Joint capsule mast cells and neuropeptides are increased within four weeks of injury and remain elevated in chronic stages of posttraumatic contractures

The purpose of this article was to determine mast cell and neuropeptide nerve fiber numbers in joint capsules in posttraumatic contractures, as elevated numbers have been implicated in other fibrotic and contracture conditions. Twelve skeletally mature rabbits had intraarticular cortical windows removed from the medial and lateral femoral condyles and the knee joint immobilized. The contralateral unoperated limb served as a control. Equal numbers of rabbits were sacrificed 4 weeks after surgery or 40 weeks after the first surgery that included 32 weeks of remobilization. Six patients with chronic posttraumatic elbow joint contractures and six age‐matched organ donor controls free of elbow contractures were also studied. Joint capsule myofibroblast, mast cell, and neuropeptide containing nerve fiber numbers were assessed with immunohistochemistry. The numbers of myofibroblasts, mast cells, and neuropeptide containing nerve fibers expressed as a percentage of total cells were significantly greater in the contracture capsules when compared to the control capsules at all time points (p < 0.0001). The range of percentages for the three components in the contracture capsules versus the controls were 41–48% versus 9–10%, 44–50% versus 11–13%, and 45–50% versus 10–12% for the acute and chronic stages of the rabbit model and the chronic stages in the human elbows, respectively. These data support the hypothesis that a myofibroblast–mast cell–neuropeptide fibrosis axis may underlie some of the pathologic changes in the joint capsule in posttraumatic contractures. Approaches designed to manipulate this axis, such as preventing degranulation of mast cells, warrant further investigation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1313–1319, 2008     

Influence of the anti‐inflammatory cytokine interleukin‐4 on human joint capsule myofibroblasts

Post‐traumatic joint contracture was reported to be associated with elevated numbers of contractile myofibroblasts (MFs) in the healing capsule. During the physiological healing process, the number of MFs declines; however, in fibroconnective disorders, MFs persist. The manifold interaction of the cytokines regulating the appearance and persistence of MFs in the pathogenesis of joint contracture remains to be elucidated. The objective of our current study was to analyze the impact of the anti‐inflammatory cytokine interleukin (IL)‐4 on functional behavior of MFs. Cells were isolated from human joint capsule specimens and challenged with three different concentrations of IL‐4 with or without its neutralizing antibody. MF viability, contractile properties, and the gene expression of both alpha‐smooth muscle actin (α‐SMA) and collagen type I were examined. Immunofluorescence staining revealed the presence of IL‐4 receptor (R)‐alpha (α) on the membrane of cultured MFs. The cytokine IL‐4 promoted MF viability and enhanced MF modulated contraction of collagen gels. Moreover, IL‐4 intervened in gene expression by up‐regulation of α‐SMA and collagen type I mRNA. These effects could be specifically lowered by the neutralizing IL‐4 antibody. On the basis of our findings we conclude that the anti‐inflammatory cytokine IL‐4 specifically regulates viability and the contractile properties of MFs via up‐regulating the gene expression of α‐SMA and collagen type I. IL‐4 may be a helpful target in developing anti‐fibrotic therapeutics for post‐traumatic joint contracture in human. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1290–1298, 2017.

     

Auxiliary proteins that facilitate formation of collagen‐rich deposits in the posterior knee capsule in a rabbit‐based joint contracture model

Post‐traumatic joint contracture is a debilitating consequence of trauma or surgical procedures. It is associated with fibrosis that develops regardless of the nature of initial trauma and results from complex biological processes associated with inflammation and cell activation. These processes accelerate production of structural elements of the extracellular matrix, particularly collagen fibrils. Although the increased production of collagenous proteins has been demonstrated in tissues of contracted joints, researchers have not yet determined the complex protein machinery needed for the biosynthesis of collagen molecules and for their assembly into fibrils. Consequently, the purpose of our study was to investigate key enzymes and protein chaperones needed to produce collagen‐rich deposits. Using a rabbit model of joint contracture, our biochemical and histological assays indicated changes in the expression patterns of heat shock protein 47 and the α‐subunit of prolyl 4‐hydroxylase, key proteins in processing nascent collagen chains. Moreover, our study shows that the abnormal organization of collagen fibrils in the posterior capsules of injured knees, rather than excessive formation of fibril‐stabilizing cross‐links, may be a key reason for observed changes in the mechanical characteristics of injured joints. This result sheds new light on pathomechanisms of joint contraction, and identifies potentially attractive anti‐fibrotic targets. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:489–501, 2016.     

Matrix metalloproteinase inhibition delays wound healing and blocks the latent transforming growth factor‐β1‐promoted myofibroblast formation and function

The ability to regulate wound contraction is critical for wound healing as well as for pathological contractures. Matrix metalloproteinases (MMPs) have been demonstrated to be obligatory for normal wound healing. This study examined the effect that the broad‐spectrum MMP inhibitor BB‐94 has when applied topically to full‐thickness skin excisional wounds in rats and its ability to inhibit the promotion of myofibroblast formation and function by the latent transforming‐growth factor‐β1 (TGF‐β1). BB‐94 delayed wound contraction, as well as all other associated aspects of wound healing examined, including myofibroblast formation, stromal cell proliferation, blood vessel formation, and epithelial wound coverage. Interestingly, BB‐94 dramatically increased the level of latent and active MMP‐9. The increased levels of active MMP‐9 may eventually overcome the ability of BB‐94 to inhibit this MMP and may explain why wound contraction and other associated events of wound healing were only delayed and not completely inhibited. BB‐94 was also found to inhibit the ability of latent TGF‐β1 to promote the formation and function of myofibroblasts. These results suggest that BB‐94 could delay wound closure through a twofold mechanism; by blocking keratinocyte migration and thereby blocking the necessary keratinocyte–fibroblast interactions needed for myofibroblast formation and by inhibiting the activation of latent TGF‐β1.     

Inhibition of COX‐2 Pathway as a Potential Prophylaxis Against Arthrofibrogenesis in a Rabbit Model of Joint Contracture

Arthrofibrosis is a common complication following total knee arthroplasty caused by pathologic fibroblast activation and excessive collagen deposition around a synovial joint leading to debilitating loss of motion. Treatment options are limited because the pathologic mechanisms remain to be characterized. Dysregulation of the inflammatory cascade may lead to communication between myofibroblasts and immune cells triggering tissue metaplasia, and excessive collagen deposition described clinically as arthrofibrosis. We explored the novel use of celecoxib (selective cyclooxygenase‐2 [COX‐2] inhibitor) to disrupt the downstream effects of the post‐traumatic inflammatory cascade and inhibit scar tissue formation in a validated rabbit model of arthrofibrosis combined with new parameters for quantifying the stiffness of the posterior capsule. Biomechanical and molecular analyses, of contracted rabbit knee posterior capsule tissue after COX‐2 inhibition revealed increased maximal passive extension and down‐regulation of collagen messenger RNA compared with controls. Histopathologic examination suggested a trend of decreased quantities of dense fibrous connective tissue with COX‐2 inhibition. These data may suggest that inhibiting the inflammatory cascade could potentially reduce pathologic myofibroblast activation, thereby reducing scar tissue formation and increasing the range of motion in arthrofibrotic joints. Implementing a multi‐modal pharmacologic approach may simultaneously target numerous cellular components contributing to the complex process of arthrofibrogenesis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2609–2620, 2019     

Blocking collagen fibril formation in injured knees reduces flexion contracture in a rabbit model

Post‐traumatic joint contracture is a frequent orthopaedic complication that limits the movement of injured joints, thereby severely impairing affected patients. Non‐surgical and surgical treatments for joint contracture often fail to improve the range of motion. In this study, we tested a hypothesis that limiting the formation of collagen‐rich tissue in the capsules of injured joints would reduce the consequences of the fibrotic response and improve joint mobility. We targeted the formation of collagen fibrils, the main component of fibrotic deposits formed within the tissues of injured joints, by employing a relevant rabbit model to test the utility of a custom‐engineered antibody. The antibody was delivered directly to the cavities of injured knees in order to block the formation of collagen fibrils produced in response to injury. In comparison to the non‐treated control, mechanical tests of the antibody‐treated knees demonstrated a significant reduction of flexion contracture. Detailed microscopic and biochemical studies verified that this reduction resulted from the antibody‐mediated blocking of the assembly of collagen fibrils. These findings indicate that extracellular processes associated with excessive formation of fibrotic tissue represent a valid target for limiting post‐traumatic joint stiffness. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1038–1046, 2017.

     

The effect of epigallocatechin-3-gallate, a constituent of green tea, on transforming growth factor-β1–stimulated wound contraction

Dermal fibrosis, or scarring, following surgical incisions, traumatic wounds and burns presents a major clinical burden. Transforming growth factor (TGF)-β1 is a major factor known to stimulate fibroblast proliferation, collagen production, and the differentiation of fibroblast to myofibroblast promoting wound contraction. Furthermore, excessive or prolonged TGF-β1 has been shown to be associated with scarring. Green tea contains high amounts of polyphenols with the major polyphenolic compound being epigallocatechin-3-gallate (EGCG). EGCG has been shown to be anti-inflammatory, anti-oxidant, and may improve wound healing and scarring, though its precise effect on TGF-β1 remains unclear. This study aimed at determining the effect of EGCG on TGF-β1 collagen contraction, gene expression and the differentiation of fibroblast to myofibroblast. EGCG appears to affect the role that TGF-β1 plays in fibroblast populated collagen gel contraction and this seems to be through both myofibroblast differentiation and connective tissue growth factor gene expression and reduces the expression of collagen type I gene regulation.     

Systemic depletion of macrophages in the subacute phase of wound healing reduces hypertrophic scar formation

Hypertrophic scars are caused by trauma or burn injuries to the deep dermis and can cause cosmetic disfigurement and psychological issues. Studies suggest that M2‐like macrophages are pro‐fibrotic and contribute to hypertrophic scar formation. A previous study from our lab showed that M2 macrophages were present in developing hypertrophic scar tissues in vivo at 3–4 weeks after wounding. In this study, the effect of systemic macrophage depletion on scar formation was explored at subacute phase of wound healing. Thirty‐six athymic nude mice that received human skin transplants were randomly divided into macrophage depletion group and control group. The former received intraperitoneal injections of clodronate liposomes while the controls received sterile saline injections on day 7, 10, and 13 postgrafting. Wound area, scar thickness, collagen abundance and collagen bundle structure, mast cell infiltration, myofibroblast formation, M1, and M2 macrophages together with gene expression of M1 and M2 related factors in the grafted skin were investigated at 2, 4, and 8 weeks postgrafting. The transplanted human skin from the control group developed contracted, elevated, and thickened scars while the grafted skin from the depletion group healed with significant less contraction and elevation. Significant reductions in myofibroblast number, collagen synthesis, and hypertrophic fiber morphology as well as mast cell infiltration were observed in the depletion group compared to the control group. Macrophage depletion significantly reduced M1 and M2 macrophage number in the depletion group 2 weeks postgrafting as compared to the control group. These findings suggest that systemic macrophage depletion in subacute phase of wound healing reduces scar formation, which provides evidence for the pro‐fibrotic role of macrophages in fibrosis of human skin as well as insight into the potential benefits of specifically depleting M2 macrophages in vivo.     

Mast cells promote fibroblast populated collagen lattice contraction through gap junction intercellular communication

The release of mast cell granules is commonly associated with inflammation and fibrosis. However, does direct communication between mast cells and fibroblasts through gap junction intercellular communication (GJIC) occur? Fibroblast populated collagen lattice (FPCL) cast with mast cells show enhanced lattice contraction. Do released granules or GJIC between mast cells and fibroblasts promote enhanced lattice contraction? Mast cells preloaded with a fluorescent dye that readily passes through gap junctions were cast in FPCL. Dye passed from mast cells into fibroblasts within these cocultured mast cell‐FPCLs. Fatty acid amide hydrolase inhibitor blocks the breakdown of oleamide, which is a potent endogenous inhibitor of GJIC. GJIC was blocked for 3 days when mast cells were pulsed for 3 hours with fatty acid amide hydrolase inhibitor. Mast cells pretreated with fatty acid amide hydrolase inhibitor cast in cocultured mast cell‐FPCLs failed to enhance cocultured lattice contraction. Mast cell‐FPCLs made with mouse fibroblasts unable to generate GJIC failed to show enhanced lattice contraction. Degranulated mast cells were equal to intact mast cells at enhancing cocultured mast cell‐FPCL contraction. The supernatant from degranulated mast cells had no effect upon FPCL contraction. Therefore, enhanced mast cell‐FPCL contraction appears to be independent of mast cell granules, but dependent upon GJIC between fibroblasts and mast cells. We speculate that mast cell–fibroblast GJIC may play a role in fibrosis.     

The mast cell stabilizer ketotifen prevents development of excessive skin wound contraction and fibrosis in red Duroc pigs

Skin wound healing in Yorkshire pigs closely approximates human wound healing. Conversely, red Duroc pigs form fibroproliferative, hypercontractile scars. As mast cells have been implicated in several fibrotic conditions, the present study used these models to evaluate the potential role of mast cells in wound contraction and fibrosis. Immediately following the creation of full‐thickness excisional wounds, the mast cell stabilizer ketotifen was used to treat both Yorkshire and red Durocs. Control red Durocs showed significantly more wound contraction than Yorkshires, both before and after reepithelialization. Ketotifen treatment significantly reduced the first phase of contraction in red Duroc wounds to a level equivalent to Yorkshire wounds, but had no detectable effect on the postepithelialization phase of contraction. Cessation of drug treatment after 10 weeks did not lead to resumption of excessive contraction in red Durocs, indicating that ketotifen blocked rather than delayed such contraction during a critical phase of healing. Ketotifen treatment also reduced the deposition of collagen within the red Duroc wounds, but did not affect Yorkshire wound contraction or collagen deposition. These results suggest that ketotifen may be an effective treatment for the reduction of excessive wound contraction and fibrosis in human cutaneous injuries, without affecting the normal healing process.     

Non‐enzymatic glycation of chondrocyte‐seeded collagen gels for cartilage tissue engineering

Collagen glycated with ribose (250 mM) in solution (pre‐glycation) and as a gel (post‐glycation) was seeded with chondrocytes and the effects of glycation on chondrocyte matrix assembly in culture were determined. Pre‐glycation enhanced GAG accumulation significantly over controls at both 2 and 4 weeks (p < 0.05), although at both time points there were no statistical differences in cell number between pre‐glycated and control gels. The increased proteoglycan accumulation was shown to be in part due to significantly increased GAG retention by the pre‐glycated constructs (p < 0.05). Total collagen content in these pre‐glycated gels was also significantly higher than unglycated gels at 4 weeks (p < 0.05). With post‐glycation of collagen gels, chondrocyte number and GAG accumulation were all significantly lower than controls (p < 0.05). Post‐glycation also inhibited GAG retention by the constructs (p < 0.05). Given these results, pre‐glycation may be an improved processing method for collagen gels for tissue engineering techniques. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1434–1439, 2008     

Myofibroblast cells are preferentially expressed early in a rabbit model of joint contracture

Studies have demonstrated increased myofibroblasts in contractures. However, the timeline is largely unknown. The aim of this study was to determine the number of myofibroblasts in contracting joint capsules at 3‐time points over a 24‐week period. Eighteen rabbits subjected to a surgical procedure designed to elicit a knee joint contracture were divided into three groups of six. Rabbits were sacrificed at each respective time point and myofibroblasts in the joint capsules were quantified using immunohistochemistry. The percent of myofibroblasts was significantly elevated in the operated limbs compared to the control limbs at 2 weeks (20% vs. 7%, respectively; p = 0.014). There was no difference in the percent of myofibroblasts between the operated and control limbs at 8 or 24 weeks (p = 0.96 and 0.07, respectively). The percent of myofibroblasts dropped from 20% at 2 weeks to 3.0% at 8 weeks (p < 0.001). The decrease from 8 to 24 weeks was not significant (p = 0.19). A large proportion of myofibroblasts are present in contracted joints at 2 weeks. By week 8, the proportion of myofibroblasts seem to return to normal. Interventions aimed at affecting the myofibroblast cell in order to prevent fibrosis should be instituted early. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:713–719, 2012     

Control of wound contraction. Basic and clinical features

Although a substantial amount of molecular and cellular data have been generated in an effort to understand the process of wound contraction and scar contracture formation, questions remain. What seems apparent is that the myofibroblast is not the only cell that generates contractile forces within wounds, but it does appear to be intrinsically linked to the development of hypertrophic scars. The supposition that the formation of scar contractures is solely the result of a continuation of wound contraction is an oversimplification. Figure 4 provides a model of the possible evolution of contractile forces during the wound healing process and their role in the development of scar contractures. Migration of fibroblasts into and through the extracellular matrix during the initial phase of wound healing, prior to the expression of alpha-SMA, appears to be a fundamental component of wound contraction. During this migration, the pulling of collagen fibrils into a streamlined pattern in their wake, and the associated production of collagenase, may facilitate a more normal arrangement of collagen. Once the wound has been repopulated and the chemotactic gradient that was established by inflammatory cells is decreased, fibroblast migration will cease. It is at this point that myofibroblasts appear and play a key role in the production of hypertrophic scars, given that their prolonged presence and over-representation are hallmarks of this pathology. One of the pivotal differences between wounds that proceed to normal scar compared with those that develop hypertrophic scars and scar contractures may be a lack (or late induction) of myofibroblast apoptotic cell death. The combined contribution of fibroblasts and myofibroblasts to abnormal extracellular matrix protein production results in an excessive and rigid scar. The isometric application of contractile forces by myofibroblasts probably contributes to the formation of the whorls, nodules, and scar contractures characteristic of hypertrophic scars. Because the prolonged presence of myofibroblasts, producing an imbalance in extracellular matrix proteins and proteases, probably exacerbates hypertrophic scars and wound contraction, accelerating the rate of apoptotic cell death to reduce the cell number to that seen in normal scar may be a useful strategy for providing effective and efficient treatment of scar contracture.     

Sodium cromolyn reduces expression of CTGF,ADAMTS1, and TIMP3 and modulates post‐injury patellar tendon morphology

The purpose of this study was to determine whether administration of a mast cell inhibitor (sodium cromolyn, SC) would influence tendon repair and extracellular matrix gene expression following acute injury. CD1 mouse patellar tendons were unilaterally injured and mast cell prevalence was determined. The effect of SC injection on tendon hypercellularity, cross‐sectional area, collagen organization, and expression of extracellular matrix‐related genes was examined. Mast cell prevalence was markedly increased in injured patellar tendons (p = 0.009), especially at 8 weeks post‐injury (p = 0.025). SC injection increased collagen organization compared to uninjected animals at 4 weeks and attenuated the development of tendon hypercellularity and tendon thickening post‐injury. Expression of CTGF, ADAMTS1, and TIMP3 in injured tendon was reduced in the SC group. SC injections moderated the structural alterations of healing tendon in association with downregulation of several genes associated with tendon fibrosis. This work corroborates previous findings pointing to a role of mast cells in tendon repair. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:678–683, 2011     

Cytokine Interferon‐γ suppresses the function of capsule myofibroblasts and induces cell apoptosis

Myofibroblasts (MFs), a contractile subset of fibroblasts, play a pivotal role in physiological wound healing and in the development of many fibroconnective disorders. The complex cytokine network regulating the function of MFs in joint stiffness is still poorly understood. In this in vitro study, we investigated the effect of the cytokine Interferon‐gamma (IFN‐γ) on MFs isolated from human joint capsules. MFs were cultivated either in the presence of increasing concentrations of IFN‐γ alone or in combination with IFN‐γ neutralizing antibodies. Cell viability, cytotoxicity, apoptosis, and mRNA gene expression of the MF markers alpha‐smooth muscle actin (α‐SMA) and collagen type I were analyzed in MF cultures. Contraction potential was analyzed in an established collagen gel contraction assay simulating the extracellular matrix. Using immunofluorescence staining, we could verify that MFs express IFN‐γ‐receptor (R)‐1 on their membrane. IFN‐γ decreased MF viability and significantly elevated the apoptosis rate in a dose‐dependent manner. IFN‐γ down‐regulated α‐SMA and collagen type I mRNA expression which was associated with a diminished MF mediated contraction of the gel matrices. These effects were suppressed by simultaneous treatment of cells with a neutralizing IFN‐γ antibody. Our experiments confirm the hypothesis that the cytokine IFN‐γ is a crucial component of the regulatory network of capsule MFs. IFN‐γ notably influences the ability of MFs to contract collagen matrices by suppressing α‐SMA gene expression. IFN‐γ is toxic for MFs in high concentrations and may negatively regulate the number of pro‐fibrotic MFs during the healing process via induction of cell apoptosis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2524–2533, 2017.

     

Combination of stromal cell‐derived factor‐1 and collagen–glycosaminoglycan scaffold delays contraction and accelerates reepithelialization of dermal wounds in wild‐type mice

While dermal substitutes can mitigate scarring and wound contraction, a significant drawback of current dermal replacement technologies is the apparent delay in vascular ingrowth compared with conventional skin grafts. Herein, we examined the effect of the chemokine stromal cell‐derived factor‐1 (SDF‐1) on the performance of a porous collagen–glycosaminoglycan dermal analog in excisional wounds in mice. C57BL/6 mice with 1 cm × 1 cm dorsal full‐thickness wounds were covered with a collagen–glycosaminoglycan scaffold, followed by four daily topical applications of 1 μg SDF‐1 or phosphate‐buffered saline vehicle. Some animals were also pretreated with five daily doses of 300 mg/kg granulocyte colony‐stimulating factor. Animals treated with SDF‐1 and no granulocyte colony‐stimulating factor reepithelialized 36% faster than vehicle controls (16 vs. 25 days), and exhibited less wound contraction on postwounding day 18 (～35% greater wound area) plus three‐fold longer neoepidermis formed than controls. Conversely, granulocyte colony‐stimulating factor promoted contraction and no epidermal regeneration. Early (postwounding Day 3) inflammatory cell infiltration in the SDF‐1‐treated group was 86% less, while the fraction of proliferating cells (positive Ki67 staining) was 32% more, when compared with controls. These results suggest that SDF‐1 simultaneously delays contraction and promotes reepithelialization and may improve the wound‐healing performance of skin substitutes.     

Development of arthrogenic joint contracture as a result of pathological changes in remobilized rat knees

This study aimed to elucidate how rats recover from immobilization‐induced knee joint contracture. Rats’ right knees were immobilized by an external fixator at a flexion of 140° for 3 weeks. After removal of the fixator, the joints were allowed to move freely (remobilization) for 0, 1, 3, 7, or 14 days (n = 5 each). To distinguish myogenic and arthrogenic contractures, the passive extension range of motion was measured before and after myotomy of the knee flexors. Knee joints were histologically analyzed and the expression of genes encoding inflammatory or fibrosis‐related mediators, interleukin‐1β (1L‐1β ), fibrosis‐related transforming growth factor‐β1 (TGF‐β1 ), and collagen type I (COL1A1 ) and III (COL3A1 ), were examined in the knee joint posterior capsules using real‐time PCR. Both myogenic and arthrogenic contractures were established within 3 weeks of immobilization. During remobilization, the myogenic contracture decreased over time. In contrast, the arthrogenic contracture developed further during the remobilization period. On day 1 of remobilization, inflammatory changes characterized by edema, inflammatory cell infiltration, and upregulation of IL‐1β gene started in the knee joint posterior capsule. In addition, collagen deposition accompanied by fibroblast proliferation, with upregulation of TGF‐β1 , COL1A1 , and COL3A1 genes, appeared in the joint capsule between days 7 and 14. These results suggest the progression of arthrogenic contracture following remobilization, which is characterized by fibrosis development, is possibly triggered by inflammation in the joint capsule. It is therefore necessary to focus on developing new treatment strategies for immobilization‐induced joint contracture. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1414–1423, 2017.

     

Extracorporeal shock waves modulate myofibroblast differentiation of adipose‐derived stem cells

Mesenchymal stem cells are precursors of myofibroblasts, cells deeply involved in promoting tissue repair and regeneration. However, since myofibroblast persistence is associated with the development of tissue fibrosis, the use of tools that can modulate stem cell differentiation toward myofibroblasts is central. Extracorporeal shock waves are transient short‐term acoustic pulses first employed to treat urinary stones. They are a leading choice in the treatment of several orthopedic diseases and, notably, they have been reported as an effective treatment for patients with fibrotic sequels from burn scars. Based on these considerations, the aim of this study is to define the role of shock waves in modulating the differentiation of human adipose‐derived stem cells toward myofibroblasts. Shock waves inhibit the development of a myofibroblast phenotype; they down‐regulate the expression of the myofibroblast marker alpha smooth muscle actin and the extracellular matrix protein type I collagen. Functionally, stem cells acquire a more fibroblast‐like profile characterized by a low contractility and a high migratory ability. Shock wave treatment reduces the expression of integrin alpha 11, a major collagen receptor in fibroblastic cells, involved in myofibroblast differentiation. Mechanistically, the resistance of integrin alpha 11‐overexpressing cells to shock waves in terms of alpha smooth muscle actin expression and cell migration and contraction suggests also a role of this integrin in the translation of shock wave signal into stem cell responses. In conclusion, this in vitro study shows that stem cell differentiation toward myofibroblasts can be controlled by shock waves and, consequently, sustains their use as a therapeutic approach in reducing the risk of skin and tissue fibrosis.