Controlled dual release of dihydrotestosterone and flutamide from polycaprolactone electrospun scaffolds accelerate burn wound healing.

Wound healing is a complex process involving multiple independent and overlapping sequential physiological mechanisms. In addition to cutaneous injury, a severe burn stimulates physiological derangements that induce a systemic hypermetabolic response resulting in impaired wound healing. Topical application of the anti-androgen drug, flutamide accelerates cutaneous wound healing, whereas paradoxically systemic dihydrotestosterone (DHT) improves burn wound healing. We developed and characterized a PCL scaffold that is capable of controlled release of androgen (DHT) and anti-androgen (F) individually or together. This study aims to investigate whether local modification of androgen actions has an impact on burn injury wound healing. In a full-thickness burn wound healing, mouse model, DHT/F-scaffold showed a significantly faster wound healing compared with F-scaffold or DHT-scaffold. Histology analysis confirmed that DHT/F-scaffold exhibited higher re-epithelization, cell proliferation, angiogenesis, and collagen deposition. Dual release of DHT and F from PCL scaffolds promoted cell proliferation of human keratinocytes and alters the keratinocyte cell cycle. Lastly, no adverse effects on androgen-dependent organs, spleen and liver were observed. In conclusion, we demonstrated DHT plus F load PCL scaffolds accelerated burn wound healing when loading alone did not. These findings point to a complex role of androgens in burn wound healing and open novel therapeutic avenues for treating severe burn patients.

Geometric framework reveals that a moderate protein, high carbohydrate intake is optimal for severe burn injury in mice.

Nutritional therapy is a cornerstone of burns management. The optimal macronutrient intake for wound healing after burn injury has not been identified, although high-energy, high-protein diets are favoured. The present study aimed to identify the optimal macronutrient intake for burn wound healing. The geometric framework (GF) was used to analyse wound healing after a 10 % total body surface area contact burn in mice ad libitum fed one of the eleven high-energy diets, varying in macronutrient composition with protein (P5-60 %), carbohydrate (C20-75 %) and fat (F20-75 %). In the GF study, the optimal ratio for wound healing was identified as a moderate-protein, high-carbohydrate diet with a protein:carbohydrate:fat (P:C:F) ratio of 1:4:2. High carbohydrate intake was associated with lower mortality, improved body weight and a beneficial pattern of body fat reserves. Protein intake was essential to prevent weight loss and mortality, but a protein intake target of about 7 kJ/d (about 15 % of energy intake) was identified, above which no further benefit was gained. High protein intake was associated with delayed wound healing and increased liver and spleen weight. As the GF study demonstrated that an initial very high protein intake prevented mortality, a very high-protein, moderate-carbohydrate diet (P40:C42:F18) was specifically designed. The dynamic diet study was also designed to combine and validate the benefits of an initial very high protein intake for mortality, and subsequent moderate protein, high carbohydrate intake for optimal wound healing. The dynamic feeding experiment showed switching from an initial very high-protein diet to the optimal moderate-protein, high-carbohydrate diet accelerated wound healing whilst preventing mortality and liver enlargement.

Dihydrotestosterone (DHT) Enhances Wound Healing of Major Burn Injury by Accelerating Resolution of Inflammation in Mice.

Androgens have been known to inhibit cutaneous wound healing in men and male mice. However, in children with major burn injuries, a synthetic androgen was reported clinically to improve wound healing. The aim of this study is to investigate the role of dihydrotestosterone (DHT) as a new therapeutic approach in treating major burn injury. In the present study, mice received systemic androgen treatment post major burn injury. Wound healing rate and body weight were monitored over 21 days. The serum level of inflammatory cytokines/chemokines were measured using multiplex immunoassays. In addition, splenocyte enumeration was performed by flow cytometry. Healing phases of inflammation, re-epithelialization, cell proliferation and collagen deposition were also examined. In results, DHT treated mice lost less weight and displayed accelerated wound healing but has no impact on hypermetabolism. Mice, after burn injury, displayed acute systemic inflammatory responses over 21 days. DHT treatment shortened the systemic inflammatory response with reduced splenic weight and monocyte numbers on day 14 and 21. DHT treatment also reduced wound infiltrating macrophage numbers. In conclusion, DHT treatment facilitates local wound healing by accelerating the resolution of inflammation, but not through alterations of post-burn hypermetabolic response.

Skin 11ß-hydroxysteroid dehydrogenase type 1 enzyme expression regulates burn wound healing and can be targeted to modify scar characteristics.

Background: Excessive scarring and fibrosis are the most severe and common complications of burn injury. Prolonged exposure to high levels of glucocorticoids detrimentally impacts on skin, leading to skin thinning and impaired wound healing. Skin can generate active glucocorticoids locally through expression and activity of the 11ß-hydroxysteroid dehydrogenase type 1 enzyme (11ß-HSD1). We hypothesised that burn injury would induce 11ß-HSD1 expression and local glucocorticoid metabolism, which would have important impacts on wound healing, fibrosis and scarring. We additionally proposed that pharmacological manipulation of this system could improve aspects of post-burn scarring. Methods: Skin 11ß-HSD1 expression in burns patients and mice was examined. The impacts of 11ß-HSD1 mediating glucocorticoid metabolism on burn wound healing, scar formation and scar elasticity and quality were additionally examined using a murine 11ß-HSD1 genetic knockout model. Slow-release scaffolds containing therapeutic agents, including active and inactive glucocorticoids, were developed and pre-clinically tested in mice with burn injury. Results: We demonstrate that 11ß-HSD1 expression levels increased substantially in both human and mouse skin after burn injury. 11ß-HSD1 knockout mice experienced faster wound healing than wild type mice but the healed wounds manifested significantly more collagen deposition, tensile strength and stiffness, features characteristic of excessive scarring. Application of slow-release prednisone, an inactive glucocorticoid, slowed the initial rate of wound closure but significantly reduced post-burn scarring via reductions in inflammation, myofibroblast generation, collagen production and scar stiffness. Conclusions: Skin 11ß-HSD1 expression is a key regulator of wound healing and scarring after burn injury. Application of an inactive glucocorticoid capable of activation by local 11ß-HSD1 in skin slows the initial rate of wound closure but significantlyimproves scar characteristics post burn injury.

The contradictory role of androgens in cutaneous and major burn wound healing.

Wound healing is a complex process involving four overlapping phases: haemostasis, inflammation, cell recruitment and matrix remodeling. In mouse models, surgical, pharmacological and genetic approaches targeting androgen actions in skin have shown that androgens increase interleukin-6 and tumor necrosis factor-α production and reduce wound re-epithelization and matrix deposition, retarding cutaneous wound healing. Similarly, clinical studies have shown that cutaneous wound healing is slower in men compared to women. However, in major burn injury, which triggers not only local wound-healing processes but also systemic hypermetabolism, the role of androgens is poorly understood. Recent studies have claimed that a synthetic androgen, oxandrolone, increases protein synthesis, improves lean body mass and shortens length of hospital stay. However, the possible mechanisms by which oxandrolone regulates major burn injury have not been reported. In this review, we summarize the current findings on the roles of androgens in cutaneous and major burn wound healing, as well as androgens as a potential therapeutic treatment option for patients with major burn injuries.

Low-protein diet accelerates wound healing in mice post-acute injury.

BACKGROUND: Wound healing processes are influenced by macronutrient intake (protein, carbohydrate and fat). The most favourable diet for cutaneous wound healing is not known, although high-protein diets are currently favoured clinically. This experimental study investigates the optimal macronutrient balance for cutaneous wound healing using a mouse model and the Geometric Framework, a nutrient modelling method, capable of analyzing the individual and interactive effects of a wide spectrum of macronutrient intake. METHODS: Two adjacent and identical full-thickness skin excisions (1 cm2) were surgically created on the dorsal area of male C57BL/6 mice. Mice were then allocated to one of 12 high-energy diets that varied in protein, carbohydrate and fat content. In select diets, wound healing processes, cytokine expression, energy expenditure, body composition, muscle and fat reserves were assessed. RESULTS: Using the Geometric Framework, we show that a low-protein intake, coupled with a balanced intake of carbohydrate and fat is optimal for wound healing. Mice fed a low-protein diet progressed quickly through wound healing stages with favourable wound inflammatory cytokine expression and significantly accelerated collagen production. These local processes were associated with an increased early systemic inflammatory response and a higher overall energy expenditure, related to metabolic changes occurring in key macronutrient reserves in lean body mass and fat depots. CONCLUSIONS: The results suggest that a low-protein diet may have a greater potential to accelerate wound healing than the current clinically used high-protein diets.

Therapeutic "Tool" in Reconstruction and Regeneration of Tissue Engineering for Osteochondral Repair.

Repairing osteochondral defects to restore joint function is a major challenge in regenerative medicine. However, with recent advances in tissue engineering, the development of potential treatments is promising. In recent years, in addition to single-layer scaffolds, double-layer or multilayer scaffolds have been prepared to mimic the structure of articular cartilage and subchondral bone for osteochondral repair. Although there are a range of different cells such as umbilical cord stem cells, bone marrow mesenchyml stem cell, and others that can be used, the availability, ease of preparation, and the osteogenic and chondrogenic capacity of these cells are important factors that will influence its selection for tissue engineering. Furthermore, appropriate cell proliferation and differentiation of these cells is also key for the optimal repair of osteochondral defects. The development of bioreactors has enhanced methods to stimulate the proliferation and differentiation of cells. In this review, we summarize the recent advances in tissue engineering, including the development of layered scaffolds, cells, and bioreactors that have changed the approach towards the development of novel treatments for osteochondral repair.

The effects of cross-linking a collagen-elastin dermal template on scaffold bio-stability and degradation.

MatriDerm is a collagen-elastin dermal template that promotes regeneration in full-thickness wound repair. Due to its noncross-linked status, MatriDerm biodegrades quickly in a wound. Facilitating vascularization and dermal repair, it is desirable for MatriDerm to remain present until the wound healing process is complete, optimizing tissue regeneration and reducing wound contraction. The aim of this study was to investigate the effect of cross-linking MatriDerm on its mechanical and biological properties and to enhance its regenerative functionality. MatriDerm was chemically cross-linked and characterized in comparison with noncross-linked MatriDerm. Scaffold properties including surface morphology, protein release and mechanical strength were assessed. Cell-scaffold interaction, cell proliferation and migration were examined using human dermal fibroblasts. Scaffold biodegradation and its impact on wound healing and contraction were studied in a mouse model. Results showed that cross-linked MatriDerm displayed a small reduction in pore size, significantly less protein loss and a threefold increase in tensile strength. A significant increase in fibroblast proliferation and migration was observed in cross-linked MatriDerm with reduced scaffold contraction in vitro. In the mouse model, noncross-linked MatriDerm was almost completely biodegraded after 14 days whereas cross-linked MatriDerm remained intact. No significant difference in wound contraction was found between scaffolds. In conclusion, cross-linked MatriDerm showed a significant increase in stability and strength, enhancing its durability and cell-scaffold interaction. in vivo analysis showed cross-linked MatriDerm had a reduced biodegradation rate with a similar host response. The extended structural integrity of cross-linked MatriDerm could potentially facilitate improved skin tissue regeneration, promoting the formation of a more pliable scar.

Advances in the Research of Bioinks Based on Natural Collagen, Polysaccharide and Their Derivatives for Skin 3D Bioprinting.

The skin plays an important role in protecting the human body, and wound healing must be set in motion immediately following injury or trauma to restore the normal structure and function of skin. The extracellular matrix component of the skin mainly consists of collagen, glycosaminoglycan (GAG), elastin and hyaluronic acid (HA). Recently, natural collagen, polysaccharide and their derivatives such as collagen, gelatin, alginate, chitosan and pectin have been selected as the matrix materials of bioink to construct a functional artificial skin due to their biocompatible and biodegradable properties by 3D bioprinting, which is a revolutionary technology with the potential to transform both research and medical therapeutics. In this review, we outline the current skin bioprinting technologies and the bioink components for skin bioprinting. We also summarize the bioink products practiced in research recently and current challenges to guide future research to develop in a promising direction. While there are challenges regarding currently available skin bioprinting, addressing these issues will facilitate the rapid advancement of 3D skin bioprinting and its ability to mimic the native anatomy and physiology of skin and surrounding tissues in the future.

Mouse models in burns research: Characterisation of the hypermetabolic response to burn injury.

OBJECTIVE: The aim of the study is to characterise burn induced hypermetabolism in a mouse model. SUMMARY BACKGROUND DATA: There are many mouse models of burn injury currently available however, their use in burns research is limited by the general assumption that post-burn hypermetabolism is difficult to study in these models. METHODS: Male Balb/c mice were subjected to either a small (1 cm2) or large (4 cm2) contact burn. The hypermetabolic response to burn injury was determined by measuring changes in basal energy expenditure. The hormonal and inflammatory mediators of hypermetabolism, and the catabolic alterations secondary to hypermetabolism were also examined. RESULTS: Post-burn hypermetabolism was induced in both models of small and large burn. However, large burns resulted in prolonged wound healing, a more pronounced and sustained increase in basal energy expenditure, and a greater stress and systemic inflammatory response with profound catabolic consequences. CONCLUSIONS: In the present study, we have successfully characterised the burn induced systemic hypermetabolic response in a mouse model of small and large burn. These models may prove useful for researchers studying the complex aetiology of hypermetabolism and interventions.

Skin wound repair: Results of a pre-clinical study to evaluate electropsun collagen-elastin-PCL scaffolds as dermal substitutes.

The gold standard treatment for severe burn injuries is autologous skin grafting and the use of commercial dermal substitutes. However, resulting skin tissue following treatment usually displays abnormal morphology and functionality including scarring, skin contracture due to the poor elasticity and strength of existing dermal substitutes. In this study, we have developed a triple-polymer scaffold made of collagen-elastin-polycaprolactone (CEP) composite, aiming to enhance the mechanical properties of the scaffold while retaining its biological properties in promoting cell attachment, proliferation and tissue regeneration. The inclusion of elastin was revealed to decrease the stiffness of the scaffold, while also decreasing hysteresis and increasing elasticity. In mice, electrospun collagen-elastin-PCL scaffolds promoted keratinocyte and fibroblast proliferation, tissue integration and accelerated early-stage angiogenesis. Only a mild inflammatory response was observed in the first 2 weeks post-subcutaneous implantation. Our data indicates that the electrospun collagen-elastin-PCL scaffolds could potentially serve as a skin substitute to promote skin cell growth and tissue regeneration after severe burn injury.

The Application of Multi-Walled Carbon Nanotubes in Bone Tissue Repair Hybrid Scaffolds and the Effect on Cell Growth In Vitro.

In this study, composite scaffolds with different multi-walled carbon nanotubes (MWCNTs) content were prepared by freeze-drying. These scaffolds were characterized by scanning electron microscope (SEM), energy dispersive spectroscopy (EDS), Fourier transform infrared spectroscopy (FTIR), porosity, hydrophilicity, mechanical strength, and degradation. The MWCNTs scaffolds were structurally sound and had porous structures that offered ample space for adherence, proliferation, and differentiation of MC3T3-E1 cells, and also supported the transport of nutrients and metabolic waste. CS/Gel/nHAp/0.3%MWCNTs scaffolds provided the best outcomes in terms of scaffold porosity, hydrophilicity, and degradation rate. However, CS/Gel/nHAp/0.6%MWCNTs scaffolds were found to support the optimal growth, homogenous distribution, and biological activity of MC3T3-E1 cells. The excellent properties of CS/Gel/nHAp/0.6%MWCNTs scaffolds for the adhesion, proliferation, and osteogenesis differentiation of MC3T3-E1 cells in vitro highlights the potential applications of this scaffold in bone tissue regeneration.

In Vitro Fabrication and Biocompatibility Assay of a Biomimetic Osteoblastic Niche.

A novel HAp-CS/Gel biomimetic osteoblastic niche was fabricated by freeze-drying, and its mechanical strength and biocompatibility were characterized. HAp-CS/Gel scaffolds in various ratios of 100:5, 100:10, and 100:20 (CS/Gel to HAp) were prepared by freeze-drying prior to chemical cross-linking followed by infrared spectrum analysis, EDS, FITR, SEM, fluorescence microscopy, MTT, and ALP experiments. Results from the infrared spectrum analysis showed that HAp doping remained the surface morphology and the architecture of scaffold with interconnected pores in the size range of 135 to 150 µm. The HAp doping ratio of 100:20 was found to be optimal based on its high porosity of 90%, better water uptake folds of 19.1. In addition, EDS and FITR analyses demonstrated that HAps were uniformly distributed on the surface of a scaffold with aggregates and particles, which has sufficient roughness for cell attachment and proliferation of osteoblasts. Under SEM and fluorescent microscopy, osteoblasts seeded onto the scaffold showed evenly distributed viable cells, which is believed to form a biomimetic niche. In the present study, we further demonstrate that osteoblasts can maintain their function and grow well on the scaffold through MTT and ALP tests. Thus, the scaffold has favorable physical properties and biocompatibility to support the proliferation and differentiation of osteoblasts and further to support the constructs of biomimetic osteoblastic niche.

A biomimetic cartilage gradient hybrid scaffold for functional tissue engineering of cartilage.

Osteochondral tissue has a complex layered structure that is not self-repairing after a cartilage defect. Therefore, constructing a biomimetic gradient scaffold that meets the specific structural requirements of osteochondral tissue is a major challenge in the field of cartilage tissue engineering. In this study, chitosan/Sodium ß-glycerophosphate/Gelatin (Cs/GP/Gel) biomimetic gradient scaffolds were prepared by regulating the mass ratio of single layer raw materials. The same ratio of Cs/GP/Gel hybrid scaffold material was used as the control. Physical properties such as water absorption, porosity and the degradation rate of the material were compared to optimize the proportion of scaffold materials. P3 Bone Mesenchymal Stem Cells (BMSCs) were inoculated on the gradient and the control scaffolds to investigate its biocompatibility. Scanning electron microscopy (SEM) results show that 3:1:2, 6:1:3.5, 9:1:5, 12:1:6.5, 15:1:8 Cs/GP/Gel gradient scaffolds had excellent three-dimensional porous structures. Channels were also shown to have been interconnected, and the walls of the pores were folded. In the longitudinal dimension, gradient scaffolds had an obvious stratified structure and pore gradient gradualism, that effectively simulated the natural physiological stratified structure of real cartilage. The diameter of the pores in the control scaffold was uniform and without any pore gradient. Gradient scaffolds had good water absorption (584.24 ± 3.79˜677.47 ± 1.70%), porosity (86.34 ± 5.10˜95.20 ± 2.86%) and degradation (86.09 ± 2.46˜92.48 ± 3.86%). After considering the physical properties assessed, the Cs/GP/Gel gradient scaffold with a ratio of 9:1:5 was found to be the most suitable material to support osteochondral tissue. BMSCs were subsequently inoculated on the proportional gradient and hybrid scaffolds culture. These cells survived, distributed and extended well on the gradient and hybrid scaffold material. The biomimetic gradient scaffold designed and prepared in this study provides an important foundation for the development of new gradient composite biomedical materials for osteochondral repair.

3D Bio-Printing of CS/Gel/HA/Gr Hybrid Osteochondral Scaffolds.

Cartilage is an important tissue contributing to the structure and function of support and protection in the human body. There are many challenges for tissue cartilage repair. However, 3D bio-printing of osteochondral scaffolds provides a promising solution. This study involved preparing bio-inks with different proportions of chitosan (Cs), Gelatin (Gel), and Hyaluronic acid (HA). The rheological properties of each bio-ink was used to identify the optimal bio-ink for printing. To improve the mechanical properties of the bio-scaffold, Graphene (GR) with a mass ratio of 0.024, 0.06, and 0.1% was doped in the bio-ink. Bio-scaffolds were prepared using 3D printing technology. The mechanical strength, water absorption rate, porosity, and degradation rate of the bio-scaffolds were compared to select the most suitable scaffold to support the proliferation and differentiation of cells. P3 Bone mesenchymal stem cells (BMSCs) were inoculated onto the bio-scaffolds to study the biocompatibility of the scaffolds. The results of SEM showed that the Cs/Gel/HA scaffolds with a GR content of 0, 0.024, 0.06, and 0.1% had a good three-dimensional porous structure and interpenetrating pores, and a porosity of more than 80%. GR was evenly distributed on the scaffold as observed by energy spectrum analyzer and polarizing microscope. With increasing GR content, the mechanical strength of the scaffold was enhanced, and pore walls became thicker and smoother. BMSCs were inoculated on the different scaffolds. The cells distributed and extended well on Cs/Gel/HA/GR scaffolds. Compared to traditional methods in tissue-engineering, this technique displays important advantages in simulating natural cartilage with the ability to finely control the mechanical and chemical properties of the scaffold to support cell distribution and proliferation for tissue repair.

Detection and Evaluation of Anti-Cancer Efficiency of Astragalus Polysaccharide Via a Tissue Engineered Tumor Model.

Polysaccharides have been known to display their anti-cancer activity via immunomodulation. The immunomodulation of RAW 246.7 macrophages by astragalus polysaccharide (APS) is herein studied in human breast cancer cells. Apart from traditional 2D culture, a novel tissue-engineered tumor model is prepared based on decellularized porcine lung scaffold. Decellularized lung scaffolds exhibit preferable biocompatibility that promote the formation and enlargement of tumor spheroids. The conditioned medium (CM, the supernatant liquid of APS-treated RAW264.7 macrophages) shows anti-cancer activity by inhibiting cell proliferation, demonstrated by a 39.25 ± 5.04% decrease in tumoroid size and 20.96 ± 2.43% reduction in cell viability, and promoting cell apoptosis by the nuclear fragmentation and increasing apoptotic-stage proportion. The cytotoxicity of CM is associated with the upregulated production of nitric oxide and tumor necrosis factor-α from RAW 246.7 cells stimulated by APS. Overall, by using this 3D tumor model to study CM, there is convincing evidence that APS can modulate macrophage function to further mediate anti-cancer activity.

Burn injury: Challenges and advances in burn wound healing, infection, pain and scarring.

Severe burn injuries are the most traumatic and physically debilitating injuries affecting nearly every organ system and leading to significant morbidity and mortality. Early burn wound excision and skin grafting are common clinical practices that have significantly improved the outcomes for severe burn injured patients by reducing mortality rate and days of hospital stay. However, slow wound healing, infection, pain, and hypertrophic scarring continue to remain a major challenge in burn research and management. In the present article, we review and discuss issues in the current treatment of burn injuries; the advances and novel strategies developed in the past decade that have improved burn management; and also, pioneer ideas and studies in burn research which aims to enhance burn wound care with a focus on burn wound infection, pain management, treatments for scarring and skin tissue engineering.

Development of a Novel Pre-Vascularized Three-Dimensional Skin Substitute Using Blood Plasma Gel.

Skin substitutes with existing vascularization are in great demand for the repair of full-thickness skin defects. In the present study, we hypothesized that a pre-vascularized skin substitute can potentially promote wound healing. Novel three-dimensional (3D) skin substitutes were prepared by seeding a mixture of human endothelial progenitor cells (EPCs) and fibroblasts into a human plasma/calcium chloride formed gel scaffold, and seeding keratinocytes onto the surface of the plasma gel. The capacity of the EPCs to differentiate into a vascular-like tubular structure was evaluated using immunohistochemistry analysis and WST-8 assay. Experimental studies in mouse full-thickness skin wound models showed that the pre-vascularized gel scaffold significantly accelerated wound healing 7 days after surgery, and resembled normal skin structures after 14 days post-surgery. Histological analysis revealed that pre-vascularized gel scaffolds were well integrated in the host skin, resulting in the vascularization of both the epidermis and dermis in the wound area. Moreover, mechanical strength analysis demonstrated that the healed wound following the implantation of the pre-vascularized gel scaffolds exhibited good tensile strength. Taken together, this novel pre-vascularized human plasma gel scaffold has great potential in skin tissue engineering.