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.

Biomechanical strain vehicles for fibroblast-directed skeletal myoblast differentiation and myotube functionality in a novel coculture.

Skeletal muscle functionality is governed by multiple stimuli, including cytokines and biomechanical strain. Fibroblasts embedded within muscle connective tissue respond to biomechanical strain by secreting cytokines that induce myoblast differentiation and, we hypothesize, regulate myotube function. A coculture was established to allow cross talk between fibroblasts in Bioflex wells and myoblasts on nondeformable coverslips situated above Bioflex wells. Cyclic short-duration strain (CSDS) modeling repetitive stress/injury, acyclic long-duration strain (ALDS) modeling manipulative therapy, and combined strain paradigms (CSDS + ALDS) were applied to fibroblasts. Nonstrained myoblasts in uniculture and coculture served as controls. After fibroblasts had induced myoblast differentiation, myotube contraction was assessed by perfusion of ACh (10(-11)-10(-3) M). CSDS-treated fibroblasts increased myotube contractile sensitivity vs. uniculture (P < 0.05). As contraction is dependent on ACh binding, expression and clustering of nicotinic ACh receptors (nAChRs) were measured. CSDS-treated fibroblasts increased nAChR expression (P < 0.05), which correlated with myotube contraction. ALDS-treated fibroblasts did not significantly affect contraction or nAChR expression. Agrin-treated myotubes were then used to design a computer algorithm to identify α-bungarotoxin-stained nAChR clusters. ALDS-treated fibroblasts increased nAChR clustering (P < 0.05), while CSDS-treated fibroblasts disrupted cluster formation. CSDS-treated fibroblasts produced nAChRs preferentially located in nonclustered regions (P < 0.05). Strain-activated fibroblasts mediate myotube differentiation with multiple functional phenotypes. Similar to muscle injury, CSDS-treated fibroblasts disrupted nAChR clusters and hypersensitized myotube contraction, while ALDS-treated fibroblasts aggregated nAChRs in large clusters, which may have important clinical implications. Cellular strategies aimed at improving muscle functionality, such as through biomechanical strain vehicles that activate fibroblasts to stabilize postsynaptic nAChRs on nearby skeletal muscle, may serve as novel targets in neuromuscular disorders.

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.

Cyclic strain upregulates VEGF and attenuates proliferation of vascular smooth muscle cells.

OBJECTIVE: Vascular smooth muscle cell (VSMC) hypertrophy and proliferation occur in response to strain-induced local and systemic inflammatory cytokines and growth factors which may contribute to hypertension, atherosclerosis, and restenosis. We hypothesize VSMC strain, modeling normotensive arterial pressure waveforms in vitro, results in attenuated proliferative and increased hypertrophic responses 48 hrs post-strain. METHODS: Using Flexcell Bioflex Systems we determined the morphological, hyperplastic and hypertrophic responses of non-strained and biomechanically strained cultured rat A7R5 VSMC. We measured secretion of nitric oxide, key cytokine/growth factors and intracellular mediators involved in VSMC proliferation via fluorescence spectroscopy and protein microarrays. We also investigated the potential roles of VEGF on VSMC strain-induced proliferation. RESULTS: Protein microarrays revealed significant increases in VEGF secretion in response to 18 hours mechanical strain, a result that ELISA data corroborated. Apoptosis-inducing nitric oxide (NO) levels also increased 43% 48 hrs post-strain. Non-strained cells incubated with exogenous VEGF did not reproduce the antimitogenic effect. However, anti-VEGF reversed the antimitogenic effect of mechanical strain. Antibody microarrays of strained VSMC lysates revealed MEK1, MEK2, phospo-MEK1T385, T291, T298, phospho-Erk1/2T202+Y204/T185+T187, and PKC isoforms expression were universally increased, suggesting a proliferative/inflammatory signaling state. Conversely, VSMC strain decreased expression levels of Cdk1, Cdk2, Cdk4, and Cdk6 by 25-50% suggesting a partially inhibited proliferative signaling cascade. CONCLUSIONS: Subjecting VSMC to cyclic biomechanical strain in vitro promotes cell hypertrophy while attenuating cellular proliferation. We also report an upregulation of MEK and ERK activation suggestive of a proliferative phenotype. Hhowever, the proliferative response appears to be aborogated by enhanced antimitogenic cytokine VEGF, NO secretion and downregulation of Cdk expression. Although exogenous VEGF alone is not sufficient to promote the quiescent VSMC phenotype, we provide evidence suggesting that strain is a necessary component to induce VSMC response to the antimitogenic effects of VEGF. Taken together these data indicate that VEGF plays a critical role in mechanical strain-induced VSMC proliferation and vessel wall remodeling. Whether VEGF and/or NO inhibit signaling distal to Erk 1/2 is currently under investigation.

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.