteinase‐1, ‐8, ‐9, and ‐13 without affecting expressions of types I and III collagen in tendon cells

Nonsteroidal antiinflammatory drugs are widely used to treat sports‐related tendon injuries or tendinopathy. This study was designed to investigate the effect of ibuprofen on expressions of types I and III collagen, as well as collagen‐degrading enzymes including matrix metalloproteinase (MMP)‐1, ‐2, ‐8, ‐9, and ‐13. Rat Achilles tendon cells were treated with ibuprofen and then underwent MTT [3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] assay. Reverse transcription‐polymerase chain reaction was used to evaluate mRNA expressions of types I and III collagen, MMP‐1, ‐2, ‐8, ‐9, and ‐13. Protein expressions of types I and III collagen, MMP‐1, ‐8, and ‐13 were determined by Western blot analysis. Gelatin zymography was used to evaluate the enzymatic activities of MMP‐2 and MMP‐9. The results revealed that ibuprofen upregulated expressions of MMP‐1, ‐8, ‐9, and ‐13, both at mRNA and protein levels. There was no effect of ibuprofen on mRNA and protein expressions of types I and III collagen. Gelatin zymography revealed that the enzymatic activity of MMP‐9 was upregulated after ibuprofen treatment. In conclusion, ibuprofen upregulates the expressions of collagenases including MMP‐1, ‐8, ‐9, and ‐13 without affecting the expressions of types I and III collagen. These findings suggest a molecular mechanism potentially accounting for the inhibition of tendon healing by ibuprofen.

Ciprofloxacin up-regulates tendon cells to express matrix metalloproteinase-2 with degradation of type I collagen

Ciprofloxacin‐induced tendinopathy and tendon rupture have been previously described, principally affecting the Achilles tendon. This study was designed to investigate the effect of ciprofloxacin on expressions of matrix metalloproteinases (MMP)‐2 and ‐9, tissue inhibitors of metalloproteinase (TIMP)‐1 and ‐2 as well as type I collagen in tendon cells. Tendon cells intrinsic to rat Achilles tendon were treated with ciprofloxacin and then underwent MTT (tetrazolium) assay. Real‐time reverse‐transcription polymerase chain reaction (RT‐PCR) and Western blot analysis were used, respectively, to evaluate the gene and protein expressions of type I collagen, and MMP‐2. Gelatin zymography was used to evaluate the enzymatic activities of MMP‐2 and ‐9. Reverse zymography was used to evaluate TIMP‐1 and ‐2. Immunohistochemical staining for MMP‐2 in ciprofloxacin‐treated tendon explants was performed. Collagen degradation was evaluated by incubation of conditioned medium with collagen. The results revealed that ciprofloxacin up‐regulated the expression of MMP‐2 in tendon cells at the mRNA and protein levels. Immunohistochemistry also confirmed the increased expressions of MMP‐2 in ciprofloxacin‐treated tendon explants. The enzymatic activity of MMP‐2 was up‐regulated whereas that of MMP‐9, TIMP‐1 or TIMP‐2 was unchanged. The amount of secreted type I collagen in the conditioned medium decreased and type I collagen was degraded after ciprofloxacin treatment. In conclusion, ciprofloxacin up‐regulates the expressions of MMP‐2 in tendon cells and thus degraded type I collagen. These findings suggest a possible mechanism of ciprofloxacin‐associated tendinopathy.

The promoting effect of pentadecapeptide BPC 157 on tendon healing involves tendon outgrowth, cell survival, and cell migration

Pentadecapeptide BPC 157, composed of 15 amino acids, is a partial sequence of body protection compound (BPC) that is discovered in and isolated from human gastric juice. Experimentally it has been demonstrated to accelerate the healing of many different wounds, including transected rat Achilles tendon. This study was designed to investigate the potential mechanism of BPC 157 to enhance healing of injured tendon. The outgrowth of tendon fibroblasts from tendon explants cultured with or without BPC 157 was examined. Results showed that BPC 157 significantly accelerated the outgrowth of tendon explants. Cell proliferation of cultured tendon fibroblasts derived from rat Achilles tendon was not directly affected by BPC 157 as evaluated by MTT assay. However, the survival of BPC 157-treated cells was significantly increased under the H(2)O(2) stress. BPC 157 markedly increased the in vitro migration of tendon fibroblasts in a dose-dependent manner as revealed by transwell filter migration assay. BPC 157 also dose dependently accelerated the spreading of tendon fibroblasts on culture dishes. The F-actin formation as detected by FITC-phalloidin staining was induced in BPC 157-treated fibroblasts. The protein expression and activation of FAK and paxillin were determined by Western blot analysis, and the phosphorylation levels of both FAK and paxillin were dose dependently increased by BPC 157 while the total amounts of protein was unaltered. In conclusion, BPC 157 promotes the ex vivo outgrowth of tendon fibroblasts from tendon explants, cell survival under stress, and the in vitro migration of tendon fibroblasts, which is likely mediated by the activation of the FAK-paxillin pathway.

Low-Level Laser Irradiation Stimulates Tenocyte Migration with Up-Regulation of Dynamin II Expression

Low-level laser therapy (LLLT) is commonly used to treat sports-related tendinopathy or tendon injury. Tendon healing requires tenocyte migration to the repair site, followed by proliferation and synthesis of the extracellular matrix. This study was designed to determine the effect of laser on tenocyte migration. Furthermore, the correlation between this effect and expression of dynamin 2, a positive regulator of cell motility, was also investigated. Tenocytes intrinsic to rat Achilles tendon were treated with low-level laser (660 nm with energy density at 1.0, 1.5, and 2.0 J/cm2). Tenocyte migration was evaluated by an in vitro wound healing model and by transwell filter migration assay. The messenger RNA (mRNA) and protein expressions of dynamin 2 were determined by reverse transcription/real-time polymerase chain reaction (real-time PCR) and Western blot analysis respectively. Immunofluorescence staining was used to evaluate the dynamin 2 expression in tenocytes. Tenocytes with or without laser irradiation was treated with dynasore, a dynamin competitor and then underwent transwell filter migration assay. In vitro wound model revealed that more tenocytes with laser irradiation migrated across the wound border to the cell-free zone. Transwell filter migration assay confirmed that tenocyte migration was enhanced dose-dependently by laser. Real-time PCR and Western-blot analysis demonstrated that mRNA and protein expressions of dynamin 2 were up-regulated by laser irradiation dose-dependently. Confocal microscopy showed that laser enhanced the expression of dynamin 2 in cytoplasm of tenocytes. The stimulation effect of laser on tenocytes migration was suppressed by dynasore. In conclusion, low-level laser irradiation stimulates tenocyte migration in a process that is mediated by up-regulation of dynamin 2, which can be suppressed by dynasore.

The effect of aging on migration, proliferation, and collagen expression of tenocytes in response to ciprofloxacin.

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.

Therapeutic Ultrasound Stimulation of Tendon Cell Migration

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.

Ciprofloxacin-mediated inhibition of tenocyte migration and down-regulation of focal adhesion kinase phosphorylation

Ciprofloxacin-induced tendinopathy and tendon rupture have been previously described, principally affecting the Achilles tendon. However, the underlying mechanism remains unclear. Tendon healing requires migration of tenocytes to the repair site, followed by proliferation and synthesis of the extracellular matrix. This study was designed to determine the effect of ciprofloxacin on migration of tenocytes intrinsic to rat Achilles tendon. Whether a correlation exits between this effect and the expression and phosphorylation of focal adhesion kinase (FAK), which is a positive regulator of cell motility, was also investigated. Using cultured tenocytes, migration was evaluated by counting the number of initial cell outgrowth from the tendon explants and by transwell filter migration assay. Tenocyte spreading was also evaluated microscopically. The serum-induced protein expression and phosphorylation of FAK were determined by Western blot analysis in synchronized tenocytes. Ciprofloxacin dose-dependently inhibited tenocytes outgrowth from the explants ex vivo, migration of tenocytes through the transwell filter, as well as cell spreading in vitro. Suppression of FAK phosphorylation was revealed by Western blot analyses. In conclusion, ciprofloxacin inhibits tenocyte migration in a process that is probably mediated by inhibition of FAK phosphorylation.

Platelet rich plasma releasate promotes proliferation of skeletal muscle cells in association with upregulation of PCNA, cyclins and cyclin dependent kinases

Abstract Platelet rich plasma (PRP) contains various cytokines and growth factors which may be beneficial to the healing process of injured muscle. The purpose of this study is to investigate the effect and molecular mechanism of PRP releasate on proliferation of skeletal muscle cells. Skeletal muscle cells intrinsic to Sprague–Dawley rats were treated with PRP releasate. Cell proliferation was evaluated by 3-[4,5-Dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and immunocytochemistry with Ki-67 stain. Flow cytometric analysis was used to evaluate the cell cycle progression. Western blot analysis was used to evaluate the protein expressions of PCNA, cyclin E1, cyclin A2, cyclin B1, cyclin dependent kinase (cdk)1 and cdk2. The results revealed that PRP releasate enhanced proliferation of skeletal muscle cells by shifting cells from G1 phase to S phase and G2/M phases. Ki-67 stain revealed the increase of proliferative capability after PRP releasate treatment. Protein expressions including cyclin A2, cyclin B1, cdk1, cdk2 and PCNA were up-regulated by PRP releasate in a dose-dependent manner. It was concluded that PRP releasate promoted proliferation of skeletal muscle cells in association with the up-regulated protein expressions of PCNA, cyclin A2, cyclin B1, cdk1 and cdk2.

Platelet-Rich Plasma Releasate Promotes Regeneration and Decreases Inflammation and Apoptosis of Injured Skeletal Muscle:

Background: Platelet-rich plasma (PRP) contains various cytokines and growth factors that may be beneficial to the healing process of injured muscle. Based on the authors’ previous study, PRP releasate can promote proliferation and migration of skeletal muscle cells in vitro, so animal studies are performed to support the use of PRP to treat muscle injury in vivo. Purpose: To investigate the effect of PRP releasate on regeneration of injured muscle, as well as its effect on inflammatory reaction and cell apoptosis, in the early stages of the muscle-healing process. Study Design: Controlled laboratory study. Methods: The gastrocnemius muscles of Sprague-Dawley rats were injured by partial transverse incision and then treated with PRP releasate. Hematoxylin and eosin stain was used to evaluate the healing process of injured muscle at 2, 5, and 10 days after injury. TUNEL assay was used to evaluate the cell apoptosis of injured muscle after PRP releasate treatment. Immunohistochemistry was used to stain the CD68-positive cells during the healing process. Muscle contractile properties, including fast-twitch and tetanic strength, were evaluated by electric stimulation. Results: The results revealed that PRP releasate treatment could enhance the muscle-healing process and decrease CD68-positive cells and apoptotic cells. Furthermore, the tetanic strength was significantly higher in injured muscle treated with PRP releasate. Conclusion: In conclusion, PRP releasate could enhance the healing process of injured muscle and decrease inflammatory cell infiltration as well as cell apoptosis. Clinical Relevance: PRP promotes skeletal muscle healing in association with decreasing inflammation and apoptosis of injured skeletal muscle. These findings provide in vivo evidence to support the use of PRP to treat muscle injury.

Effects of Low-Level Laser Therapy on Matrix Metalloproteinase and Collagen Expression of Tendon Cells

Objective: Low-level laser therapy (LLLT) is commonly used to treat sports-related tendinopathy or tendon injury. However, scientific evidence of the effects and underlying molecular mechanisms of tendinopathy treatment remained limited. Tendon healing requires tenocyte migration to the repair site, followed by proliferation and synthesis of the extracellular matrix, including collagens. The study was designed to determine the effect of laser on the metabolism of collagen for injuried tendon repair, furthermore, become a foundation of the LLLT for tendon repair. Methods: Tendon cells from rat Achilles tendons were obtained to investigate the effect of LLLT on collagen metabolism in tendon repair. LLLT was performed with a 600-nm laser in continuous mode with an output power of 50 mW at different periods with increasing energy densities. Real-time polymerase chain reaction (PCR) and western blot analysis were used to determine the gene expression and production of matrix metalloproteinase (MMP) and collagen. Results: The mRNA expression of MMP-9 was down-regulated by LLLT dose-dependently, the mRNA expression of type I collagen was up-regulated at an energy density of 1.0 J/cm^2, and type III collagen was up-regulated dose-dependently. Densitometry analysis using western blot indicated that protein expression of MMP-8 and MMP-9 was dose-dependently down-regulated by laser treatment and type I collagen was up-regulated at an energy density of 1.0 J/cm^2. Conclusions: LLLT dose-dependently down-regulates the expression of MMP-9 and up-regulates the expressions of type I collagen at an energy density of 1.0 J/cm^2.