Roles of cutaneous cell-cell communication in wound healing outcome: An emphasis on keratinocyte-fibroblast crosstalk.

Tissue repair is a very complex event and involves a continuously orchestrated sequence of signals and responses from platelets, fibroblasts, epithelial, endothelial and immune cells. The details of interaction between these signals, which are mainly growth factors and cytokines, have been widely discussed. However, it is still not clear how activated cells at wound sites lessen their activities after epithelialization is completed. Termination of the wound healing process requires a fine balance between extracellular matrix (ECM) deposition and degradation. Maintaining this balance requires highly accurate epithelial-mesenchymal communication and correct information exchange between keratinocytes and fibroblasts. As it has been reported in the literature, a disruption in epithelialization during the process of wound healing increases the frequency of developing chronic wounds or fibrotic conditions, as seen in a variety of clinical cases. Conversely, the potential stop signal for wound healing should have a regulatory role on both ECM synthesis and degradation to reach a successful wound healing outcome. This review briefly describes the potential roles of growth factors and cytokines in controlling the early phase of wound healing and predominantly explores the role of releasable factors from epithelial-mesenchymal interaction in controlling during and the late stage of the healing process. Emphasis will be given on the crosstalk between keratinocytes and fibroblasts in ECM modulation and the healing outcome following a brief discussion of the wound healing initiation mechanism. In particular, we will review the termination of acute dermal wound healing, which frequently leads to the development of hypertrophic scarring.

Studying the in vivo application of a liquid dermal scaffold in promoting wound healing in a mouse model.

Lack of matrix deposition is one of the main factors that complicates the healing process of wounds. The aim of this study was to test the efficacy and safety of a liquid dermal scaffold, referred to as MeshFill (MF) that can fill the complex network of tunnels and cavities which are usually found in chronic wounds and hence improve the healing process. We evaluated in vitro and in vivo properties of a novel liquid dermal scaffold in a delayed murine full-thickness wound model. We also compared this scaffold with two commercially available granular collagen-based products (GCBP). Liquid dermal scaffold accelerated wound closure significantly compared with no-treated control and collagen-based injectable composites in a delayed splinted wound model. When we compared cellular composition and count between MF, no treatment and GCBP at the histology level, it was found that MF was the most analogous and consistent with the normal anatomy of the skin. These findings were matched with the clinical outcome observation. The flowable in situ forming scaffold is liquid at cold temperature and gels after application to the wound site. Therefore, it would conform to the topography of the wound when liquid and provides adequate tensile strength when solidified. This patient-ready gelling dermal scaffold also contains the nutritional ingredients and therefore supports cell growth. Applying an injectable liquid scaffold that can fill wound gaps and generate a matrix to promote keratinocytes and fibroblasts migration, can result in improvement of the healing process of complex wounds.

Promotion of Burn Wound Healing by Local Application of Adipose-Derived Mesenchymal Stem Cells: An Experimental Study.

Background:The burn wound is one of the health problems in the world that affects physical and mental health. Today, adipose-derived mesenchymal stem cells (ADSCs) have received medical attention for their accessibility and the ability to reproduce and repair. The present study was designed to investigate the effect of ADSCs on burn wound healing. Methods : The present experimental study was performed on 36 male Wistar rats divided into 1 control group and 3 experimental groups. The second-degree burns with a radius of 10 mm were induced after anesthesia. ADSCs and Dulbecco's Modified Eagle Medium (DMEM) were injected into the dermis around the burn area in the ADSCs and DMEM groups, respectively. Silver sulfadiazine (SSD) ointment was applied topically once daily as the SSD group. The control group did not receive any treatment. The rats were evaluated for 21 days. Wound healing rate, histopathological parameters, and the number of fibroblasts were evaluated by the immunofluorescence technique and vascular endothelial growth factor and transforming growth factor ß (TGF-ß) gene expression by reverse transcription-polymerase chain reaction. The results were entered into SPSS software (SPSS Inc) and analyzed with 1-way analysis of variance and repeated measures analysis. Results: The number of fibroblasts, the number of vessels, TGF-ß, and VEGF gene expression in the burn area were significantly higher in the ADSCs group than in the SSD, DMEM, and control groups. The results also showed that the amount of inflammation was significantly lower in the ADSCs group compared with the control group (p<0.001). Moreover, the percentage of wound recovery was significantly higher in the ADSCs group compared with other groups (p<0.001). Conclusion: ADSCs accelerate and improve burn wound healing by affecting fibroblasts, keratinocytes, and inflammatory cells as well as increasing the expression of the TGF-ß and VEGF genes, and thus increase in angiogenesis.

Myeloid Adherent Cells Are Involved in Hair Loss in the Alopecia Areata Mouse Model.

Alopecia areata (AA), which is defined as an autoimmune hair loss disease, has a serious impact on the quality of life for patients with AA worldwide. In this study, to our knowledge, a previously unreported method of AA induction in C3H mice has been established and validated. Using this method, we showed that dermal injection of 1-3 million of a mixture of skin cells freshly isolated from AA-affected skin induces AA in more than 80% of healthy mice. Contrary to the previous protocol, the induction of AA by this approach does not need any surgical AA skin grafting, cell manipulation, or high number of activated T cells. We also showed that dermal injection of adherent myeloid cells (mainly CD11b+) in healthy mice is as potent as a mixture of none adherent CD3+ T cells and CD19+ B cells in the induction of AA. Interestingly, most of the mice (7 out of 8) that received non-adherent cells developed AA universalis, whereas most of the mice (5 out of 7) that received adherent cells developed patchy AA. Finally, we found a high number of stage-specific embryonic antigen-expressing cells whose expression in monocytes in an inflammatory disease causes the release of inflammatory cytokines, TNF-α and IL-1ß, from these cells in AA-affected skin.

Increased expression of PD-L1 and PD-L2 in dermal fibroblasts from alopecia areata mice.

Alopecia areata (AA) is a common autoimmune disorder affecting millions of people worldwide, which manifests as a sudden, non-scarring hair loss. The expression of a pro-inflammatory cytokine, interferon-gamma (INF-γ), has been well established to be involved in the development of AA. As IFN-γ and other cytokines are also known to up-regulate programmed cell death ligand 1 and 2 (PD-L1 and PD-L2), which both negatively control immune responses, we asked whether or not a high number of infiltrated T cells, seen in AA lesions, can modulate the expression of PD-L1 and PD-L2 in skin cells. From a series of experiments, we showed that a significantly higher number of PD-L1 or PD-L2 positive cells affect the skin in AA mice, compared to the skin of non-AA mice. The number of PD-L1 positive cells was well correlated with the number of infiltrated T cells, especially CD8+ T cells. We also found that the expression of PD-L1 and PD-L2 was co-localized with type 1 pro-collagen, CD90 and vimentin, which are biomarkers for dermal fibroblasts. Further studies revealed that releasable factors from activated, but not inactivated, lymphocytes significantly increase the expressions of both PD-L1 and PD-L2 in cultured dermal fibroblasts. In conclusion, our findings suggest that the expression of PD-L1 and PD-L2 in dermal fibroblasts is up-regulated by activated T cells in AA-affected skin, and as such, these regulatory molecules may not exert a negative control of the immune activation seen in AA lesions.

Keratinocyte-Releasable Factors Stimulate the Expression of Granulocyte Colony-Stimulating Factor in Human Dermal Fibroblasts.

Interaction between keratinocytes and fibroblasts plays a critical role in maintaining skin integrity under both normal and pathological conditions. We have previously demonstrated that keratinocyte-releasable factors influence the expression of key extracellular matrix components, such as collagen and matrix metalloproteinases in dermal fibroblasts. In this study, we utilized DNA microarray analysis to examine the effects of keratinocyte-releasable factors on the expression of several cytokines in human dermal fibroblasts. The results revealed significantly higher granulocyte colony-stimulating factor (G-CSF) expression in fibroblasts co-cultured with keratinocytes relative to mono-cultured cells, which was verified by RT-PCR and western blot. G-CSF is an important hematopoietic factor also thought to play a beneficial role in wound healing through stimulating keratinocyte proliferation. To partially characterize the keratinocyte-releasable factors responsible for stimulating G-CSF production, keratinocyte-conditioned medium (KCM) was subjected to thermal treatment and ammonium sulfate precipitation before treating fibroblasts. The results showed that keratinocyte-releasable G-CSF-stimulating factors remain stable at 56°C and upon 50% ammonium sulfate precipitation. Knowing that keratinocytes release IL-1, which stimulates G-CSF expression in various immune cells, several experiments were conducted to ask whether this might also be the case for fibroblasts. The results showed that the addition of recombinant IL-1 markedly increased G-CSF expression in fibroblasts; however, IL-1 receptor antagonist only partially abrogated KCM-stimulated G-CSF expression, indicating the role of additional keratinocyte-releasable factors. These findings underline the importance of cross-talk between keratinocytes and fibroblasts, suggesting that communication between these cells in vivo modulates the production of cytokines required for cutaneous wound healing and maintenance. J. Cell. Biochem. 118: 308-317, 2017. © 2016 Wiley Periodicals, Inc.

Intraperitoneal injection of IDO-expressing dermal fibroblasts improves the allograft survival.

Indoleamine 2,3-dioxygenase (IDO) is an immunosuppressive enzyme with tolerogenic effects on different immune cells. Our group has previously shown that co-transplantation of IDO-expressing fibroblasts with donor tissues can delay immune rejection by inducing local immunosuppression. In this study, we have employed a systemic approach to improve allograft survival without using any immunosuppressive medication. To achieve this, 10 million lentiviral transduced IDO-expressing donor derived fibroblasts were injected into the peritoneal cavity of allograft recipients. We showed that IDO-fibroblast therapy increases the survival of both islets and skin allografts and decreases the infiltration of immune cells in subcutaneous transplanted skins. Indirect pathway of allo-reactive T cell activation was suppressed more than the direct pathway. Injected IDO-fibroblasts were found in peritoneal cavity and mesenteric lymph nodes of the recipient mice. In conclusion, IDO-expressing fibroblast therapy proved to be a novel approach in improving the allogeneic graft survival.

Kynurenic acid downregulates IL-17/1L-23 axis in vitro.

Exploring the function of interleukin (IL) 17 and related cytokine interactions have been proven useful toward understanding the role of inflammation in autoimmune diseases. Production of the inflammatory cytokine IL-23 by dendritic cells (DC's) has been shown to promote IL-17 expression by Th17 cells. It is well established that Th17 cells play an important role in several autoimmune diseases including psoriasis and alopecia. Our recent investigations have suggested that Kynurenine-rich environment can shift a pro-inflammatory response to an anti-inflammatory response, as is the case in the presence of the enzyme Indoleamine 2,3 dioxygenase (IDO), the rate-limiting enzyme in tryptophan degradation and Kynurenine (Kyn) production. In this study, we sought to explore the potential role of kynurenic acid (KynA), in modulating the expression of IL-23 and IL-17 by DCs and CD4+ cells, respectively. The result of flow cytometry demonstrated that the frequency of IL-23-producing DCs is reduced with 100 µg/ml of KynA as compared with that of LPS-stimulated DCs. KynA (100 µg/ml) addition to activated T cells significantly decreased the level of IL-17 mRNA and frequency of IL-17+ T cells as compared to that of concanavalin (Con) A-activated T cells. To examine the mechanism of the suppressive role of KynA on IL-23/IL-17 in these cells, cells were treated with 3 µM G-protein-coupled receptor35 (GPCR35) inhibitor (CID), for 60 min. The result showed that the reduction of both adenylate cyclase (AC) and cyclic adenosine monophosphate (cAMP) by KynA is involved in suppression of LPS-induced IL-23p19 expression. Since GPCR35 is also detected on T cells; therefore, it is concluded that KynA plays an important role in modulating the expression of IL-23 and IL-17 in DCs and Th17 cells through inhibiting GPCR35 and downregulation of both AC and cAMP.

Kynurenine Modulates MMP-1 and Type-I Collagen Expression Via Aryl Hydrocarbon Receptor Activation in Dermal Fibroblasts.

Dermal fibrosis is characterized by a high deposition of extracellular matrix (ECM) and tissue cellularity. Unfortunately all means of treating this condition are unsatisfactory. We have previously reported the anti-fibrotic effects of Kynurenine (Kyn), a tryptophan metabolite, in fibrotic rabbit ear model. Here, we report the mechanism by which Kyn modulates the expression of key ECM components in dermal fibroblasts. The results showed that Kyn activates aryl hydrocarbon receptor (AHR) nuclear translocation and up-regulates cytochrome-P450 (CYP1A-1) expression, the AHR target gene. A specific AHR antagonist, 6,2',4'-trimethoxyflavone, inhibited the Kyn-dependent modulation of CYP1A-1, MMP-1, and type-I collagen expression. Establishing the anti-fibrogenic effect of Kyn and its mechanism of action, we then developed nano-fibrous Kyn slow-releasing dressings and examined their anti-fibrotic efficacy in vitro and in a rat model. Our results showed the feasibility of incorporating Kyn into PVA/PLGA nanofibers, prolonging the Kyn release up to 4 days tested. Application of medicated-dressings significantly improved the dermal fibrosis indicated by MMP-1 induction, alpha-smooth muscle actin and type-I collagen suppression, and reduced tissue cellularity, T-cells and myofibroblasts. This study clarifies the mechanism by which Kyn modulates ECM expression and reports the development of a new slow-releasing anti-fibrogenic dressing. J. Cell. Physiol. 231: 2749-2760, 2016. © 2016 Wiley Periodicals, Inc.

IDO-Expressing Fibroblasts Protect Islet Beta Cells From Immunological Attack and Reverse Hyperglycemia in Non-Obese Diabetic Mice.

Indoleamine 2,3-dioxygenase (IDO) induces immunological tolerance in physiological and pathological conditions. Therefore, we used dermal fibroblasts with stable IDO expression as a cell therapy to: (i) Investigate the factors determining the efficacy of this cell therapy for autoimmune diabetes in non-obese diabetic (NOD) mice; (ii) Scrutinize the potential immunological mechanisms. Newly diabetic NOD mice were randomly injected with either 10 × 10(6) (10M) or 15 × 10(6) (15M) IDO-expressing dermal fibroblasts. Blood glucose levels (BGLs), body weight, plasma kynurenine levels, insulitis severity, islet beta cell function, autoreactive CD8(+) T cells, Th17 cells and regulatory T cells (Tregs) were then investigated in these mice. IL-1ß and cleaved caspase-3 levels were assessed in islets co-cultured with IDO-expressing fibroblasts. BGLs in 83% mice treated with 15M IDO-expressing fibroblasts recovered to normal up to 120 days. However, only 17% mice treated with 10M IDO-expressing cells were reversed to normoglycemia. A 15M IDO-expressing fibroblasts significantly reduced infiltrated immune cells in islets and recovered the functionality of remaining islet beta cells in NOD mice. Additionally, they successfully inhibited autoreactive CD8(+) T cells and Th17 cells as well as increased Tregs in different organs of NOD mice. Islet beta cells co-cultured with IDO-expressing fibroblasts had reduced IL-1ß levels and cell apoptosis. Both cell number and IDO enzymatic activity contributes to the efficiency of IDO cell therapy. Optimized IDO-expressing fibroblasts successfully reverse the progression of diabetes in NOD mice through induction of Tregs as well as inhibition of beta cell specific autoreactive CD8(+) T cells and Th17 cells. J. Cell. Physiol. 231: 1964-1973, 2016. © 2016 Wiley Periodicals, Inc.

Identification of a Hematopoietic Cell Dedifferentiation-Inducing Factor.

It has long been realized that hematopoietic cells may have the capacity to trans-differentiate into non-lymphohematopoietic cells under specific conditions. However, the mechanisms and the factors for hematopoietic cell trans-differentiation remain unknown. In an in vitro culture system, we found that using a conditioned medium from proliferating fibroblasts can induce a subset of hematopoietic cells to become adherent fibroblast-like cells (FLCs). FLCs are not fibroblasts nor other mesenchymal stromal cells, based on their expression of type-1 collagen, and other stromal cell marker genes. To identify the active factors in the conditioned medium, we cultured fibroblasts in a serum-free medium and collected it for further purification. Using the fractions from filter devices of different molecular weight cut-offs, and ammonium sulfate precipitation collected from the medium, we found the active fraction is a protein. We then purified this fraction by using fast protein liquid chromatography (FPLC) and identified it by mass spectrometer as macrophage colony-stimulating factor (M-CSF). The mechanisms of M-CSF-inducing trans-differentiation of hematopoietic cells seem to involve a tyrosine kinase signalling pathway and its known receptor. The FLCs express a number of stem cell markers including SSEA-1 and -3, OCT3/4, NANOG, and SOX2. Spontaneous and induced differentiation experiments confirmed that FLCs can be further differentiated into cell types of three germ layers. These data indicate that hematopoietic cells can be induced by M-CSF to dedifferentiate to multipotent stem cells. This study also provides a simple method to generate multipotent stem cells for clinical applications.

Tolerogenic effect of mouse fibroblasts on dendritic cells.

There is controversy about the immunomodulatory effect of fibroblasts on dendritic cells (DCs). To clarify this issue, in this study, we have evaluated different features of fibroblast-primed DCs including their ability to express co-inhibitory and co-stimulatory molecules, pro-inflammatory and anti-inflammatory cytokines and their ability to induce T-cell proliferation. We also examined migratory capacity of DCs to lymphatic tissues and present fibroblast-derived antigens after encountering fibroblasts. The results of our in vitro study showed that both co-inhibitory (programmed death ligand 1 and ligand 2 and B7H4) and co-stimulatory (CD86) molecules were up-regulated when DCs were co-cultured with fibroblasts. In an animal model, we showed that intra- peritoneal injection (IP) of both syngeneic and allogeneic fibroblasts significantly increased both total DC count and expression level of co-inhibitory and co-stimulatory molecules on DCs. Priming of DCs with syngeneic and allogeneic fibroblasts reduced the proliferation of CD4(+) and CD8(+) T cells. Even activation of fibroblast- primed DCs failed to restore their ability to induce T-cell proliferation. Likewise, priming of DCs with fibroblasts blocked the ability of ovalbumin-pulsed DCs to induce proliferation of ovalbumin-specific CD4(+) T cells. Compared with non-activated DCs, fibroblast-primed DCs had significantly higher expression levels of interleukin-10 and indoleamine 2, 3 dioxygenase. Fibroblast-primed DCs had a significantly reduced interleukin-12 expression level compared with that of activated DCs. After priming with fibroblasts, DCs were able to migrate to lymphatic tissues and present fibroblast-derived antigens (ovalbumin). In conclusion, after priming with fibroblasts, DCs gain tolerogenic features. This finding suggests the potential role of fibroblasts in the maintenance of immune tolerance.

A new method for skin grafting in murine model.

Skin transplantation provides an excellent potential model to investigate the immunology of allograft rejection and tolerance induction. Despite the theoretical ease of performing skin transplantation, as well as the potential of directly observing the reaction to the transplanted tissue, the poor reliability of skin transplantation in the mouse has largely precluded the use of this model. Furthermore, there is controversy regarding the most appropriate skin graft donor site due to poor success of back skin transplantation, as compared with the thinner ear or tail skin. This study demonstrates a reliable method to successfully perform skin grafts in a mouse model, as well as the clinical and histologic outcome of syngeneic grafts. A total of 287 grafts were performed (in 126 mice) utilizing donor skin from the ear, tail or back. No graft failure or postoperative mortality was observed. Comparison of this technique with two previously established protocols of skin transplantation (5.0 absorbable Suture + tissue glue technique and no-suture technique) demonstrates the significant improvement in the engraftment success of the new technique. In summary, a new technique for murine skin grafting demonstrates improved reliability across donor site locations and strains, increasing the potential for investigating interventions to alter the rejection process.

Methotrexate modulates the expression of MMP-1 and type 1 collagen in dermal fibroblast.

Methotrexate (MTX), an anti-metabolite and anti-inflammatory drug, has been used to effectively manage and prevent keloids, but its mechanism(s) of action has not been elucidated. Our study sought to evaluate the effect of MTX on the production of key extra cellular matrix components, collagen, and matrix metalloproteinase-1 (MMP-1), produced by fibroblasts and involved in development of fibrosis. The proliferation and viability of cultured human dermal fibroblasts in response to different concentrations of MTX were determined using cell counting and MTT assay, respectively. Western blot analysis was used to determine the levels of both intracellular and secreted type 1 collagen and MMP-1. The results showed no significant changes in the proliferation of fibroblasts treated with 50 ng/ml of MTX as compared to that of control. Under the same experimental conditions, the level of secreted and intracellular type I collagen was markedly reduced and, conversely, the level of MMP-1 increased in treated neonatal, adult, and hypertrophic scar fibroblasts as compared with those of controls. The possible involvement of MTX-induced extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in MMP-1 production was also studied and the result showed an increase in phosphorylated ERK 1/2 in response to MTX treatment. In summary, the findings of this study revealed that MTX significantly reduced collagen production in different strains of fibroblasts derived from neonatal, adult, and hypertrophic scar tissues, while under the same experimental conditions, it increased the expression of MMP-1. As such, our findings validate and identify a potential mechanism through which MTX functions as an anti-fibrogenic factor in treating fibroproliferative disorders.

Effects of kynurenine on CD3+ and macrophages in wound healing.

As prolongation of the inflammation phase in a healing process frequently leads to wound impairment, here we queried whether kynurenine (Kyn) could modulate this phase of wound healing. To address this, a protein microarray, quantitative polymerase chain reaction (qPCR), flow cytometry for immune cells and immune cell proliferation in the presence and absence of Kyn were conducted and compared. The result of a protein microarray revealed that the expression of 12 pro-inflammatory cytokines and chemokines was modulated in Kyn-treated mouse splenocytes as compared with those of control. These findings were then evaluated by conducting a qPCR for the gene expression of these factors and showed a significant reduction in the gene expression of majority of these cytokines and chemokines (interleukin [IL]-2, IL-17, C-X-C motif chemokine ligand [CXCL] 10, CXCL1, C-C motif ligand [CCL] 12, CXCL9, CCL4, CXCL2, and CCL5) in response to Kyn treatment. To test the anti-inflammatory effect of Kyn in an animal model, dorsal surface wounds were generated in a mouse model and wounds received daily topical application of either nothing (control), dermal cream (second control), or Kyn cream using uninjured skin tissue as another control. The wounded tissues were harvested on days 3, 6, and 10 postwounding. As anticipated, the results of fluorescence-activated cell sorting analysis revealed that upon wounding, the number of total infiltrated CD3+ cells and macrophages (CD11b+) significantly increased on day 3, peaked on day 6, and reduced on day 10 post-wounding. Interestingly, as compared with those of uninjured and dermal cream alone-treated wounds, Kyn treatment significantly reduced the number of infiltrated CD3+ cells, but not CD11b+ cells, at different time intervals examined. These findings collectively suggest that Kyn, as a small molecule, can potentially be used to overcome the difficulties associated with persistency of inflammation in healing wounds.

Three-dimensional scaffolds reduce islet amyloid formation and enhance survival and function of cultured human islets.

Islet transplantation provides a promising approach for treatment of type 1 diabetes mellitus. Amyloid formation and loss of extracellular matrix are two nonimmune factors contributing to death of isolated human islets. We tested the effects of two types of three-dimensional scaffolds, collagen matrix (CM) and fibroblast-populated collagen matrix (FPCM), on amyloid formation, viability, and function of isolated islets. Islets from cadaveric donors were cultured in FPCM, CM, or two-dimensional plate (2D) for 7 days. After 7 days, compared with the 2D culture condition, CM and FPCM markedly reduced amyloid formation of cultured islets and decreased apoptotic ß-cell rate by ∼75%. IL-1ß and Fas levels were also reduced in scaffold-embedded islets. Furthermore, ß/α cell ratios were increased by ∼18% and ∼36% in CM- and FPCM-embedded islets, respectively. Insulin content and insulin response to elevated glucose were also enhanced by both three-dimensional scaffolds. Moreover, culture in CM and FPCM (but not 2D) preserved insulin, GLUT-2, and PDX-1 mRNA expression. FPCM-embedded islets had significantly higher insulin response and lower amyloid formation than CM-embedded islets. These findings suggest that three-dimensional scaffolds reduce amyloid formation and improve viability and function of human islets in vitro, and that CM and fibroblasts have additive effects in enhancing islet function and reducing amyloid formation. Using this strategy is likely to improve outcome in human islet transplantation.

Topical application of aminopeptidase N-neutralizing antibody accelerates wound closure.

Upon release from keratinocytes, 14-3-3 sigma (also known as stratifin) acts on the dermal fibroblast and modulates its production of extracellular matrix proteins. Subsequent to the recent identification as a receptor responsible for stratifin-mediated matrix turnover in dermal fibroblasts, aminopeptidase N has been implicated in the regulation of epidermal-dermal communication and expression of key matrix proteases and adhesion molecules. In light of the growing importance of aminopeptidase N in modulation of the fibroblast phenotype, the present study evaluates the potential of targeting the ectoenzyme in cutaneous repair, and demonstrates that neutralization of aminopeptidase N led to acceleration of wound closure. This was attributed to at least in part an increase of collagen deposition and fibroblast contractility in the granulation tissue. These findings confirmed the important role of aminopeptidase N in post-injury tissue remodeling and wound contraction.

Topical application of a film-forming emulgel dressing that controls the release of stratifin and acetylsalicylic acid and improves/prevents hypertrophic scarring.

Here, we evaluate the efficacy of an emulgel dressing to control the release of an antifibrogenic factor, stratifin (SFN), along with an anti-inflammatory drug, acetylsalicylic acid (ASA), to be used as a wound dressing with hypertrophic scar reducing features. Emulgel dressings were prepared by dispersing positively charged submicron vesicles in carboxymethyl cellulose gel. Release kinetics of SFN/ASA and toxicity for primary skin cells were assessed in vitro. Antifibrogenic efficacy of medicated emulgel dressings was tested on a rabbit ear fibrotic model. Following topical application on the wounds, emulgels formed an occlusive film and controlled the release of SFN and ASA for 7 and 24 hours, respectively. Wounds treated with SFN/ASA-containing emulgel dressings showed an 80% reduction in scar elevation compared with untreated controls. Topical formulations were nontoxic for cultured human keratinocytes and fibroblasts. Inflammation was significantly controlled in treated wounds, as shown by a reduced number of infiltrated CD3(+) T cells (p < 0.001) and macrophages. SFN/ASA-treated wounds showed a significantly higher (p < 0.001) expression of matrix metalloproteinase-1, resulting in reduced collagen deposition and less scarring. Film-forming emulgel dressings that control the release of antifibrogenic and anti-inflammatory factors provide an excellent treatment option for postburn hypertrophic scar management.

Novel, Blended Polymeric Microspheres for the Controlled Release of Methotrexate: Characterization and In Vivo Antifibrotic Studies.

Low dose methotrexate (MTX) is known to effectively decrease type I collagen production in dermal fibroblasts, while increasing the matrix metalloproteinase-1 (MMP-1) production in vitro. For in vivo use as an antifibrotic agent on wounds, a linear and extended controlled release formulation of MTX is required. The objective of this study was to optimize the fabrication of MTX-loaded polymeric microspheres with such properties, and to test the efficacy for the prevention of fibrosis in vivo. Poly lactic-co-glycolic acid (PLGA), Poly (L-lactic acid) (PLLA) and the diblock copolymer, methoxypolyethylene glycol-block-poly (D, L-lactide) (MePEG-b-PDLLA), were used to fabricate microspheres, which were then characterized in terms of size, drug encapsulation efficiency, and in vitro release profiles. The optimized formulation (PLGA with diblock copolymer) showed high drug encapsulation efficiency (>80%), low burst release (~10%) and a gradual release of MTX. The amphipathic diblock copolymer is known to render the microsphere surface more biocompatible. In vivo, these microspheres were effective in reducing fibrotic tissue which was confirmed by quantitative measurement of type I collagen and α-smooth muscle actin expression, demonstrating that MTX can be efficiently encapsulated in PLGA microspheres to provide a delayed, gradual release in wound beds to reduce fibrosis in vivo.

Antibacterial Thermosensitive Silver-Hydrogel Nanocomposite Improves Wound Healing.

Bacterial infection and poor cell recruitment are among the main factors that prolong wound healing. To address this, a strategy is required that can prevent infection while promoting tissue repair. Here, we have created a silver nanoparticle-based hydrogel composite that is antibacterial and provides nutrients for cell growth, while filling cavities of various geometries in wounds that are difficult to reach with other dressings. Silver nanoparticles (AgNPs) were synthesized by chemical reduction and characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), and inductively coupled plasma-mass spectroscopy (ICP-MS). Using varying concentrations of AgNPs (200, 400, and 600 ppm), several collagen-based silver-hydrogel nanocomposite candidates were generated. The impact of these candidates on wound healing was assessed in a rat splinted wound model, while their ability to prevent wound infection from a contaminated surface was assessed using a rat subcutaneous infection model. Biocompatibility was assessed using the standard MTT assay and in vivo histological analyses. Synthesized AgNPs were spherical and stable, and while hydrogel alone did not have any antibacterial effect, AgNP-hydrogel composites showed significant antibacterial activity both in vitro and in vivo. Wound healing was found to be accelerated with AgNP-hydrogel composite treatment, and no negative effects were observed compared to the control group. The formulations were non-cytotoxic and did not differ significantly in hematological and biochemical factors from the control group in the in vivo study. By presenting promising antibacterial and wound healing activities, silver-hydrogel nanocomposite offers a safe therapeutic option that can be used as a functional scaffold for an acceleration of wound healing.