Abstract
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.
Myofascial release (MFR) is a common technique in manual medicine used to treat patients with various soft tissue disorders. The technique involves slowly applying an external mechanical load that overcomes the fascia or tendon's intrinsic tension to lengthen the collagen fibers.
1 Myofascial release is designed to actively stretch and elongate the fascia and underlying soft tissue to release areas of decreased fascial motion. Documented clinical outcomes associated with MFR include improved physiologic function, reduction of pain, and increased range of motion in the affected joint.
2,3 To our knowledge, clinical findings that directly implicate MFR in enhanced wound healing have not been documented. However, clinical studies using vacuum compression therapy, which uses similar mechanical stretch parameters, suggests a potential correlation between biomechanical strain and enhanced wound healing.
4-7
Fibroblasts play an important role in wound healing and in the regulation of the local inflammatory response.
8,9 As the primary cell type in connective tissue, fibroblasts maintain and restore the tissue's structural homeostasis through the synthesis, degradation, and reorganization of the extracellular matrix.
10 In addition, the ability of fibroblasts to respond to mechanical stimuli by modifying gene expression
11-13 makes them likely targets for the mechanotransduction of MFR. In previous studies
12,13 we used 2-dimensional fibroblast tissue constructs to identify the mechanisms behind the clinical efficacy of MFR. Our previous findings revealed that modeled MFR applied in vitro inhibits the cytotoxic effects of fibroblasts, alters fibroblast actin architecture, and induces the expression of various anti-inflammatory and growth factors.
12,13 We have also shown that MFR downregulates inhibitory factors on protein kinase C and phosphoinositide 3-kinase to sensitize fibroblasts to nitric oxide and improve wound healing.
14 These findings highlight mechanisms that may support the clinical effectiveness of MFR.
In vivo, fibroblasts exist in a 3-dimensional extracellular matrix, which serves as a vital biomechanical feedback system that dictates signaling and adaptive response in wound repair.
15 In our previous studies
12,15 we used 2-dimensional fibroblast tissue constructs on which a modeled MFR of a single strain magnitude and duration setting was applied. Various rates of manual manipulative treatment can induce different degrees of sympathetic efferent nerve stimulation and blood flow.
16 Recent data from our laboratory have also shown that in a 3-dimensional environment, fibroblast cytokine secretion is dependent on MFR strain duration and magnitude,
17 suggesting a correlation between physiologic response and dosed MFR. Results from our 2-dimensional studies
14 provide evidence to support improved wound-healing rates using a single MFR treatment; however, the cellular mechanisms that link MFR to wound closure were not identified, owing to the single strain parameter tested and the lack of dimensionality.
In the present study, we improved on our strain model and in vitro tissue system by investigating the effects of MFR strain duration and magnitude using a 3-dimensional bioengineered tendon (BET) wound model. We hypothesized that a unique MFR strain regimen would result in unique wound-healing responses. We expected the results of this study to help elucidate the potential efficacy of MFR on muscle fascia and tendon wound repair.
All experiments were conducted using commercially available normal human dermal fibroblasts. Cells were cultured at 37°C, 5% CO2, and 100% humidity in Dulbecco's modified Eagle's medium supplemented with 2% fetal bovine serum and 1% penicillin-streptomycin. The medium was replaced with fresh prewarmed growth medium every 2 days. Confluent cultures (acquired within 7-10 days) were passaged at a ratio of 1:3, and all experiments used cell passages between 4 and 10.
Normal human fibroblasts at a concentration of 1000 cells/μL were mixed in a solution of 70% Purecol collagen type I (Advanced Biomatrix, Inc) and 30% 5× Dulbecco's modified Eagle's medium to create a collagen-fibroblast gel. A loading channel was created for the gel using a linear Trough Loader (Flexcell International Corp) placed beneath the Tissue Train Plates (Flexcell International Corp). These 6-well formatted plates consisted of flexible elastomeric well bottoms that were attached to nondeformable nylon mesh anchors at each end of the long axis. To create a cylindrical structure attached at the 2 anchors, 200 μL of the collagen-fibroblast gel were then added to the loading channel and allowed to polymerize for 2 hours in a humidified 37°C incubator. After the gel matrix polymerized, the vacuum was released, thereby allowing the BETs to be free from all attachments except at the 2 anchor points. Fresh culture medium supplemented with 2% fetal bovine serum was then added to each well, and the BETs were allowed to acclimate for 24 to 48 hours before undergoing MFR.
To study the effect of fibroblasts in remodeling of the overall structure of BETs, we developed fibroblast-free BETs made entirely of Purecol collagen. These fibroblast-free BETs revealed the difference in structure of BETs in the presence and absence of fibroblasts (
Figure 1A).
For the strain duration groups, strain was slowly applied to elongate the tissue to 2.5% of its resting length per second to reach a maximum 6% strain (ie, to 106% of its initial resting length). The BET was then held at 6% elongation for 1, 2, 3, 4, or 5 minutes. The strain was then slowly released back to baseline at a rate of −1.5% per second. For the strain magnitude groups, a slow-loading strain was similarly applied until a maximum magnitude of 3%, 6%, 9%, or 12% elongation was reached. The maximum strain was held for 90 seconds and then allowed to slowly release back to baseline using the identical unloading rate noted above. The nonstrained BETs served as controls.
The current study investigated the influences of MFR strain duration and magnitude on BET wound healing in vitro and found that lower-magnitude and longer-duration MFR strain is associated with accelerated wound closure rates. Longer-duration MFR caused a rapid change in wound morphology as shown by decreased overall wound size measured at 3 hours after MFR. In contrast, larger-magnitude MFR exacerbated the original wound by further enlarging the site of injury.
The ability of fibroblasts to respond to mechanical stimuli and their involvement in wound repair, inflammation, and matrix contraction
12,13 identify them as likely targets for mechanistic studies in manual medicine. Our laboratory has been studying the response of fibroblasts to mechanical stimuli, particularly MFR in both 2- and 3-dimensional matrices.
14,17 In the present study, we used a 3-dimensional tissue construct to more closely mimic a physiologically relevant system. The 3-dimensional extracellular matrix found in vivo provides an important environment for biomechanical feedback mechanisms in regulating fibroblast proliferation, migration, differentiation, and signaling responses.
18-20 The matrix also affects the diffusion of regulatory soluble mediators through ligand binding within the matrix that can alter the local concentration gradient of inflammatory cytokine and growth factors secreted by fibroblasts.
21,22 Immediately after excising the BETs with a circular cutting apparatus, the resulting wound took on an elliptical shape and had a smaller area than the cutting apparatus. Bush et al
23 found that the natural line of tension in skin, also known as a Langer line,
24,25 causes wounds to collapse when the extracellular matrix is disrupted, resulting in a smaller elliptical wound. The wound in our model behaved similarly to skin tissue, suggesting that fibroblasts may have been contracting or remodeling the extracellular matrix to generate tension in the BETs (
Figure 1B). This phenomenon was not present in fibroblast-free BETs (
Figure 1A), suggesting that fibroblasts are required for active remodeling of the extracellular matrix.
Myofascial release altered the wound closure rate in dose-dependent manners. In the strain magnitude group, faster wound closure time correlated with lower strain magnitudes (3% and 6%) and reduction in the minor axis of the wound (
Table 1). This phenomenon was unrelated to the changes in the width of the overall structure of the BET as observed with the 12% magnitude group, which showed narrowing of the BET, exacerbating of the wound size, and lengthening of the major axis of the wound (
Figure 3 and
Table 1). Smith et al
26 found that extraneous mechanical strain can lead to disassembly of larger-diameter fibrils. In addition, a strain magnitude greater than 10% causes thinning of collagen fibril diameter, whereas 0% to 5% strain has no effect.
27 This finding offers a possible explanation as to why we observed a narrowing of the BET width in the 12% magnitude group, whereas a magnitude of 3% to 9% resulted in no measurable changes.
We found that a longer duration of MFR resulted in rapid changes in the shape and measurable wound area (
Figure 4, Figure 6, Figure 7, and
eTable 2). These rapid responses to architectural change suggest that this phenomenon cannot be attributed to gene activation or modification, owing to the short time that had elapsed since MFR. However, the possibility of MFR activating an ectocytotic process to secrete membrane-bound matrix vesicles or to modify existing matrix protein architecture cannot be ruled out. This process has been shown to occur as rapidly as 5 minutes after stress-relaxation.
28 The release of matrix vesicles is typically observed when fibroblasts differentiate into the contractile phenotype (myofibroblast) to induce extracellular matrix remodeling. It is possible that MFR held for longer than 2 minutes may simulate the mechanical stress signals that are generated by myofibroblasts to trigger early matrix remodeling. Another potential explanation could be the ability of mechanical strain to decrease cell/extracellular matrix attachment. In vivo, fibroblasts bind to multiple collagen filaments that cross-link numerous fibers to dictate the tissues' resting texture, tension, and shape. Mechanical tension is known to affect cell orientation and to decrease cytoskeleton rigidity and viscosity and extracellular matrix contact points.
11-13,29 The reduction in fibroblast contact with the extracellular matrix and the decrease in cell shape stability could cause the wound to collapse, resulting in a change in wound shape. Studies investigating focal adhesion kinase and integrin interaction with the extracellular matrix are in progress in our laboratory. We are also interested in histologic analysis to quantify the reorganization of the preexisting extracellular matrix protein as well as to quantify the newly synthesized collagen.
The limitations of using in vitro models to correlate with in vivo wound-healing responses include the lack of blood and tissue fluid dynamics as well as the lack of other cell types (eg, neutrophils, macrophages, epithelial cells) that significantly contribute to chemotaxis, inflammation, and wound healing. Age, physique, and countless other patient variables also play important roles in manual treatments because each patient's body type possesses distinct fascial and muscle tone and density. These physical variations may also affect how the soft tissue opposes the biomechanical maneuver applied by the physician. These factors were beyond the scope of this study. This model allowed us to negate the effects of age, tissue integrity, and external environmental factors to allow the application of a reproducible MFR protocol and to focus on the direct effects of strain on fibroblast-mediated wound healing.
The results of the current study provide evidence to suggest that different parameters of MFR induce unique wound-healing responses and an optimal MFR strain magnitude and duration exists that results in optimal wound healing. The BET wound-healing rates were hastened when a lower magnitude and longer duration of MFR strain were applied. Larger magnitudes of MFR strain resulted in enlarging the wound size and exacerbating the site of injury. If the results of the current study are clinically translatable, they suggest that MFR significantly affects tendon wound healing in manners that are dependent on both duration and magnitude. These data provide preliminary evidence to suggest that there may be an optimal biomechanical range at which MFR yields the maximum beneficial outcomes, thus supporting a need for individualized MFR. Clinical studies have been vital in establishing the efficacy of manual manipulative treatments; however, such studies have not yet looked at variables such as magnitude and duration of MFR. Thus, we cannot rule out the importance of quantifying the techniques used (eg, pressure, duration, frequency) and investigating the effects of various parameters of strain, because it may help to refine and optimize the practice of manual therapy.
Financial Disclosures: None reported.
Support: Funding for this study was provided by the American Osteopathic Association (grant 1121640).