The Effects and Molecular Mechanisms of Low-Level Laser on Migration, Proliferation and Collagen Metabolism of Tenocytes---Potential Mechanisms of Laser Therapy for Promoting Tendon Healing

Low-level laser (LLL) therapy has been used for treating soft tissue musculoskeletal injuries, chronic pain, fractures, and wound healing. Many studies have revealed the effectiveness of LLL therapy to treat patients with tendinopathy. However, scientific evidence of the effects and underlying molecular mechanisms for laser to treat tendinopathy remained limited. Tendon structure consists mainly of dense collagen arranged in a linear fashion and the basic cellular component, i.e. fibroblasts (tendon cells; tenocytes). The tenocyte is the source of collagen production, protein mediators of repair, and matrix proteoglycans. For an injured tendon, the healing process requires tenocytes migrate into the repaired site, proliferate actively, and are responsible for the abundant deposition of extracellular matrix in the tissue. Therefore, adequate tendon healing requires tenocye proliferation, migration as well as production of an appropriate extracellular matrix (mainly type I collagen). However, to our knowledge, there are few reports related to the effect and underlying molecular mechanism of LLL therapy on tendon healing. Researches to investigating the aforementioned issues are of much clinical relevance and worthy to de performed Therefore, this 3-year research project is aimed to investigate the effects and molecular mechanisms of LLL on migration, proliferation and collagen metabolism of tenocytes: 1. The purpose of the first year project is to evaluate the effect and molecular mechanism of LLL on tenocyte migration with or without the presence of ibuprofen. Tenocytes intrinsic to Sprague-Dawley rats will be harvested and treated with low-level laser (LLL)(660 nm). In vitro wound closure model is used to document the effect of LLL on wound healing in vitro. Transwell filter migration assay and cell spreading assay are used to evaluate the cell migratory ability. RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis are used to evaluate the gene and protein expressions of α-smooth muscle (SM) actin, paxillin, focal adhesion kinase (FAK) as well as phosphorylated FAK. Then ibuprofen will be added to LLL treated cells to evaluate the combined effect of LLL and ibuprofen on tenocyte migration. 2. The purpose of the second year project is to evaluate the effect and molecular mechanism of LLL on tenocyte proliferation with or without the presence of ibuprofen. Tenocyte proliferation after LLL treatment will be assessed by MTT assay. Expressions of cell-cycle check-point proteins such as p21, proliferating cell nuclear antigen and retinoblastoma protein will be evaluated by RT-PCR and/or Western-blot analysis. Flow cytometry will be used to evaluate the change of phases of cell cycle of tenocytes after LLL treatment. Then ibuprofen will be added to LLL treated cells to evaluate if the inhibitory effect of ibuprofen on tenocyte can be counteracted by LLL treatment. 3. The purpose of the third year project is to evaluate the effect and molecular mechanism of LLL on expressions of collagen and matrix metalloproteinases of tenocytes with or without the presence of ibuprofen. RT-PCR will be used to evaluate the gene expressions of types I and III collagen, MMP-1, -2, -8, -9, and -13 of LLL-treated tenocytes. Protein expressions of types I and III collagen, MMP-1, -8, and -13 will be determined by Western-blot analysis. Gelatin zymography will be used to evaluate the enzymatic activities of MMP-2 and MMP-9. Enzyme-linked immunosorbent assay will be used to measure the concentration of TGF-β1 in culture supernatant of tendon cells. Then ibuprofen will be added to LLL treated cells to evaluate if the inhibitory effect of ibuprofen on tenocyte can be counteracted by LLL treatment. The result from this research project may provide better understanding of the effects and underlying mechanisms of LLL on tendon cells. Meanwhile, it can document whether the inhibitory effect of ibuprofen on tenocyte can be counteracted by LLL.

Project ID:PC10101-1789
External Project ID:NSC99-2314-B182-013-MY3