Optimized Modeled Myofascial Release Enhances Wound Healing in 3-Dimensional Bioengineered Tendons: Key Roles for Fibroblast Proliferation and Collagen Remodeling.

OBJECTIVE: The purpose of this study was to evaluate the mechanisms of action of optimized myofascial release (MFR) on wound healing using a 3-dimensional human tissue construct. METHODS: Bioengineered tendons were cultured on a deformable matrix, wounded using a steel cutting tip, then strained in an acyclic manner with a modeled MFR paradigm at 103% magnitude for 5 minutes. Imaging and measurements of the width and wound size were performed daily, and the average tissue width of the entire bioengineered tendon was measured, and wound size and major and minor axes of the elliptical wound were additionally measured. Assessments of actin and collagen were performed by immunofluorescence, and Gomori's trichrome staining and fibroblast nuclei deposition was quantified using the CellProfiler analysis software. RESULTS: Optimized modeled MFR treatment significantly reduced the wound size and increased both collagen density and cell deposition at the wound site. All measures of wound healing improvements required the presence of proliferating fibroblasts. CONCLUSION: Myofascial release-induced cell deposition and collagen density at wound sites required actively proliferating fibroblasts. If clinically translatable, our results support a mechanism by which MFR improves patient wound healing.

Modeled Osteopathic Manipulative Treatments: A Review of Their in Vitro Effects on Fibroblast Tissue Preparations.

A key osteopathic tenet involves the body's ability to self-heal. Osteopathic manipulative treatment (OMT) has been evolved to improve this healing capacity. The authors' in vitro work has focused on modeling 2 common OMT modalities: myofascial release (MFR) and counterstrain. Their studies have evaluated the effects of these modalities on wound healing, cytokine secretion, and muscle repair. The key components of the host response to mechanical forces are fibroblasts, which are the main fascial cells that respond to different types of strain by secreting anti-inflammatory chemicals and growth factors, thus improving wound healing and muscle repair processes. The purpose of this review is to discuss the cellular and molecular mechanisms by which MFR and other OMT modalities work, in particular, the role of strained fibroblasts in inflammation, wound healing, and muscle repair and regeneration. Changing MFR parameters, such as magnitude, duration, direction, and frequency of strain, might uniquely affect the physiologic response of fibroblasts, muscle contraction, and wound healing. If such results are clinically translatable, the mechanisms underlying the clinical outcomes of OMT modalities will be better understood, and these treatments will be more widely accepted as evidence-based, first-line therapies.

Duration and magnitude of myofascial release in 3-dimensional bioengineered tendons: effects on wound healing.

CONTEXT: Myofascial release (MFR) is one of the most commonly used manual manipulative treatments for patients with soft tissue injury. However, a paucity of basic science evidence has been published to support any particular mechanism that may contribute to reported clinical efficacies of MFR. OBJECTIVE: To investigate the effects of duration and magnitude of MFR strain on wound healing in bioengineered tendons (BETs) in vitro. METHODS: The BETs were cultured on a deformable matrix and then wounded with a steel cutting tip. Using vacuum pressure, they were then strained with a modeled MFR paradigm. The duration of MFR dose consisted of a slow-loading strain that stretched the BETs 6% beyond their resting length, held them for 0, 1, 2, 3, 4, or 5 minutes, and then slowly released them back to baseline. To assess the effects of MFR magnitude, the BETs were stretched to 0%, 3%, 6%, 9%, or 12% beyond resting length, held for 90 seconds, and then released back to baseline. Repeated measures of BET width and the wound's area, shape, and major and minor axes were quantified using microscopy over a 48-hour period. RESULTS: An 11% and 12% reduction in BET width were observed in groups with a 9% (0.961 mm; P<.01) and 12% (0.952 mm; P<.05) strain, respectively. Reduction of the minor axis of the wound was unrelated to changes in BET width. In the 3% strain group, a statistically significant decrease (-40%; P<.05) in wound size was observed at 24 hours compared with 48 hours in the nonstrain, 6% strain, and 9% strain groups. Longer duration of MFR resulted in rapid decreases in wound size, which were observed as early as 3 hours after strain. CONCLUSION: Wound healing is highly dependent on the duration and magnitude of MFR strain, with a lower magnitude and longer duration leading to the most improvement. The rapid change in wound area observed 3 hours after strain suggests that this phenomenon is likely a result of the modification of the existing matrix protein architecture. These data suggest that MFR's effect on the extracellular matrix can potentially promote wound healing.

In vitro biomechanical strain regulation of fibroblast wound healing.

CONTEXT: Strain-directed therapy such as vacuum compression and manual manipulative therapies are clinically effective, but their cellular and molecular mechanisms are not well understood. OBJECTIVE: To determine the effects of modeled myofascial release (MFR) on fibroblast wound healing and to investigate the potential role of nitric oxide (NO) in mediating these responses. METHODS: Using an in vitro scratch wound strain model, the authors investigated human fibroblast wound healing characteristics in response to injurious repetitive motion strain (RMS) and MFR. Secretion of NO was induced with interleukin-1ß and sodium nitroprusside and inhibited with NO synthase inhibitor L-N(G)-monomethyl arginine citrate (L-NMMA) to determine the effects of NO on wound healing. Protein microarray was also performed to evaluate the expression of intracellular protein and activation of protein kinase G (PKG), extracellular signal-regulated kinase (ERK1/2), protein kinase C (PKC), and phosphoinositide 3-kinase (PI3K), the downstream effectors in the NO pathway. RESULTS: Fibroblasts that received RMS resulted in reduced wound closure rates (vs nonstrain, P<.05), which are partially attenuated by a single dose of MFR. Interleukin-1ß and exogenous NO did not appear to have an effect on nonstrained fibroblast wound healing. However, strained fibroblasts appeared to express increased sensitivity to NO. The authors also observed a 12.2% increase in NO secretion, an increase in PKG activation, and a downregulation of PKC and PI3K inhibitory domain in the combined strain group. CONCLUSION: If clinically translatable, these data suggest that mechanical strain such as vacuum compression therapy and manual manipulative therapy may modify PKC and PI3K to sensitize fibroblasts to NO and improve wound healing by promoting cell proliferation and migration by means of PKC and PKG signaling.

Dosed myofascial release in three-dimensional bioengineered tendons: effects on human fibroblast hyperplasia, hypertrophy, and cytokine secretion.

OBJECTIVE: The purpose of this study was to investigate potential differences of magnitudes and durations associated with dosed myofascial release (MFR) on human fibroblast proliferation, hypertrophy, and cytokine secretions. METHODS: Bioengineered tendons (BETs) attached to nylon mesh anchors were strained uniaxially using a vacuum pressure designed to model MFR varying in magnitudes (0%, 3%, 6%, 9%, and 12% elongation) and durations (0.5 and 1-5 minutes). Conditioned media were analyzed for cytokine secretion via protein microarray (n = 2). Bioengineered tendons were weighted and fibroblasts extracted from the BET were assessed for total cell protein and proliferation via double-stranded DNA quantification (n = 5). All data were compared by a 1-way analysis of variance with post hoc Dunnett test and Student t test. RESULTS: Changing MFR magnitude and duration did not have an effect on total fibroblast cellular protein or DNA accumulation. However, we observed a stepwise increase in BET weight with higher-magnitude MFR treatments. Longer durations of MFR resulted in progressive increase in the secretions of angiogenin, interleukin (IL)-3, IL-8, growth colony-stimulating factor, and thymus activation-regulated chemokine. Alternatively, increasing strain magnitude induced secretions of IL-1ß, monocyte chemoattractant cytokine, and regulated and normal T cell expressed and secreted chemotactic cytokine. CONCLUSION: Cellular proliferation and hypertrophy were not significantly changed by any treatment. However, the change in total BET dry weight suggests that production of extracellular matrix protein may be up-regulated. Different MFR parameters induce secretions of a unique subset of cytokines and growth factors that can be further enhanced by increasing the magnitude and duration of treatment. If clinically translatable, these results suggest that variations to manual therapy biomechanical parameters may differentially affect physiological responses in vivo.

Mechanical strain applied to human fibroblasts differentially regulates skeletal myoblast differentiation.

Cyclic short-duration stretches (CSDS) such as those resulting from repetitive motion strain increase the risk of musculoskeletal injury. Myofascial release is a common technique used by clinicians that applies an acyclic long-duration stretch (ALDS) to muscle fascia to repair injury. When subjected to mechanical strain, fibroblasts within muscle fascia secrete IL-6, which has been shown to induce myoblast differentiation, essential for muscle repair. We hypothesize that fibroblasts subjected to ALDS following CSDS induce myoblast differentiation through IL-6. Fibroblast conditioned media and fibroblast-myoblast cocultures were used to test fibroblasts' ability to induce myoblast differentiation. The coculture system applies strain to fibroblasts only but still allows for diffusion of potential differentiation mediators to unstrained myoblasts on coverslips. To determine the role of IL-6, we utilized myoblast unicultures ± IL-6 (0-100 ng/ml) and cocultures ± α-IL-6 (0-200 µg/ml). Untreated uniculture myoblasts served as a negative control. After 96 h, coverslips (n = 6-21) were microscopically analyzed and quantified by blinded observer for differentiation endpoints: myotubes per square millimeter (>3 nuclei/cell), nuclei/myotube, and fusion efficiency (%nuclei within myotubes). The presence of fibroblasts and fibroblast conditioned media significantly enhanced myotube number (P < 0.05). However, in coculture, CSDS applied to fibroblasts did not reproduce this effect. ALDS following CSDS increased myotube number by 78% and fusion efficiency by 96% vs. CSDS alone (P < 0.05). Fibroblasts in coculture increase IL-6 secretion; however, IL-6 secretion did not correlate with enhanced differentiation among strain groups. Exogenous IL-6 in myoblast uniculture failed to induce differentiation. However, α-IL-6 attenuated differentiation in all coculture groups (P < 0.05). Fibroblasts secrete soluble mediators that have profound effects on several measures of myoblast differentiation. Specific biophysical strain patterns modify these outcomes, and suggest that myofascial release after repetitive strain increases myoblast differentiation and thus may improve muscle repair in vivo. Neutralization of IL-6 in coculture significantly reduced differentiation, suggesting fibroblast-IL-6 is necessary but not sufficient in this process.

In vitro modeling of repetitive motion injury and myofascial release.

OBJECTIVE: In this study we modeled repetitive motion strain (RMS) and myofascial release (MFR) in vitro to investigate possible cellular and molecular mechanisms to potentially explain the immediate clinical outcomes associated with RMS and MFR. METHOD: Cultured human fibroblasts were strained with 8h RMS, 60s MFR and combined treatment; RMS+MFR. Fibroblasts were immediately sampled upon cessation of strain and evaluated for cell morphology, cytokine secretions, proliferation, apoptosis, and potential changes to intracellular signaling molecules. RESULTS: RMS-induced fibroblast elongation of lameopodia, cellular decentralization, reduction of cell to cell contact and significant decreases in cell area to perimeter ratios compared to all other experimental groups (p<0.0001). Cellular proliferation indicated no change among any treatment group; however RMS resulted in a significant increase in apoptosis rate (p<0.05) along with increases in death-associated protein kinase (DAPK) and focal adhesion kinase (FAK) phosphorylation by 74% and 58% respectively, when compared to control. These responses were not observed in the MFR and RMS+MFR group. Of the 20 cytokines measured there was a significant increase in GRO secretion in the RMS+MFR group when compared to control and MFR alone. CONCLUSION: Our modeled injury (RMS) appropriately displayed enhanced apoptosis activity and loss of intercellular integrity that is consistent with pro-apoptotic dapk-2 and FAK signaling. Treatment with MFR following RMS resulted in normalization in apoptotic rate and cell morphology both consistent with changes observed in dapk-2. These in vitro studies build upon the cellular evidence base needed to fully explain clinical efficacy of manual manipulative therapies.

In vitro modeling of repetitive motion strain and manual medicine treatments: potential roles for pro- and anti-inflammatory cytokines.

Despite positive clinical outcomes documented post-treatment with a variety of manual medicine treatments (MMT), the underlying cellular mechanisms responsible remain elusive. We have developed an in vitro human fibroblast cell system used to model various biomechanical strains that human fibroblasts might undergo in response to repetitive motion strain (RMS) and MMT. Our data utilizing this system suggest that RMS induces disruption of cell-cell and cell-matrix contacts, which appear are reversed when a modeled MMT is also added to the treatment protocol. Similarly, while RMS induces secretion of several inflammatory cytokines, modeled MMT attenuates this secretory response. In terms of strain direction, fibroblasts strained equiradially exhibit unique cytokine secretory profiles vs. those strained heterobiaxially. Taken together, these data suggest that this cell model may prove useful in identifying the cellular mechanisms by which various fascial strains used clinically to treat somatic dysfunctions yield positive clinical outcomes such as reduced pain, reduced analgesic use and improved range of motion.

Importance of strain direction in regulating human fibroblast proliferation and cytokine secretion: a useful in vitro model for soft tissue injury and manual medicine treatments.

OBJECTIVE: Manual medicine treatments (MMTs) rely on biophysical techniques that use manually guided forces in numerous strain directions to treat injuries and somatic dysfunctions. Although clinical outcomes post-MMT are positive, the underlying cellular mechanisms responsible remain elusive. We previously described an in vitro model of strain-induced tissue injury and MMTs. Using this model, the current study sought to determine if strain direction (equibiaxial [EQUI] vs heterobiaxial [HETERO]) differentially regulates human fibroblast function. METHODS: Fibroblasts were strained EQUI at 10% beyond their resting length for 48 hours followed by assessment of cell morphology, proliferation, and cytokine secretion via protein cytokine array and enzyme-linked immunosorbent assay (ELISA). These observations were then compared with those obtained previously for HETERO fibroblasts. RESULTS: No alterations in cell morphology were seen in EQUI fibroblasts despite our report of such changes in HETERO cells. Fibroblasts secretion profiles for 60 cytokines (via cytokine protein array) showed that in EQUI strained cells, fractalkine significantly increased (121%), whereas macrophage-derived chemoattractant/chemokine and pulmonary and activation-regulated chemokine significantly decreased (32% and 10%, respectively) compared with nonstrained cells (P < .05). The EQUI fibroblasts when compared with HETERO fibroblasts exhibited a significant decrease in proliferation (22%), inflammatory interleukin 6 secretion (75%, measured by ELISA), and macrophage-derived chemoattractant/chemokine secretion (177%, measured by ELISA, P < .05). CONCLUSIONS: These divergent observations in HETERO vs EQUI strained fibroblasts may underlie the relative efficacies of MMTs carried out in different tissue strain directions. We are currently modeling MMTs such as myofascial release to further investigate this.

Modeled repetitive motion strain and indirect osteopathic manipulative techniques in regulation of human fibroblast proliferation and interleukin secretion.

CONTEXT: Clinical studies have supported the efficacy of a variety of osteopathic manipulative techniques. However, an evidence base for the cellular mechanisms underlying these clinical findings is lacking. OBJECTIVE: To investigate human fibroblast proliferation and interleukin secretory profiles in response to modeled repetitive motion strain (RMS) and modeled indirect osteopathic manipulative techniques (IOMT). The authors hypothesized that the RMS model would increase fibroblast proliferation and proinflammatory interleukin secretion, while the IOMT model would reverse these effects. METHODS: Human fibroblasts were exposed in vitro to one of three conditions: (1) an 8-hour RMS; (2) a 60-second IOMT; or (3) an 8-hour RMS followed by a 60-second IOMT. Data on fibroblast proliferation and interleukins present in conditioned media were obtained immediately after RMS, at 24 hours after RMS (24RMS), at 24 hours after IOMT (24IOMT), and at 24 hours after RMS and IOMT (24RMS+IOMT). Cytokine protein array and enzyme-linked immunosorbent assay were used in data analysis. Fibroblast proliferation was also measured colorimetrically with a cell proliferation assay. RESULTS: Fibroblasts that underwent RMS secreted several proinflammatory interleukins 24 hours after strain cessation, with substantially increased secretion of IL-1alpha, IL-1beta, IL-2, IL-3, IL-6, and IL-16. At 24 hours after strain cessation, fibroblasts subjected to RMS also secreted increased amounts of the anti-inflammatory IL-1ra, and they displayed 15% less proliferation, compared with baseline cells (P<.05). Fibroblasts that underwent IOMT, when analyzed at 24 hours after IOMT, did not display increased interleukin secretion or proliferation. However, they did display a 44% reduction in proinflammatory IL-3 secretion when compared with baseline cells (P<.05). The use of 24RMS+IOMT did not induce interleukin secretion in fibroblasts that were analyzed 24 hours after the combined exposure. However, cells in the 24RMS+IOMT group did display a 46% reduction in proinflammatory IL-6 secretion compared with RMS alone (24RMS; P<.05), as well as a 51% increase in proliferation compared with the 24RMS group (P<.05). CONCLUSION: An in vitro strain model that simulates RMS has different effects on fibroblast proliferation and interleukin secretion than does an in vitro model that simulates IOMT. Modeled RMS appears to cause a reduction in fibroblast proliferation and a delayed inflammatory response. Modeled IOMT not only fails to induce this response, it also reverses inflammatory effects in cells that have been strained repetitively. Data from the present study suggest that fibroblast proliferation and expression/secretion of proinflammatory and anti-inflammatory interleukins may contribute to the clinical efficacy of indirect osteopathic manipulative techniques.

In vitro biophysical strain model for understanding mechanisms of osteopathic manipulative treatment.

CONTEXT: Normal physiologic movement, pathologic conditions, and osteopathic manipulative treatment (OMT) are believed to produce effects on the shape and proliferation of human fibroblasts. Studies of biophysically strained fibroblasts would be useful in producing a model of the cellular mechanisms underlying OMT. OBJECTIVE: To investigate the effects of acyclic in vitro biophysical strain on normal human dermal fibroblasts and observe potential changes in cellular shape and proliferation, as well as potential changes in cellular production of nitric oxide, interleukin (IL) 1beta, and IL-6. DESIGN AND METHODS: Randomized controlled trial. Human fibroblasts were subjected in vitro to control conditions (no strain) or biophysical strain of various magnitudes (10%-30% beyond resting length) and durations (12-72 hours). After control or strain stimuli, fibroblasts were analyzed for potential changes in cell shape, proliferative capacity, nitric oxide secretion, and cytokine (IL-1beta, IL-6) secretion. RESULTS: Low strain magnitudes (<20%) induced mild cellular rounding and pseudopodia truncation. High strain magnitudes (>20%) decreased overall cell viability and the mitogenic response, and induced cell membrane decomposition and pseudopodia loss. No basal or strain-induced secretion of IL-1beta was observed. Interleukin 6 concentrations increased two-fold, while nitric oxide levels increased three-fold, in cells strained at 10% magnitude for 72 hours (P<.05). CONCLUSION: Human fibroblasts respond to in vitro strain by secreting inflammatory cytokines, undergoing hyperplasia, and altering cell shape and alignment. The in vitro biophysical strain model developed by the authors is useful for simulating a variety of injuries, determining in vivo mediators of somatic dysfunction, and investigating the underlying mechanisms of OMT.