Anteroposterior patterning of the zebrafish ear through Fgf- and Hh-dependent regulation of hmx3a expression.

In the zebrafish, Fgf and Hh signalling assign anterior and posterior identity, respectively, to the poles of the developing ear. Mis-expression of fgf3 or inhibition of Hh signalling results in double-anterior ears, including ectopic expression of hmx3a. To understand how this double-anterior pattern is established, we characterised transcriptional responses in Fgf gain-of-signalling or Hh loss-of-signalling backgrounds. Mis-expression of fgf3 resulted in rapid expansion of anterior otic markers, refining over time to give the duplicated pattern. Response to Hh inhibition was very different: initial anteroposterior asymmetry was retained, with de novo duplicate expression domains appearing later. We show that Hmx3a is required for normal anterior otic patterning, and that otic patterning defects in hmx3a-/- mutants are a close phenocopy to those seen in fgf3-/- mutants. However, neither loss nor gain of hmx3a function was sufficient to generate full ear duplications. Using our data to infer a transcriptional regulatory network required for acquisition of otic anterior identity, we can recapitulate both the wild-type and the double-anterior pattern in a mathematical model.

The Safety and Tolerability of Topically Delivered Kynurenic Acid in Humans. A Phase 1 Randomized Double-Blind Clinical Trial.

Scarring is a consequence of biological tissue repair following trauma. Currently, there are no generally agreed ways to prevent scarring. Recently, kynurenic acid has shown to be a potent modulator of extracellular matrix deposition and remodeling. Kynurenic acid can reduce matrix deposition and other fundamental characteristics of fibrosis in vitro and in vivo. Specifically, kynurenic acid has shown to increase matrix metalloproteinase-1 activity and subsequently reduce collagen deposition in a rabbit ear scar model. In the present study kynurenic acid cream in different concentrations was topically applied on healthy skin on volunteers to assess skin reactions and skin sensitivity in both acute and chronic application settings. Skin reactions were assessed, and concentrations for kynurenic acid were assessed both form serum and urine. Results showed to acute or delayed skin reactions. Kynurenic acid was not detectable in blood at any time point, and only trace elements of kynurenic acid were found in urine. This study supports safety and tolerability of topically administered FS2 when using a liposomal, compounding base carrier.

Evaluation of Detergent-Free and Detergent-Based Methods for Decellularization of Murine Skin.

Acute and chronic wounds contribute to increased morbidity and mortality in affected people and impose significant financial burdens on healthcare systems. For these challenging wounds, acellular dermal matrices (ADMs) have been used as a biological wound coverage. Unlike engineered dermal matrices, ADMs are prepared through the removal of cells from skin, while preserving the extracellular matrix structure and function. In this study, our primary objective was to develop a detergent-free method for decellularization of the skin to mitigate chemical stress on matrix molecules. Then, we performed a set of in vitro and in vivo experiments to compare this method with nonionic and anionic detergent methods. All decellularization methods satisfactorily removed cells and supported fibroblast growth and migration in vitro. Sulfated glycosaminoglycan content was reduced significantly (p < 0.05) only in the ionic detergent treatment group. In contrast to the detergent-free method, all detergent-based methods significantly reduced scaffold mechanical strength and elastin content (p < 0.05). Three weeks after transplantation, the results showed reepithelialization, angiogenesis, and migration of host cell into scaffolds with no induction of immunogenic reaction in all ADM groups tested. In our study, the detergent-free method showed better preservation of matrix composition and biomechanical properties, but after transplantation, all methods of ADM preparation resulted in equally biofunctional matrices as wound coverage.

Polyvinyl alcohol-graft-polyethylene glycol hydrogels improve utility and biofunctionality of injectable collagen biomaterials.

Collagen-based materials have become a staple in both research and the clinic. In wound care, collagen-based materials comprise a core gamut of biological dressings and therapeutic strategies. In research, collagen-based materials are employed in everything from 3D cultures to bioprinting. Soluble collagen is well characterized to undergo fibrillation at neutral pH and 37 °C. To remain stable, a neutralized collagen solution must be maintained at 4 °C. These physical characteristics of collagen impose limitations on its utility. In our previous work, we identified that the incorporation of a simple polyvinyl alcohol:borate hydrogel could improve the rate of collagen gel fibrillation. In this work we sought to further investigate the interactions of polyvinyl alcohol blend variants, as surfactant-like polymers, in comparison with known non-polymer surfactants. To conduct our investigations scaffold variants were created using increasing concentrations of polyvinyl alcohol, differing combinations of polymers, and non-polymer surfactants Tweens 20 and 80, and TritonX-100. Activation energy for collagen fibrillation was found to significantly decrease in the presence of polyvinyl alcohols (p < 0.01) at and above 0.4%w/v concentration. Further, addition of polyvinyl alcohol-graft-polyethylene glycol had the greatest enhancement (2.02 fold) on the fibrillation kinetics (p < 0.01), wetting properties and the stability of the collagen scaffolds post-freeze drying. Our results demonstrated that the addition of polyvinyl alcohol hydrogels to a collagen solution could stabilize collagen solution such that the solution could easily be lyophilized (at pH 7) and then reconstituted with water. Cells cultured in polyvinyl alcohol scaffolds also exhibited more organized F-actin, as well as a reduced abundance of pro-collagen and α-smooth actin. In conclusion, our results demonstrate for the first time that polyvinyl alcohol, preferably polyvinyl alcohol-graft-polyethylene glycol, directly affects the physical properties of collagen and the physiology of cells cultured within improving the utility of the combined material for both research and clinic needs.

Immunoprotection and Functional Improvement of Allogeneic Islets in Diabetic Mice, Using a Stable Indoleamine 2,3-Dioxygenase Producing Scaffold.

BACKGROUND: We have previously shown that an immunomodulatory enzyme, indoleamine 2,3-dioxygenase (IDO) in dermal fibroblasts generates a tryptophan-deficient environment that selectively inhibits proliferation and induces apoptosis of bystander CD4+ and CD8+ T cells, but not pancreatic islets. Because these immune cells are involved in islet allograft rejection, we hypothesized that transplantation of islets embedded in a novel 3-dimensional composite scaffold within which stable IDO-expressing fibroblasts serve as source of local immunosuppression would lead to normoglycemia in a streptozotocin-induced diabetic mouse model. METHODS: Islet grafts were prepared by embedding stable IDO-expressing fibroblasts and allogeneic islets into a protease-resistant composite scaffold. Islets function and survival were evaluated in vitro using immunohistochemistry. Allografts were transplanted under the kidney capsule of streptozotocin-induced diabetic mice; viability, function, and criteria for graft take were evaluated. Flow cytometry was performed to determine specific intragraft, draining lymph nodes and spleen T-cell population, and splenocytes alloantigen responsiveness of graft recipients. RESULTS: The results of a series of in vitro experiments revealed that IDO-expressing fibroblasts do not compromise islet function or survival. The expression of IDO suppressed the proliferation of alloantigen-stimulated splenocytes. The in vivo experiments revealed that local IDO expression delivered by lentiviral vector prolonged islet allograft survival (51.0 ± 2.9 days) by increasing the population of FOXP3+ regulatory T cells at the graft site and graft-draining lymph nodes and preventing T-cell infiltration. CONCLUSIONS: This study shows that incorporation of islets within our novel matrix that is equipped with stable IDO-expressing fibroblasts prolongs allograft survival.

An in-situ forming skin substitute improves healing outcome in a hypertrophic scar model.

Wound repair requires a sequential series of biological events that begins with the deposition of a temporary scaffold within which cells can repair the skin. Without a scaffold, repair is essentially impossible. Aberrant wound healing, such as hypertrophic scarring or nonhealing, has a tremendous burden on healthcare and quality of life. Timely wound closure dramatically reduces the risk of infection and scarring. Cellular skin substitutes are opportune to meet this need. Our goal was to create an in-situ forming scaffold that can be easily combined with cells to rapidly form a dermal substitute within the wound bed. In this study, we evaluated the application of a polyvinyl alcohol-collagen-glycosaminoglycan-based biohybrid scaffold system in full-thickness wounds on a rabbit fibrotic ear model. Punch wounds (6 mm) were either untreated or filled with an acellular scaffold, a scaffold containing xenofibroblasts, or a scaffold containing xenofibroblasts expressing indoleamine 2,3-dioxygenase (IDO). Results demonstrated that (1) both acellular and IDO-expressing fibroblast in-situ forming scaffolds significantly reduced scar elevation index (1.24±0.05 and 1.25±0.03; p<0.05) and improved overall healing quality compared with xenofibroblast scaffolds and untreated wounds; (2) IDO-expressing fibroblast scaffolds significantly reduced T-cell infiltration into the scaffold-engrafted area (p<0.05); and (3) both IDO-expressing and acellular in-situ forming scaffolds demonstrated increased vessel-like and nerve-like structures (p<0.05). The results demonstrated that the use of the in-situ forming scaffold, and even more so when delivering IDO-expressing cells, improved healing outcome in full-thickness hypertrophic rabbit ear wounds.

Anti-scarring properties of different tryptophan derivatives.

Hypertrophic scars are associated with prolonged extracellular matrix (ECM) production, aberrant ECM degradation and high tissue cellularity. Routinely used antifibrotic strategies aim to reduce ECM deposition and enhance matrix remodeling. Our previous study investigating the antifibrotic effects of indoleamine2, 3 dioxygenase (IDO) led to the identification of kynurenine (Kyn) as an antiscarring agent. A topical antifibrogenic therapy using Kyn is very attractive; however, it is well established that Kyn passes the blood brain barrier (BBB) which causes complications including excitatory neuronal death. Here we investigated the antiscarring properties of kynurenic acid (KynA), a downstream end product of Kyn that is unlikely to pass the BBB, as an effective and safe replacement for Kyn. Our results indicated that while not having any adverse effect on dermal cell viability, KynA significantly increases the expression of matrix metalloproteinases (MMP1 and MMP3) and suppresses the production of type-I collagen and fibronectin by fibroblasts. Topical application of cream containing KynA in fibrotic rabbit ear significantly decreased scar elevation index (1.13±0.13 vs. 1.61±0.12) and tissue cellularity (221.38±21.7 vs. 314.56±8.66 cells/hpf) in KynA treated wounds compared to controls. KynA treated wounds exhibited lower levels of collagen deposition which is accompanied with a significant decrease in type-I collagen and fibronectin expression, as well as an increase in MMP1 expression compared to untreated wounds or wounds treated with cream only. The results of this study provided evidence for the first time that KynA is promising candidate antifibrogenic agent to improve healing outcome in patients at risk of hypertrophic scarring.

Postelectrospinning modifications for alginate nanofiber-based wound dressings.

Alginate nanofibers have been attractive for potential tissue regeneration applications due to a combination of their moisture retention ability and large surface area available in a nonwoven nanofiber form. This study aims to address several challenges in alginate nanofiber application, including the lack of structural stability in aqueous environment and limited cell attachment as compared to commercial wound dressings, via examining crosslinking techniques. In addition to the commonly performed divalent ion crosslinking, a glutaraldehyde double-crosslinking step and polylysine addition were applied to an electrospun alginate nanofiber nonwoven mat. With optimization of the electrospinning solution, nanofiber morphology was maintained after the two-stage crosslinking process. Extensibility of the nanofiber mat reduced after the crosslinking process. However, both aqueous stability and cell attachment improved after the postspinning modifications, as shown through degradation tests in phosphate buffered saline solutions and fibroblast cell culture studies, respectively.

Embedding islet in a liquid scaffold increases islet viability and function.

INTRODUCTION: Islet transplantation is a promising strategy to restore efficient insulin regulation in type 1 diabetes mellitus patients. However, shortage of islet donors, poor islet survival and toxicity of immunosuppressants often reduce the graft functional lifetime. METHODS: We previously showed that a fibroblast populated-collagen matrix (CM) significantly improved engrafted islet viability/function. However, this composite was prone to gradual biodegradation and contraction. Moreover, to avoid use of systemic immunosuppressants, we proposed the use of a local immunosuppressive enzyme, indoleamine-2,3-dioxygenase (IDO). We developed a novel bioengineered crosslinked CM (CCM) to provide optimal matrix biomimetic. Viability and insulin secretory function of islets embedded within fibroblast populated CCM (FP-CCM) was evaluated in vitro and in vivo. IDO expression was transduced in fibroblasts by a lentiviral vector carrying IDO gene and islet viability was evaluated in the presence and absence of IDO producing cells. RESULTS: Islet survival/function markedly improved within FP-CCM. Furthermore, our data shows that local lentiviral induction of IDO delivered by FP-CCM is nontoxic to the embedded islets. CONCLUSIONS: This promising finding offers a new approach to improving islet transplant outcome.

Immuno-regulatory function of indoleamine 2,3 dioxygenase through modulation of innate immune responses.

Successful long-term treatment of type-1 diabetes mainly relies on replacement of ß-cells via islet transplantation. Donor shortage is one of the main obstacles preventing transplantation from becoming the treatment of choice. Although animal organs could be an alternative source for transplantation, common immunosuppressive treatments demonstrate low efficacy in preventing xenorejection. Immunoprotective effects of indoleamine 2,3-dioxygenase (IDO) on T-cell mediated allorejection has been extensively studied. Our studies revealed that IDO expression by fibroblasts, induced apoptosis in T-cells while not affecting non-immune cell survival/function. Since macrophages play a pivotal role in xenograft rejection, herein we investigated the effect of IDO-induced tryptophan deficiency/kynurenine accumulation on macrophage function/survival. Moreover, we evaluated the local immunosuppressive effect of IDO on islet-xenograft protection. Our results indicated that IDO expression by bystander fibroblasts significantly reduced the viability of primary macrophages via apoptosis induction. Treatment of peritoneal macrophages by IDO-expressing fibroblast conditioned medium significantly reduced their proinflammatory activity through inhibition of iNOS expression. To determine whether IDO-induced tryptophan starvation or kynurenine accumulation is responsible for macrophage apoptosis and inhibition of their proinflammatory activity, Raw264.7 cell viability and proinflammatory responses were evaluated in tryptophan deficient medium or in the presence of kynurenine. Tryptophan deficiency, but not kynurenine accumulation, reduced Raw264.7 cell viability and suppressed their proinflammatory activity. Next a three-dimensional islet-xenograft was engineered by embedding rat islets within either control or IDO-expressing fibroblast-populated collagen matrix. Islets morphology and immune cell infiltration were then studied in the xenografts transplanted into the C57BL/6 mouse renal sub-capsular space. Local IDO significantly decreased the number of infiltrating macrophages (11 ± 1.47 vs. 70.5 ± 7.57 cells/HPF), T-cells (8.75 ± 1.03 vs. 75.75 ± 5.72 cells/HPF) and iNOS expression in IDO-expressing xenografts versus controls. Islet morphology remained intact in IDO-expressing grafts and islets were strongly stained for insulin/glucagon compared to control. These findings support the immunosuppressive role of IDO on macrophage-mediated xeno-rejection.

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.

Localized controlled release of stratifin reduces implantation-induced dermal fibrosis.

Localized controlled release of anti-fibrogenic factors can potentially prevent tissue fibrosis surrounding biomedical prostheses, such as vascular stents and breast implants. We have previously demonstrated that therapeutic intervention with topically applied stratifin in a rabbit ear fibrotic model not only prevents dermal fibrosis but also promotes more normal tissue repair by regulating extracellular matrix deposition. In this work, the anti-fibrogenic effect of a controlled release form of stratifin was investigated in the prevention of fibrosis induced by dermal poly(lactic-co-glycolic acid) (PLGA) microsphere/poly(vinyl alcohol) (PVA) hydrogel implants. Pharmacodynamic effects were evaluated by histopathological examination of subcutaneous tissue surrounding implanted composites. Controlled release of stratifin from PLGA microsphere/PVA hydrogel implants significantly moderated dermal fibrosis and inflammation by reducing collagen deposition (30%), total tissue cellularity (48%) and infiltrated CD3(+) immune cells (81%) in the surrounding tissue compared with the stratifin-free implants. The controlled release of stratifin from implants markedly increased the level of matrix metalloproteinase-1 expression in the surrounding tissue, which resulted in less collagen deposition. These stratifin-eluting PLGA/PVA composites show promise as coatings to decrease the typical fibrosis exhibited around implanted biomedical prostheses, such as breast implants and vascular stents.

SPARC/SFN interaction, suppresses type I collagen in dermal fibroblasts.

We previously suggested that keratinocyte releasable factors might modulate the wound healing process by regulating the expression of key extracellular matrix components such as collagenase (matrix metalloproteinase-1) and type I collagen in fibroblasts. The first one, we called it keratinocyte-derived anti-fibrogenic factor (KDAF), identified as stratifin (SFN) also named 14-3-3σ, revealing a strong collagenase activity. However, the second factor, which we named keratinocyte-derived collagen-inhibiting factor(s) (KD-CIF) that has shown to control the synthesis of type I collagen, was not known. Upon conducting a series of systematic protein purification methods followed by mass spectroscopy, two proteins: secreted protein acidic rich in cystein (SPARC) and SFN were identified in keratinocyte-conditioned media. Using co-immunoprecipitation and 3D modeling, we determined that SFN and SPARC form a complex thereby controlling the type I collagen synthesis and expression in fibroblasts. The levels of these proteins in fibrotic tissues (animal and human) were also evaluated and a differential expression of these proteins between normal and fibrotic tissue confirmed their potential role in development of fibrotic condition. In conclusion, this study describes for the first time an interaction between SPARC and SFN that may have implications for the regulation of matrix deposition and prevention of dermal fibrotic conditions such as hypertrophic scars and keloid.

MAP kinase mediates silica-induced fibrotic nodule formation and collagen accumulation in fibroblasts.

It is well known that silica generates fibrosis around them in animals and human. However, the pathogenesis and mechanism of silica-induced fibrosis are still poorly understood. Here, we established a new strategy through which the effects of silica on fibrotic nodule formation, key extracellular matrix accumulation, and the mechanism involved were explored. To achieve this, human dermal fibroblasts were directly exposed to silica gel for various durations. Fibrotic nodule formation was evaluated by their microscopic appearance, type-1 procollagen, and fibronection expression in cell lysate and MMP-1 and-3 in conditioned media were analyzed by Western blotting. The results show an easily formation of nodule-like structures around silica gel in an in vitro-cultured system. The findings further revealed that silica gel stimulates collagen and fibronectin expression, while down-regulates matrix metalloproteinase-1 and -3 (MMP-1 and MMP-3) released in conditioned medium. To explore the mechanism involved, P38 and ERK1/2 Mitogen-Activated Protein Kinase (MAPK) signaling pathways were evaluated. Result showed that silica inhibits P38 and extracellular signal-regulated kinases (ERK1/2) MAP kinase phosphorylation. The addition of ERK1/2 inhibitor increases silica-stimulated type-1 collagen expression, reduces MMP-1 release and further enhances silica-induced nodule formation in dermal fibroblasts. These findings indicate that the inhibition of ERK1/2 MAPK signaling pathway may contribute to silica-caused fibrosis. In summary, our findings suggest that silica can directly cause fibrotic phenotype when fibroblasts contact with silica particles independent of any inflammation and other factors may exist in an in vivo condition.

Local expression of indoleamine 2,3-dioxygenase suppresses T-cell-mediated rejection of an engineered bilayer skin substitute.

Engineered skin substitutes (ESSs) comprising both keratinocytes and fibroblasts can afford many advantages over the use of autologous keratinocyte grafts for the treatment of full-thickness and partial-thickness burns. In this study, we investigated the efficacy of a novel ESS containing both genetically altered fibroblasts that express the immunosuppressive factor indoleamine 2,3-dioxygenase (IDO) and primary keratinocytes from a nonautologous source to confer immune protection of xenogeneic cells cultured in a bilayer ESS. The results show that engraftment of IDO expressing skin substitutes on the back of rats significantly improves healing progression over 7 days compared with both nontreated and non-IDO-expressing skin substitutes (p<0.001). Immuno-staining of CD3 and CD31 suggests that IDO-expressing skin substitutes significantly suppress T cell infiltration (p<0.001) and improve neovascularization by four-fold (12.6±1.2 vs. 3.0±1.0 vessel-like structure/high power field), respectively. In conclusion, we found that IDO expression can improve the efficacy of nonautologous ESS for the purpose of wound healing by mitigating T-cell infiltration as well as promoting vascularization of the graft.

Local expression of indoleamine 2,3 dioxygenase in syngeneic fibroblasts significantly prolongs survival of an engineered three-dimensional islet allograft.

OBJECTIVE: The requirement of systemic immunosuppression after islet transplantation is of significant concern and a major drawback to clinical islet transplantation. Here, we introduce a novel composite three-dimensional islet graft equipped with a local immunosuppressive system that prevents islet allograft rejection without systemic antirejection agents. In this composite graft, expression of indoleamine 2,3 dioxygenase (IDO), a tryptophan-degrading enzyme, in syngeneic fibroblasts provides a low-tryptophan microenvironment within which T-cells cannot proliferate and infiltrate islets. RESEARCH DESIGN AND METHODS: Composite three-dimensional islet grafts were engineered by embedding allogeneic mouse islets and adenoviral-transduced IDO-expressing syngeneic fibroblasts within collagen gel matrix. These grafts were then transplanted into renal subcapsular space of streptozotocin diabetic immunocompetent mice. The viability, function, and criteria for graft take were then determined in the graft recipient mice. RESULTS: IDO-expressing grafts survived significantly longer than controls (41.2 +/- 1.64 vs. 12.9 +/- 0.73 days; P < 0.001) without administration of systemic immunesuppressive agents. Local expression of IDO suppressed effector T-cells at the graft site, induced a Th2 immune response shift, generated an anti-inflammatory cytokine profile, delayed alloantibody production, and increased number of regulatory T-cells in draining lymph nodes, which resulted in antigen-specific impairment of T-cell priming. CONCLUSIONS: Local IDO expression prevents cellular and humoral alloimmune responses against islets and significantly prolongs islet allograft survival without systemic antirejection treatments. This promising finding proves the potent local immunosuppressive activity of IDO in islet allografts and sets the stage for development of a long-lasting nonrejectable islet allograft using stable IDO induction in bystander fibroblasts.