Modeled Repetitive Motion Strain and Myofascial Release (MFR): Potential Roles in Fibroblast Wound Healing Thanh Cao, BA; Michael Hicks, BS; Paul Standley, PhD Department of Basic Medical Sciences, University of Arizona- College of Medicine, Phoenix, AZ Introduction Human fibroblast (HF) migration into a wound bed is followed by secretion of extracellular matrix and growth factors as well as fibroblast differentiation into contractile myofibroblasts. Manual therapies such as myofascial release (MFR) have been shown clinically effective in treating acute ankle sprain by improving recovery time and reducing reoccurring injury. Additionally, vacuum compression therapy is effective in treating diabetic foot and ischemic ulcers by accelerating wound healing. Taken together, these data suggest that biomechanical strain affects wound healing rates. We have modeled repetitive motion strain (RMS) in vitro to induce somatic dysfunction in HF culture. HF respond with increased secretion of growth factors, pro-inflammatory cytokines and increased apoptosis. In this study we used modeled RMS and MFR to investigate their potential effects on wound healing in an in vitro fibroblast wound construct. Secretion of nitric oxide is vital for the progression of wound healing as it has been shown to modify proliferation, apoptosis and migration of fibroblasts in concentration- dependant manners. In this study we will also investigate the potential roles of NO in mediating strain-regulated fibroblast wound healing. Methods HF were plated on 6-well collagen coated BioFlex plates with DMEM supplemented with 2% FBS. A 2-mm scratch wound was then applied to 80% confluent HF monolayers, followed by one of four strain profiles: no strain (control), 8 hours cyclic RMS, acyclically strained for 60 sec. (MFR), and combined RMS+MFR Phase contrast images were taken pre-strain / post wounding (time = 0) and again at 24 and 48 hours post strain. Images were analyzed by Adobe and ImageJ software to calculate the area of injury and consequent wound closure rates. Exogenous IL-1β and SNP were used to manipulate NO secretions. Fibroblast tissue constructs were stained for α- actin expression in order to assess potential fibroblast-to-myofibroblast differentiation. Antibody microarray services from Kinexus were used to track potential changes in 800 signaling proteins. Results HF subjected to RMS resulted in an 83% decrease in wound closure rate at 48 hrs when compared to non- strained group while treatment with MFR alone resulted in a ~30% reduction in wound closure (Figures 1A and 1B). RMS treated HF constructs subsequently subjected to MFR showed improvement in wound closure by ~160% when compared to RMS alone (Figures 1A and 1B). Condition media (CM) from fibroblasts previously strained by RMS impaired wound closure of non- strained fibroblast by 30% when compared to non- strained condition media. This effect was not observed with HF treated with CM from MFR and RMS+MFR groups (Figure 1C). Biomechanical strain induces fibroblast sensitivity to nitric oxide with concomitant increase in nitric oxide secretion (Figure 3A-C). HF cultures express a low level of α-actin-containing myofibroblasts. However, neither strain, IL-1  nor nitric oxide modify fibroblast-to-myofibroblast differentiation as assessed by  -actin expression (Figures 2B and 4A-F). Mechanical strain increases expression of PKG1, and RAF1 (Figure 5). Discussion Condition media experiments showed that strained fibroblasts release soluble mediators sufficient to impair wound healing in non-strained fibroblasts. IL-1β alone was insufficient to induce this response in the absence of strain. MFR improved wound healing in RMS-injured fibroblast constructs. Mechanical strain sensitizes HF to varying nitric oxide levels which appears to be concentration-dependent. This increased sensitivity may result from increased PKG1α/β and RAF1 expression. Mechanical strain has been shown to induce fibroblast nitric oxide secretion with known roles in autocrine mediated pro- and anti-mitogenic and apoptotic responses. Results from this study suggest that MFR post-RMS may accelerate wound healing by NO- mediated regulation of PKC and PKG migratory and proliferative pathways. These data suggest that short duration of slow loading sustained stretch post mechanical injury, such as those used in MFR and vacuum compression therapies, may potentially increase wound healing rate by accelerating fibroblast ability to infiltrate into the wound bed. Non Strain Time 0 Time 48 B MFR Figure 1: (A) Representative photomicrographs (40X) of human fibroblast constructs immediately after wounding (Time = 0) and 48 post-strain. The effects of modeled strain paradigms (B) and conditioned media crossover (C) on fibroblast wound healing. Different letters denote significant differences among groups (p<0.05). Figure 4: Representative images of α-actin stained (A) smooth muscle cells, (B) non-strained fibroblasts and (C-F) non-strained fibroblast treated with SNP at 0, 5, 10 and 25  M. Figure 2: Strain regulation of nitric oxide secretion (A) and myofibroblast differentiation (B). Strain Sensitive Fibroblast Wound Healing Effects of Conditioned Media on Wound Closure Acknowledgements This study was supported by the American Osteopathic Association and the NIH National Center for Complimentary and Alternative Medicine Citruline + NO g Nitric Oxide synthase Sodium nitroprusside (SNP) IL-1  + LNMMA - migration apoptosis antiproliferation etc. RMSRMS+MFR Manipulation of Nitric Oxide 0  M5  M 10  M25  M Intracellular Signaling Proteins Figure 3: (A-C): Strained and unstrained HF wound closure rates in response to SNP at 0, 5, 10, 25  M. (D): IL-1β effects on non- strained HF wound healing. A. B. C. A.B. D. B. C. A. A.. B. C. D. E. F. Figure 5: Nitric oxide-induced intracellular proteins potentially required for strain-mediated migration, proliferation and wound closure; data represent percent changes between groups listed.