Myofibroblasts contribute to but are not necessary for wound contraction.

Wound contraction facilitates tissue repair. The correct balance between too little contraction, which leads to non-healing wounds, and too much contraction, which leads to contractures, is important for optimal healing. Thus, understanding which cells cause wound contraction is necessary to optimize repair. Wound contraction is hypothesized to develop from myofibroblast (cells which express alpha-smooth muscle actin; ACTA2) contractility, while the role of fibroblast contractility is unknown. In this study, we utilized ACTA2 null mice to determine what role fibroblasts play in wound contraction. Human scar contractures were immunostained for ACTA2, beta-cytoplasmic actin (ACTB), and gamma-cytoplasmic actin (ACTG1). Full-thickness cutaneous wounds were created on dorsum of ACTA2(+/+) mice and strain-matching ACTA2(+/-) and ACTA2(-/-) mice. Wound contraction was quantified. Tissue was harvested for histologic, immunohistochemical and protein analysis. Compared with surrounding unwounded skin, human scar tissue showed increased expression of ACTA2, ACTB, and ACTG1. ACTA2 was focally expressed in clusters. ACTB and ACTG1 were widely, highly expressed throughout scar tissue. Wound contraction was significantly retarded in ACTA2(-/-) mice, as compared to ACTA2(+/+) controls. Control mice had increased epithelialization, cell proliferation, and neovascularization. ACTA2(-/-) mice had lower levels of apoptosis, and fewer total numbers of cells. Smaller amount of collagen deposition and immature collagen organization in ACTA2(-/-) mice demonstrate that wounds were more immature. These data demonstrate that myofibroblasts contribute to but are not necessary for wound contraction. Mechanisms by which fibroblasts promote wound contraction may include activation of contractile signaling pathways, which promote interaction between non-muscle myosin II and ACTB and ACTG1.

Sternal talon offers a solution for secondary sternum osteosynthesis in patients with nonunion.

BACKGROUND: Median sternotomy may be associated with postoperative complications such as nonunion after conventional metal wire closure. The Sternal Talon device (KLS Martin, Jacksonville, FL) has recently been introduced as an alternative for osteosynthesis after median sternotomy and may also be beneficial for patients with persistent sternal nonunion. METHODS: A consecutive series of 24 patients underwent Sternal Talon repair for sternal nonunion or acute mediastinitis, or both, after sternal wire closure. Patient data--including demographics, surgical history, and indication for operation, as well as outcomes--were obtained and analyzed by retrospective chart review. RESULTS: The average patient age was 61.3 years and 23 patients were men (95.8%). The most common median sternotomy procedure was coronary artery bypass grafting (CABG) in 19 patients (79.2%). Secondary closure using the Sternal Talon was indicated for sternal nonunion or infection, or both, in all patients. Eight patients underwent simultaneous muscle flap procedures during the placement of the Sternal Talon (33.3%). Sternal union was eventually achieved in 23 of 24 patients (95.8%). Subsequent reoperation was required in 4 patients (16.7%). CONCLUSIONS: The data presented suggest that the osteosynthesis using the Sternal Talon device is a safe and effective modality for treating symptomatic sternal nonunion or acute dehiscence associated with infection (mediastinitis.).

The nucleic acid scavenger polyamidoamine third-generation dendrimer inhibits fibroblast activation and granulation tissue contraction.

BACKGROUND: Pathologic cutaneous scarring affects over 40 million people worldwide and costs billions of dollars annually. Understanding mechanisms of fibroblast activation and granulation tissue contraction is the first step toward preventing pathologic scarring. The authors hypothesize that nucleic acids increase fibroblast activation and cause granulation tissue contraction and that sequestration of nucleic acids by application of a nucleic acid scavenger dendrimer, polyamidoamine third-generation dendrimer, will decrease pathologic scarring. METHODS: In vitro experiments were performed to assess the effect of nucleic acids on pathologic scar-associated fibroblast activity. The effect of nucleic acids on cytokine production and migration on mouse fibroblasts was evaluated. Immunofluorescence microscopy was used to determine the effect of nucleic acids on the differentiation of human primary fibroblasts into myofibroblasts. Using a murine model, the effect of polyamidoamine third-generation dendrimer on granulation tissue contraction was evaluated by gross and histologic parameters. RESULTS: Mouse fibroblasts stimulated with nucleic acids had increased cytokine production (i.e., transforming growth factor-ß, monocyte chemotactic protein 1, interleukin-10, tumor necrosis factor-α, and interferon-γ), migration, and differentiation into myofibroblasts. Polyamidoamine third-generation dendrimer blocked cytokine production, migration, and differentiation into myofibroblasts. Using a murine model of granulation tissue contraction, polyamidoamine third-generation dendrimer decreased wound contraction and angiogenesis. Collagen deposition in polyamidoamine third-generation dendrimer-treated tissues was aligned more randomly and whorl-like compared with control tissue. CONCLUSIONS: The data demonstrate that nucleic acid-stimulated fibroblast activation and granulation tissue contraction are blocked by polyamidoamine third-generation dendrimer. Sequestration of pathogen-associated molecular patterns may be an approach for preventing pathologic scarring.

A detailed evaluation of the anatomical variations of the profunda artery perforator flap using computed tomographic angiograms.

BACKGROUND: The profunda artery perforator flap is a new option for breast reconstruction in appropriate patients. While the basic anatomy is known, detailed profunda perforator anatomy has never fully been described and we present new data that will aid dissection. METHODS: Fifty consecutive lower extremity computed tomography angiogram scans (100 legs) were retrospectively analyzed to acquire profunda artery perforator measurements. Patient medical records were then examined to ascertain patient information. Data were then analyzed using simple descriptive statistics and bivariate linear regressions with repeated measures. RESULTS: Bilateral thighs from 50 consecutive angiograms were included for a total of 100 thighs. Females comprised 30 (60 percent) of the patients and the cohort average age was 59.1 years old. All thighs had at least two perforators, with 85 percent having three or more. On average, perforators were located 6.2 cm below the gluteal crease, and were evenly distributed between the medial and lateral halves of the thigh. The average perforator diameter at origin off profunda was 2.7 mm. There was significantly greater diameter in vessels in the lateral thigh (p<0.001), in patients with higher Body Mass Index (BMI) (p<0.05), and in patients with decreased age (p<0.05). Males were more likely to have perforators that shared a common trunk off the profunda artery (p<0.05). CONCLUSIONS: At least two profunda perforators exist in each thigh with an average diameter suitable for microvascular transfer, although larger perforators are observed laterally and in younger patients with higher BMI.

"Supersize" panniculectomy: indications, technique, and results.

BACKGROUND: Obesity remains a significant health problem associated with considerable morbidity and mortality. Panniculectomy in the obese patient population aims at treating complications related to excess abdominal skin in an attempt to improve quality of life, increase mobility, and potentially prepare the patient for subsequent bariatric surgery or enrollment in a weight-loss program. We describe the indications and outcomes of "supersize" panniculectomy in the extreme obesity patient population. PATIENTS AND METHODS: A Duke University institutional review board-approved retrospective chart review of patients who underwent a "supersize" panniculectomy by a single surgeon during a 6-year period was conducted. Data on patient demographics, operative indication, preoperative imaging, concomitant operations, and postoperative complications were collected. RESULTS: Twenty-six patients underwent a "supersize" panniculectomy for indications including immobility secondary to excess abdominal skin, panniculitis, ventral hernia, and presence of a gynecologic tumor. The mean pannus resection weight was 15.6 kg and the mean follow-up was 15.7 months. Twelve patients underwent preoperative abdominal computed tomographic imaging. Eleven patients underwent concomitant surgical procedures at the time of their panniculectomy. The overall wound complication rate for the "supersize" panniculectomy was 42.3% (11/26 patients). However, the rate of major complications, defined as those complications requiring a return to the operating room, was only 11.5% (3/26 patients). CONCLUSIONS: "Supersize" panniculectomy is defined as a panniculectomy in the obese patient population with a resected pannus specimen weight greater than or equal to 10 kg, and a pannus formation that extends to the mid-thigh level or below. Despite the obstacles and reported high complication rates, the incidence of major complications in this series justifies the operative intervention in patients with an otherwise therapy-resistant "supersize" pannus. A preoperative computed tomographic imaging may rule out an underlying hernia in most cases and is recommended by the authors.

Changes in dermal histomorphology following surgical weight loss versus diet-induced weight loss in the morbidly obese patient.

INTRODUCTION: Patients with postgastric bypass and diet-induced weight loss present to the plastic surgeon for various body contouring procedures. Gross differences in skin dermal elasticity may exist between these populations; however, studies evaluating histologic differences are lacking. This prospective study aims to evaluate histomorphologic differences in morbidly obese patients following surgical versus diet-induced (nonsurgical) weight loss. Further, we aim to elicit if postoperative complications are correlated with the mechanism of weight loss and potential histomorphologic differences. METHODS: Defined infraumbilical skin specimens were collected during abdominal contouring procedures following weight loss achieved through surgical or nonsurgical means. Specimens were stained for elastic fiber content and morphology, collagen deposition, and inflammation. All sections underwent evaluation for quality and quantity of elastic fibers, collagen architecture, and presence of inflammation in the context of age-matched controls. Histomorphological results were compared between the 2 groups and subanalyzed according to clinical variables and postbody contouring wound complications. RESULTS: Between July 2008 and December 2010, 30 consecutive patients with significant weight loss (17 surgical, 13 nonsurgical) underwent a panniculectomy (n = 15), abdominoplasty (n = 13), and lower body lift (n = 2), with an average age of 48.3 ± 11.10 years and a body mass index of 39.23 ± 13.65 kg/m. Demographic and clinical variables were not statistically significant between the 2 groups. Blinded histologic evaluation revealed a trend toward normal elastic fiber appearance (P = 0.255), increased wound complications (P = 0.546), and mild inflammation (P = 0.462) in the surgical group. Analysis of dermal histomorphology correlating with wound complications was not statistically significant at follow-up (4.76 ± 5.55 months). Interestingly, there was a persistent inflammatory component in both groups when compared with age-matched controls. CONCLUSIONS: Although the differences in histomorphology between the surgical and nonsurgical weight loss groups did not reach statistical significance, the results demonstrated an existence of weight loss-induced histomorphological skin changes that may impact future studies. The study did not demonstrate a relationship between dermal histomorphology and postoperative wound complications, suggesting that aberrant healing in body contouring procedures involves a multifactorial process.

Wound contraction is attenuated by fasudil inhibition of Rho-associated kinase.

BACKGROUND: Dermal scarring and scar contracture result in restriction of movement. There are no effective drugs to prevent scarring. RhoA and Rho-associated kinase have emerged as regulators of fibrosis and contracture. Fasudil, a Rho-associated kinase inhibitor, has been demonstrated to have antifibrotic effects in models of liver, renal, and cardiac fibrosis. The role of fasudil in preventing dermal scarring and contractures has not been studied. The authors used a rat model of dermal wound healing to assess the effects of fasudil with regard to the prevention of scarring. METHODS: Human scar tissue and surrounding normal skin were immunostained for RhoA and Rho-associated kinase. Full-thickness wounds were created on Wistar-Han rats, and fasudil (30 mg/kg/day) or saline was continuously delivered subcutaneously. Wound contraction was measured by gravitational planimetry. After 21 days, tissue was harvested for Masson's trichrome, hematoxylin and eosin, Ki-67, and CD31 staining. Fibroblast-populated collagen lattices were used to assess the mechanistic effects of fasudil on contractility. Myofibroblast formation was assessed in the presence of fasudil. RESULTS: Human scar tissue in the remodeling phase of repair showed increased expression of RhoA and Rho-associated kinase in scar tissue compared with surrounding normal tissue. Fasudil inhibited wound contraction as compared with controls. Hematoxylin and eosin and Masson's trichrome were similar between groups. Fasudil did not alter angiogenesis or proliferation. Fasudil inhibited fibroblast contractility and myofibroblast formation in vitro. CONCLUSIONS: There is growing evidence that the RhoA/Rho-associated kinase pathway plays an important role in wound healing and scar contracture. The authors present data showing that inhibition of Rho-associated kinase hinders fibroblast contractility and may be beneficial in preventing scar contracture.

Angiotensin-II mediates nonmuscle myosin II activation and expression and contributes to human keloid disease progression.

Aberrant fibroblast migration in response to fibrogenic peptides plays a significant role in keloid pathogenesis. Angiotensin II (Ang II) is an octapeptide hormone recently implicated as a mediator of organ fibrosis and cutaneous repair. Ang II promotes cell migration but its role in keloid fibroblast phenotypic behavior has not been studied. We investigated Ang II signaling in keloid fibroblast behavior as a potential mechanism of disease. Primary human keloid fibroblasts were stimulated to migrate in the presence of Ang II and Ang II receptor 1 (AT1), Ang II receptor 2 (AT2) or nonmuscle myosin II (NMM II) antagonists. Keloid and the surrounding normal dermis were immunostained for NMM IIA, NMM IIB, AT2 and AT1 expression. Primary human keloid fibroblasts were stimulated to migrate with Ang II and the increased migration was inhibited by the AT1 antagonist EMD66684, but not the AT2 antagonist PD123319. Inhibition of the promigratory motor protein NMM II by addition of the specific NMM II antagonist blebbistatin inhibited Ang II-stimulated migration. Ang II stimulation of NMM II protein expression was prevented by AT1 blockade but not by AT2 antagonists. Immunostaining demonstrated increased NMM IIA, NMM IIB and AT1 expression in keloid fibroblasts compared with scant staining in normal surrounding dermis. AT2 immunostaining was absent in keloid and normal human dermal fibroblasts. These results indicate that Ang II mediates keloid fibroblast migration and possibly pathogenesis through AT1 activation and upregulation of NMM II.

The modified Patient and Observer Scar Assessment Scale: a novel approach to defining pathologic and nonpathologic scarring.

BACKGROUND: Scarring is a highly prevalent and multifactorial process, yet no studies to date have attempted to distinguish pathologic from nonpathologic scarring. METHODS: This article defines and proposes methods of classifying pathologic scarring as it pertains to clinical presentation. RESULTS: The authors propose a new scar scale that incorporates pain and functional impairment. CONCLUSIONS: The modified Patient and Observer Scar Assessment Scale is the first of its kind to factor in the functional deficits pain and pruritus of scarring into measurements of associated morbidity. This scale has great potential in evaluating patient response to treatment and analyzing clinical outcomes.

Temporal spatial expression and function of non-muscle myosin II isoforms IIA and IIB in scar remodeling.

Scar contracture is believed to be caused by the cell contractility during the remodeling phase of wound healing. Cell contractility is mediated by non-muscle myosin II (NMMII) and actin, but the temporal-spatial expression profile of NMMII isoforms A and B (IIA and IIB) during the remodeling phase and the role of NMMII in scar fibroblast tissue remodeling are unknown. Human scar tissue immunostained for IIA and IIB showed that both isoforms were highly expressed in scar tissue throughout the remodeling phase of repair and expression levels returned to normal after the remodeling phase. Human scar tissue immunostained for ß-, γ- and α-smooth muscle actin showed that all isoforms were consistently expressed throughout the remodeling phase of repair. The ß- and γ-smooth muscle actin were widely expressed throughout the dermis, but α-smooth muscle actin was only locally expressed within the dermis. In vitro, fibroblasts explanted from scar tissue were shown to express more IIA than fibroblasts explanted from normal tissue and scar fibroblasts contracted collagen lattices to a greater extent than normal fibroblasts. Blebbistatin was used to demonstrate the function of NMMII in collagen lattice contraction. In normal tissue, fibroblasts are stress-shielded from external tensile stress by the extracellular matrix. After dermal injury and during remodeling, fibroblasts are exposed to a matrix of increased stiffness. The effect of matrix stiffness on IIA and IIB expression was examined. IIA expression was greater in fibroblasts cultured in collagen lattices with increasing stiffness, and in fibroblasts cultured on glass slides compared with polyacrylamide gels with stiffness of 1 kPa. In conclusion, NMMII and actin isoform expression changes coordinately with the remodeling phase of repair, and NMMII is increased as matrix stiffness increases. As NMMII expression increases, so does the fibroblast contractility.

Calcium-dependent signaling in Dupuytren's disease.

BACKGROUND: Previous studies suggest that Dupuytren's disease is caused by fibroblast and myofibroblast contractility. Cell contractility in smooth muscle cells is caused by calcium-dependent and calcium-independent signaling mechanisms. In the calcium-dependent pathway, calcium/calmodulin activates myosin light chain kinase (MLCK). In this study, the effects of calcium/calmodulin inhibition with the FDA-approved drug fluphenazine on Dupuytren's fibroblast contractility and MLCK expression were tested. METHODS: Fibroblast lines from the palmar fascia of patients with Dupuytren's disease were explanted and used for in vitro study. The effect of fluphenazine on Dupuytren's fibroblast migration was determined using a scratch migration assay, and contractility was determined using fibroblast-populated collagen lattice (FPCL) assays. Immunohistochemical staining of MLCK in different samples of Dupuytren's tissue and normal fascia were compared. RESULTS: Fluphenazine demonstrated a dose-dependent inhibition of Dupuytren's fibroblast migration, with the maximum inhibition of migration observed at 20 µM (69.8 ± 1.9%). Fluphenazine also inhibited FPCL contraction in a dose-dependent manner. Maximal inhibition was observed at a fluphenazine concentration of 20 µM (52.5 ± 6.1%). Immunohistological staining illustrated that MLCK was predominantly expressed throughout the cytoplasm of select fibroblasts within Dupuytren's nodules, yet was absent in the fibroblasts of Dupuytren's cords and normal palmar fascia. CONCLUSIONS: Fluphenazine inhibits Dupuytren's fibroblast contractility and migration through inhibition of MLCK in vitro. However, the inconsistent expression of MLCK throughout Dupuytren's tissue suggests that calcium-dependent signaling may not be a primary mode of contracture formation. Fluphenazine inhibition of MLCK is not likely to be a target for the treatment of Dupuytren's disease.

Validation of a Novel Rodent Model to Test Anti-scarring Therapeutics.

UNLABELLED: Scar contracture is a debilitating disease that affects many people worldwide. There are currently no effective preventative medi- cal treatments. A pivotal step to attaining the goal of developing a treatment is the testing of anti-scarring agents in preclinical hierarchi- cal animal models of human scarring. METHODS: A 2-cm x 2-cm, full- thickness, excisional wound was created in the rats' mid-scapular area. Three experiments were performed. The first experiment determined the optimal dressing in wound contraction. The second experiment de- veloped upon the results of the first experiment, and determined how anatomic site of osmotic pump implantation affected wound healing. The third experiment determined how the size of osmotic pump affect- ed wound healing. Wound healing parameters including rate of wound contraction, systemic and local toxicity, proliferation, collagen archi- tecture, and collagen production were assessed. RESULTS: The results of the present study showed that covering the wound with TegadermTM (3M Health Care, St. Paul, MN) alone had the most linear wound con- traction rate with the smallest standard error of the mean. Implanta- tion of all osmotic pump sizes, when implanted intraperitoneally, was tolerated and did not interfere with wound healing. In contrast, sub- cutaneous implanted pumps caused significant discomfort in the rats. CONCLUSION: Implantation of an osmotic pump intraperitoneal in the rat excisional wound model, where the wound is covered with Tegaderm, provides for a reproducible, accurate, preclinical animal model to study anti-scar contracture treatments. .

Pseudomonas aeruginosa PilY1 binds integrin in an RGD- and calcium-dependent manner.

PilY1 is a type IV pilus (tfp)-associated protein from the opportunistic pathogen Pseudomonas aeruginosa that shares functional similarity with related proteins in infectious Neisseria and Kingella species. Previous data have shown that PilY1 acts as a calcium-dependent pilus biogenesis factor necessary for twitching motility with a specific calcium binding site located at amino acids 850-859 in the 1,163 residue protein. In addition to motility, PilY1 is also thought to play an important role in the adhesion of P. aeruginosa tfp to host epithelial cells. Here, we show that PilY1 contains an integrin binding arginine-glycine-aspartic acid (RGD) motif located at residues 619-621 in the PilY1 from the PAK strain of P. aeruginosa; this motif is conserved in the PilY1s from the other P. aeruginosa strains of known sequence. We demonstrate that purified PilY1 binds integrin in vitro in an RGD-dependent manner. Furthermore, we identify a second calcium binding site (amino acids 600-608) located ten residues upstream of the RGD. Eliminating calcium binding from this site using a D608A mutation abolished integrin binding; in contrast, a calcium binding mimic (D608K) preserved integrin binding. Finally, we show that the previously established PilY1 calcium binding site at 851-859 also impacts the protein's association with integrin. Taken together, these data indicate that PilY1 binds to integrin in an RGD- and calcium-dependent manner in vitro. As such, P. aeruginosa may employ these interactions to mediate host epithelial cell binding in vivo.

Antigen delivery by alpha(2)-macroglobulin enhances the cytotoxic T lymphocyte response.

alpha(2)M* targets antigens to APCs for rapid internalization, processing, and presentation. When used as an antigen-delivery vehicle, alpha(2)M* amplifies MHC class II presentation, as demonstrated by increased antibody titers. Recent evidence, however, suggests that alpha(2)M* encapsulation may also enhance antigen-specific CTL immunity. In this study, we demonstrate that alpha(2)M*-delivered antigen (OVA) enhances the production of specific in vitro and in vivo CTL responses. Murine splenocytes expressing a transgenic TCR specific for CTL peptide OVA(257-264) (SIINFEKL) demonstrated up to 25-fold greater IFN-gamma and IL-2 secretion when treated in vitro with alpha(2)M*-OVA compared with soluble OVA. The frequency of IFN-gamma-producing cells was increased approximately 15-fold, as measured by ELISPOT. Expansion of the OVA-specific CD8+ T cell population, as assayed by tetramer binding and [3H]thymidine incorporation, and OVA-specific cell-mediated cytotoxicity, as determined by a flow cytometric assay, were also enhanced significantly by alpha(2)M*-OVA. Furthermore, significant CTL responses were observed at antigen doses tenfold lower than those required with OVA alone. Finally, we also observed enhanced humoral and CTL responses by naïve mice following intradermal immunization with alpha(2)M*-OVA. These alpha(2)M*-OVA-immunized mice demonstrated increased protection against a s.c.-implanted, OVA-expressing tumor, as demonstrated by delayed tumor growth and prolonged animal survival. The observation that alpha(2)M*-mediated antigen delivery elicits specific CTL responses suggests the cross-presentation of antigen onto MHC class I. These results support alpha(2)M* as an effective antigen-delivery system that may be particularly useful for vaccines based on weakly immunogenic subunits or requiring dose sparing.

Incorporation of low molecular weight molecules into alpha(2)-macroglobulin by nucleophilic exchange.

alpha(2)-Macroglobulin (alpha(2)M) is a proteinase inhibitor that functions by a trapping mechanism which has been exploited such that the receptor-recognized, activated form (alpha(2)M( *)) can be employed to target antigens to antigen-presenting cells. Another potential use of alpha(2)M( *) is as a drug delivery system. In this study we demonstrate that guanosine triphosphate, labeled with Texas red (GTP-TR) formed complexes with alpha(2)M( *) following activation by proteolytic or non-proteolytic reactions. Optimal incorporation occurred with 20 microM GTP-TR, pH 8.0 for 5h at 50 degrees C. NaCl concentration (100 or 200 mM) had little effect on incorporation at this pH or temperature, but was significant at sub-optimum temperature and pH values. Maximum incorporation was 1.2 mol GTP-TR/mol alpha(2)M( *). PAGE showed that 70-90% of the GTP-TR is bound in a SDS/2-mercaptoethanol resistant manner. Guanosine, adenosine, and imidazole competed with GTP-TR to form complexes with alpha(2)M( *).