Periostin modulates myofibroblast differentiation during full-thickness cutaneous wound repair.

The matricellular protein periostin is expressed in the skin. Although periostin has been hypothesized to contribute to dermal homeostasis and repair, this has not been directly tested. To assess the contribution of periostin to dermal healing, 6 mm full-thickness excisional wounds were created in the skin of periostin-knockout and wild-type, sex-matched control mice. In wild-type mice, periostin was potently induced 5-7 days after wounding. In the absence of periostin, day 7 wounds showed a significant reduction in myofibroblasts, as visualized by expression of α-smooth muscle actin (α-SMA) within the granulation tissue. Delivery of recombinant human periostin by electrospun collagen scaffolds restored α-SMA expression. Isolated wild-type and knockout dermal fibroblasts did not differ in in vitro assays of adhesion or migration; however, in 3D culture, periostin-knockout fibroblasts showed a significantly reduced ability to contract a collagen matrix, and adopted a dendritic phenotype. Recombinant periostin restored the defects in cell morphology and matrix contraction displayed by periostin-deficient fibroblasts in a manner that was sensitive to a neutralizing anti-ß1-integrin and to the FAK and Src inhibitor PP2. We propose that periostin promotes wound contraction by facilitating myofibroblast differentiation and contraction.

Creating 3D angiogenic growth factor gradients in fibrous constructs to guide fast angiogenesis.

Fast angiogenesis in 3D fibrous constructs that mimic the morphology of the extracellular matrix remains challenging due to limited porosity in the densely packed constructs. We investigated whether mimicking the in vivo chemotaxis microenvironment for native blood vessel formation would stimulate angiogenesis in the fibrous constructs. The chemotaxis microenvironment was created by introducing 3D angiogenic growth factor gradients into the constructs. We have developed a technique that can quickly fabricate (∼40 min) such 3D gradients by simultaneously electrospinning polycaprolactone (PCL) fibers, encapsulating gradient amount of bFGF (stabilized by heparin) into poly(lactide-co-glycolide) (PLGA) microspheres, and electrospraying the microspheres into PCL fibers. Gradient formation was confirmed by fluorescence microscopy. Gradients with different steepnesses were obtained by modulating the initial concentration of the bFGF solution. All of the constructs were able to sustainedly release bioactive bFGF over a 28 day period. The release kinetics was dependent on the bFGF loading and steepness of the gradient. In vitro cell migration study demonstrated that bFGF gradients significantly increased the depth of cell migration. To assess the efficacy of bFGF gradients in inducing angiogenesis, we implanted constructs subcutaneously using mouse model. bFGF gradients significantly promoted cell penetration into the constructs. After 10 days of implantation, a high density of mature blood vessels (positive to both CD31 and α-SMA) were formed in the constructs. Vessel density was increased with the increase in steepness of the bFGF gradient. These gradient constructs may have potential to engineer vascularized tissues for various applications.

The impact of learner involvement in emergency department patient assessments on short-term return visits requiring hospital admission: a cross-sectional study.

PURPOSE: Learners, either medical students or residents, often perform the initial assessment of patients visiting the emergency department (ED). It is unclear, however, if learners affect the rate of short-term unscheduled return visits. The objective of this study was to determine if the involvement of learners in ED visits increases the rate of return visits. METHODS: This was a retrospective cross-sectional analysis of ED visit data at a single tertiary care centre over a 1-year period. Return visits were defined as those presenting within 72 h of discharge from an initial non-admit ED visit and resulting in an admission on the second visit. A generalized linear mixed model was used to determine the odds ratios of return visits, adjusting for prespecified co-variates, with and without learners involved during the initial visit. Secondary analyses assessed for associations between learner level of training, program of study and return visits. RESULTS: Return visits occurred after 658 (1.3%) of 51,149 encounters involving learners and 701 (0.8%) of 83,310 encounters with no learner involvement. Involvement of learners in ED initial visits was not associated with increased odds of return visits (adjusted OR 1.13 [95% CI 0.71-1.81]), although the point estimates were heterogeneous over learner level of training, with clerkship students (medical student years 3 and 4) and senior residents (post-graduate years 4 and 5) trending towards reduced odds of a return visit. Resident program of study did not independently predict return visits. CONCLUSIONS: This study demonstrated that the involvement of learners in ED patient assessments is not associated with increased odds of short-term unscheduled return visits.

RéSUMé: OBJECTIF: Les apprenants, qu'ils soient étudiants en médecine ou résidents, procèdent souvent à l'évaluation initiale des patients qui se rendent au service des urgences (SU). Il n'est pas clair, cependant, si les apprenants ont une incidence sur le taux de visites de retour imprévues à court terme. L'objectif de cette étude était de déterminer si la participation des apprenants dans les visites aux urgences augmentait le taux de retour des visites. MéTHODES: Il s'agissait d'une analyse transversale rétrospective des données sur les visites à l'urgence dans un seul centre de soins tertiaires sur une période d'un an. Les visites de retour ont été définies comme celles qui se sont présentées dans les 72 heures suivant la sortie d'une première visite à l'urgence sans admission et qui ont donné lieu à une admission lors de la deuxième visite. Un modèle linéaire mixte généralisé a été utilisé pour déterminer les rapports de cotes des visites de retour, en tenant compte des co-variables préétablies, avec et sans participation des apprenants pendant la visite initiale. Des analyses secondaires ont évalué les associations entre le niveau de formation de l'apprenant, le programme d'études et les visites de retour. RéSULTATS: Les visites de retour ont eu lieu après 658 (1,3%) des 51 149 rencontres impliquant des apprenants et 701 (0,8%) des 83 310 rencontres sans participation de l'apprenant. L'implication des apprenants dans les visites initiales à l'urgence n'était pas associée à une probabilité accrue de visites ultérieures (OR ajusté = 1,13 [IC à 95% 0,71­1,81]), bien que les estimations ponctuelles aient été hétérogènes selon le niveau de formation de l'apprenant, les étudiants en externat (années d'études en médecine 3 et 4) et les résidents seniors (années 4 et 5 des études supérieures) ont tendance à réduire les risques de visite de retour. Le programme d'études des résidents n'a pas prédit de manière indépendante les visites de retour. CONCLUSIONS: Cette étude a démontré que la participation des apprenants aux évaluations des patients à l'urgence n'est pas associée à une probabilité accrue de visites de retour imprévues à court terme.

Periostin and CCN2 Scaffolds Promote the Wound Healing Response in the Skin of Diabetic Mice.

IMPACT STATEMENT: Nonhealing skin wounds remain a significant burden on health care systems, with diabetic patients 20 times as likely to undergo a lower extremity amputation due to impaired healing. Novel treatments that suppress the proinflammatory signature and induce the proliferative and remodeling phases are needed clinically. We demonstrate that the addition of periostin and CCN2 in a scaffold form increases closure rates of full-thickness skin wounds in diabetic mice, concomitant with enhanced angiogenesis. Our results demonstrate the efficacy of periostin- and CCN2-containing biomaterials to stimulate wound closure, which could represent a novel method for the treatment of diabetic skin wounds.

Genetic Deletion of Galectin-3 Does Not Impair Full-Thickness Excisional Skin Healing.

Galectin-3 has been linked to the regulation of several molecular processes essential during acute cutaneous wound healing, but a comprehensive study of the role of galectin-3 has yet to be performed. With known roles in macrophage polarization, myofibroblast differentiation, re-epithelialization, and angiogenesis, we hypothesized that genetic deletion of galectin-3 would significantly impair healing of excisional skin wounds in mice. In wild-type mice, galectin-3 expression correlated temporally with the inflammatory phase of healing. Conversely, genetic deletion of galectin-3 did not alter gross wound healing kinetics even though it resulted in delayed re-epithelialization. Wound composition was not altered up to 15 days after wounding in knockout mice, and isolated dermal fibroblast function in vitro was unchanged. We further explored, spatially, the expression of galectin-3 in human chronic wound tissue in relation to the immune cell infiltrate. We show a decreased mRNA and protein abundance in the wound edge tissue, whereas markers of neutrophils, M1 and M2 macrophages are expressed abundantly. Both transforming growth factor-ß1 and tumor necrosis factor-α decrease galectin-3 mRNA abundance in chronic wound edge dermal fibroblasts in vitro, providing a potential mechanism for this decreased expression in chronic wounds.

Inflammatory microenvironment and tumor necrosis factor alpha as modulators of periostin and CCN2 expression in human non-healing skin wounds and dermal fibroblasts.

Non-healing skin wounds remain a significant clinical burden, and in recent years, the regulatory role of matricellular proteins in skin healing has received significant attention. Periostin and CCN2 are both upregulated at day 3 post-wounding in murine skin, where they regulate aspects of the proliferative phase of repair including mesenchymal cell infiltration and myofibroblast differentiation. In this study, we examined 1) the wound phenotype and expression patterns of periostin and CCN2 in non-healing skin wounds in humans and 2) the regulation of their expression in wound fibroblasts by tumor necrosis factor α (TNFα) and transforming growth factor-ß1 (TGF-ß1). Chronic skin wounds had a pro-inflammatory phenotype, characterized by macrophage infiltration, TNFα immunoreactivity, and neutrophil infiltration. Periostin, but not CCN2, was significantly suppressed in non-healing wound edge tissue at the mRNA and protein level compared with non-involved skin. In vitro, human wound edge fibroblasts populations were still able to proliferate and contract collagen gels. Compared to cells from non-involved skin, periostin and α-SMA mRNA levels increased significantly in the presence of TGF-ß1 in wound cells and were significantly decreased by TNFα, but not those of Col1A2 or CCN2. In the presence of both TGF-ß1 and TNFα, periostin and α-SMA mRNA levels were significantly reduced compared to TGF-ß1 treated wound cells. Effects of TGF-ß1 and TNFα on gene expression were also more pronounced in wound edge cells compared to non-involved fibroblasts. We conclude that variations in the expression of periostin and CCN2, are related to an inflammatory microenvironment and the presence of TNFα in human chronic wounds.

Detection of galectin-3 and localization of advanced glycation end products (AGE) in human chronic skin wounds.

The matricellular protein galectin-3 (Gal-3) is upregulated in excisional skin repair in rats where it has been shown to modulate the inflammatory phase of repair. Recent research into kidney pathology has implicated Gal-3 as a receptor for advanced glycation end products (AGE), resulting in the binding and clearance of these molecules. AGEs are thought to contribute to defective skin repair in diabetic patients as well as a result of the normal aging process. However, the distribution and localization of Gal-3 and AGEs has never been performed in human chronic skin wound tissue. Using immunohistochemistry, the localization of Gal-3 and AGEs in tissue isolated from chronic wounds and non-involved skin from the same patient was investigated. Of the 16 patients from which tissue was isolated, 13 had type II diabetes, one had type I diabetes and 2 patients without diabetes were also examined. In non-involved dermis, Gal-3 was detected strongly in the epidermis and in the vasculature. However, at the wound edge and in the wound bed, the level of Gal-3 labelling was greatly reduced in both the epidermis and vasculature. Labelling of serial sections for Gal-3 and AGE demonstrated that where Gal-3 immunoreactivity is reduced in the epidermis and vasculature, there is a concomitant increase in the level of AGE staining. Interestingly, similar labelling patterns were evident in diabetic and non-diabetic patients. The results from our study demonstrate an inverse correlation between Gal-3 and AGEs localization, suggesting that Gal-3 may protect against accumulation of AGEs in wound healing.

Inhibition of Rac and ROCK signalling influence osteoblast adhesion, differentiation and mineralization on titanium topographies.

Reducing the time required for initial integration of bone-contacting implants with host tissues would be of great clinical significance. Changes in osteoblast adhesion formation and reorganization of the F-actin cytoskeleton in response to altered topography are known to be upstream of osteoblast differentiation, and these processes are regulated by the Rho GTPases. Rac and RhoA (through Rho Kinase (ROCK)). Using pharmacological inhibitors, we tested how inhibition of Rac and ROCK influenced osteoblast adhesion, differentiation and mineralization on PT (Pre-treated) and SLA (sandblasted large grit, acid etched) topographies. Inhibition of ROCK, but not Rac, significantly reduced adhesion number and size on PT, with adhesion size consistent with focal complexes. After 1 day, ROCK, but not Rac inhibition increased osteocalcin mRNA levels on SLA and PT, with levels further increasing at 7 days post seeding. ROCK inhibition also significantly increased bone sialoprotein expression at 7 days, but not BMP-2 levels. Rac inhibition significantly reduced BMP-2 mRNA levels. ROCK inhibition increased nuclear translocation of Runx2 independent of surface roughness. Mineralization of osteoblast cultures was greater on SLA than on PT, but was increased by ROCK inhibition and attenuated by Rac inhibition on both topographies. In conclusion, inhibition of ROCK signalling significantly increases osteoblast differentiation and biomineralization in a topographic dependent manner, and its pharmacological inhibition could represent a new therapeutic to speed bone formation around implanted metals and in regenerative medicine applications.

Functional significance of periostin in excisional skin repair: is the devil in the detail?

In the past year, three papers have been published exploring the role of the matricellular protein periostin in excisional skin repair. These papers all show a delay in wound closure and the kinetics of this delay are strikingly similar across the three reports. The similarities between these papers end, however, when each investigates the mechanism through which periostin influences skin repair. Three proposed mechanisms have been identified: (1) myofibroblast differentiation, (2) keratinocyte proliferation and (3) fibroblast proliferation and migration. The aim of this commentary is to compare and contrast the three studies performed to date in an attempt to decipher the role of periostin in the repair of full-thickness skin wounds.

Deconstructing fibrosis research: do pro-fibrotic signals point the way for chronic dermal wound regeneration?

Chronic wounds are characterized by inadequate matrix synthesis, no re-epithelialization, infection and ultimately no wound resolution. In contrast, fibrosis is characterized by overproduction of matrix and excess matrix contraction. As research in the fields of chronic wounds and fibrosis surges forward, important parallels can now be drawn between the dysfunctions in fibrotic diseases and the needs of chronic wounds. These parallels exist at both the macroscopic level and at the molecular level. Thus in finding the individual factors responsible for the progression of fibrotic diseases, we may identify new therapeutic targets for the resolution of chronic wounds. The aim of this review is to discuss how recent advances in fibrosis research have found a home in the treatment of chronic wounds and to highlight the benefits that can be obtained for chronic wound treatments by employing a translational approach to molecules identified in fibrosis research.