Development of a Static Avascular and Dynamic Vascular Human Skin Equivalent Employing Collagen/Keratin Hydrogels.

One of the primary complications in generating physiologically representative skin tissue is the inability to integrate vasculature into the system, which has been shown to promote the proliferation of basal keratinocytes and consequent keratinocyte differentiation, and is necessary for mimicking representative barrier function in the skin and physiological transport properties. We created a 3D vascularized human skin equivalent (VHSE) with a dermal and epidermal layer, and compared keratinocyte differentiation (immunomarker staining), epidermal thickness (H&E staining), and barrier function (transepithelial electrical resistance (TEER) and dextran permeability) to a static, organotypic avascular HSE (AHSE). The VHSE had a significantly thicker epidermal layer and increased resistance, both an indication of increased barrier function, compared to the AHSE. The inclusion of keratin in our collagen hydrogel extracellular matrix (ECM) increased keratinocyte differentiation and barrier function, indicated by greater resistance and decreased permeability. Surprisingly, however, endothelial cells grown in a collagen/keratin extracellular environment showed increased cell growth and decreased vascular permeability, indicating a more confluent and tighter vessel compared to those grown in a pure collagen environment. The development of a novel VHSE, which incorporated physiological vasculature and a unique collagen/keratin ECM, improved barrier function, vessel development, and skin structure compared to a static AHSE model.

Comparison of Intact Fish Skin Graft and Allograft as Temporary Coverage for Full-Thickness Burns: A Non-Inferiority Study.

The extent and depth of burn injury may mandate temporary use of cadaver skin (allograft) to protect the wound and allow the formation of granulation tissue while split-thickness skin grafts (STSGs) are serially harvested from the same donor areas. However, allografts are not always available and have a high cost, hence the interest in identifying more economical, readily available products that serve the same function. This study evaluated intact fish skin graft (IFSG) as a temporary cover to prepare the wound bed for STSG application. Thirty-six full-thickness (FT) 5 × 5 cm burn wounds were created on the dorsum of six anesthetized Yorkshire pigs on day -1. To mimic the two-stage clinical situation, on day 0, wounds were excised down to a bleeding wound bed and a temporary cover (either IFSG or cadaver porcine skin) was applied; then, on day 7, wounds were debrided to a viable wound bed prior to the application of autologous 1.5:1 meshed STSG (mSTSG). Rechecks were performed on days 14, 21, 28, 45, and 60 with digital images, non-invasive measurements, and punch biopsies. The IFSG created a granulated wound bed receptive to the application of an mSTSG. FT burn wounds treated with an IFSG had similar outcome measures, including contraction rates, trans-epidermal water loss (TEWL) measurements, hydration, and blood perfusion levels, compared to cadaver skin-treated burn wounds. Pathology scoring indicated significant differences between the allograft- and IFSG-treated wounds on day 7, with the IFSG having increased angiogenesis, granulation tissue formation, and immune cells. Pathology scoring indicated no significant differences once mSTSGs were applied to wounds. The IFSG performed as well as cadaver skin as a temporary cover and was not inferior to the standard of care, suggesting the potential to transition IFSGs into clinical use for burns.

Biological function of Extracellular Vesicles (EVs): a review of the field.

Extracellular vesicles (EVs) theranostic potential is under intense investigation. There is a wealth of information highlighting the role that EVs and the secretome play in disease and how these are being utilized for clinical trials and novel therapeutic possibilities. However, understanding of the physiological and pathological roles of EVs remain incomplete. The challenge lies in reaching a consensus concerning standardized quality-controlled isolation, storage, and sample preparation parameters. Interest in circulating EV cargo as diagnostic and prognostic biomarkers is steadily growing. Though promising, various limitations need to be addressed before there can be successful, full-scale therapeutic use of approved EVs. These limitations include obtaining or manufacturing from the appropriate medium (e.g., from bodily fluid or cell culture), loading and isolating EVs, stability, and storage, standardization of processing, and determining potency. This review highlights specific topics, including circulation of abnormal EVs contribute to human disease and the theranostic potential of EVs. Theranostics is defined as a combination of the word's therapeutics and diagnostics and describes how a specific medicine or technique can function as both. Key findings include, (1) EVs and the secretome are future theranostics which will be utilized as both biomarkers for diagnosis and as therapeutics, (2) basic and translational research supports clinical trials utilizing EVs/secretome, and (3) additional investigation is required to fully unmask the theranostic potential of EVs/secretome in specific diseases and injuries.

Model to Inhibit Contraction in Third-Degree Burns Employing Split-Thickness Skin Graft and Administered Bone Marrow-Derived Stem Cells.

Third-degree burns typically result in pronounced scarring and contraction in superficial and deep tissues. Established techniques such as debridement and grafting provide benefit in the acute phase of burn therapy, nevertheless, scar and contraction remain a challenge in deep burns management. Our ambition is to evaluate the effectiveness of novel cell-based therapies, which can be implemented into the standard of care debridement and grafting procedures. Twenty-seven third-degree burn wounds were created on the dorsal area of Red Duroc pig. After 72 h, burns are surgically debrided using a Weck knife. Split-thickness skin grafts (STSGs) were then taken after debridement and placed on burn scars combined with bone marrow stem cells (BM-MSCs). Biopsy samples were taken on days 17, 21, and 45 posttreatment for evaluation. Histological analysis revealed that untreated control scars at 17 days are more raised than burns treated with STSGs alone and/or STSGs with BM-MSCs. Wounds treated with skin grafts plus BM-MSCs appeared thinner and longer, indicative of reduced contraction. qPCR revealed some elevation of α-SMA expression at day 21 and Collagen Iα2 in cells derived from wounds treated with skin grafts alone compared to wounds treated with STSGs + BM-MSCs. We observed a reduction level of TGFß-1 expression at days 17, 21, and 45 in cells derived from wounds treated compared to controls. These results, where the combined use of stem cells and skin grafts stimulate healing and reduce contraction following third-degree burn injury, have a potential as a novel therapy in the clinic.

Advances in Immunomodulation and Immune Engineering Approaches to Improve Healing of Extremity Wounds.

Delayed healing of traumatic wounds often stems from a dysregulated immune response initiated or exacerbated by existing comorbidities, multiple tissue injury or wound contamination. Over decades, approaches towards alleviating wound inflammation have been centered on interventions capable of a collective dampening of various inflammatory factors and/or cells. However, a progressive understanding of immune physiology has rendered deeper knowledge on the dynamic interplay of secreted factors and effector cells following an acute injury. There is a wide body of literature, both in vitro and in vivo, abstracted on the immunomodulatory approaches to control inflammation. Recently, targeted modulation of the immune response via biotechnological approaches and biomaterials has gained attention as a means to restore the pro-healing phenotype and promote tissue regeneration. In order to fully realize the potential of these approaches in traumatic wounds, a critical and nuanced understanding of the relationships between immune dysregulation and healing outcomes is needed. This review provides an insight on paradigm shift towards interventional approaches to control exacerbated immune response following a traumatic injury from an agonistic to a targeted path. We address such a need by (1) providing a targeted discussion of the wound healing processes to assist in the identification of novel therapeutic targets and (2) highlighting emerging technologies and interventions that utilize an immunoengineering-based approach. In addition, we have underscored the importance of immune engineering as an emerging tool to provide precision medicine as an option to modulate acute immune response following a traumatic injury. Finally, an overview is provided on how an intervention can follow through a successful clinical application and regulatory pathway following laboratory and animal model evaluation.

Characterization of a Human Platelet Lysate-Loaded Keratin Hydrogel for Wound Healing Applications In Vitro.

One of the promising approaches to facilitate healing and regenerative capacity includes the application of growth-factor-loaded biomaterials. Human platelet lysate (hPL) derived from platelet-rich plasma through a freeze-thaw process has been used as a growth factor rich therapeutic in many regenerative applications. To provide sustained local delivery of the hPL-derived growth factors such as epidermal growth factor (EGF), the hPL can be loaded into biomaterials that do not degrade rapidly in vivo. Keratin (KSO), a strong filamentous protein found in human hair, when formulated as a hydrogel, is shown to sustain the release of drugs and promote wound healing. In the current study, we created a KSO biomaterial that spontaneously forms a hydrogel when rehydrated with hPL that is capable of controlled and sustained release of pro-regenerative molecules. Our study demonstrates that the release of hPL is controlled by changing the KSO hydrogel and hPL-loading concentrations, with hPL loading concentrations having a greater effect in changing release profiles. In addition, the 15% KSO concentration proved to form a stable hydrogel, and supported cell proliferation over 3 days without cytotoxic effects in vitro. The hPL-loaded keratin hydrogels show promise in potential applications for wound healing with the sustained release of pro-regenerative growth factors with easy tailoring of hydrogel properties.

Impact of dermal matrix thickness on split-thickness skin graft survival and wound contraction in a single-stage procedure.

Optimal treatment of full-thickness skin injuries requires dermal and epidermal replacement. To spare donor dermis, dermal substitutes can be used ahead of split-thickness skin graft (STSG) application. However, this two-stage procedure requires an additional general anaesthetic, often prolongs hospitalisation, and increases outpatient services. Although a few case series have described successful single-stage reconstructions, with application of both STSG and dermal substitute at the index operation, we have little understanding of how the physical characteristics of dermal substitutes affects the success of a single-stage procedure. Here, we evaluated several dermal substitutes to optimise single-stage skin replacement in a preclinical porcine model. A porcine full-thickness excisional wound model was used to evaluate the following dermal substitutes: autologous dermal graft (ADG; thicknesses 0.15-0.60 mm), Integra (0.4-0.8 mm), Alloderm (0.9-1.6 mm), and chitosan-based hydrogel (0.1-0.2 mm). After excision, each wound was treated with either a dermal substitute followed by STSG or STSG alone (control). Endpoints included graft take at postoperative days (PODs) 7 and 14, wound closure at POD 28, and wound contracture from POD 28-120. Graft take was highest in the STSG alone and hydrogel groups at POD 14 (86.9% ± 19.5% and 81.3% ± 12.3%, respectively; P < .001). There were no differences in graft take at POD 7 or in wound closure at POD 28, though highest rates of wound closure were seen in the STSG alone and hydrogel groups (93.6% ± 9.1% and 99.8% ± 0.5%, respectively). ADG-treated wounds demonstrated the least amount of wound contracture at each time point. Increase dermal substitute thickness was associated with worse percent graft take at PODs 14 and 28 (Spearman ρ of -0.50 and -0.45, respectively; P < .001). In this preclinical single-stage skin reconstruction model, thinner ADG and hydrogel dermal substitutes outperformed thicker dermal substitutes. Both substitute thickness and composition affect treatment success. Further preclinical and clinical studies to optimise this treatment modality are warranted.

Assessing multimodal optical imaging of perfusion in burn wounds.

A critical need exists for early, accurate diagnosis of burn wound severity to help identify the course of treatment and outcome of the wound. Laser speckle imaging (LSI) is a promising blood perfusion imaging approach, but it does not account for changes in tissue optical properties that can occur with burn wounds, which are highly dynamic environments. Here, we studied optical property dynamics following burn injury and debridement and the associated impact on interpretation of LSI measurements of skin perfusion. We used spatial frequency domain imaging (SFDI) measurements of tissue optical properties to study the impact of burn-induced changes in these properties on LSI measurements. An established preclinical porcine model of burn injury was used (n = 8). SFDI and LSI data were collected from burn wounds of varying severity. SFDI measurements demonstrate that optical properties change in response to burn injury in a porcine model. We then apply theoretical modeling to demonstrate that the measured range of optical property changes can affect the interpretation of LSI measurements of blood flow, but this effect is minimal for most of the measured data. Collectively, our results indicate that, even with a dynamic burn wound environment, blood-flow measurements with LSI can serve as an appropriate strategy for accurate assessment of burn severity.

ASCs derived from burn patients are more prone to increased oxidative metabolism and reactive oxygen species upon passaging.

BACKGROUND: Patients with severe burn injury (over 20% of the total body surface area) experience profound hypermetabolism which significantly prolongs wound healing. Adipose-derived stem cells (ASCs) have been proposed as an attractive solution for treating burn wounds, including the potential for autologous ASC expansion. While subcutaneous adipocytes display an altered metabolic profile post-burn, it is not known if this is the case with the stem cells associated with the adipose tissue. METHODS: ASCs were isolated from discarded burn skin of severely injured human subjects (BH, n = 6) and unburned subcutaneous adipose tissue of patients undergoing elective abdominoplasty (UH, n = 6) and were analyzed at passages 2, 4, and 6. Flow cytometry was used to quantify ASC cell surface markers CD90, CD105, and CD73. Mitochondrial abundance and reactive oxygen species (ROS) production were determined with MitoTracker Green and MitoSOX Red, respectively, while JC-10 Mitochondrial Membrane Potential Assays were also performed. Mitochondrial respiration and glycolysis were analyzed with a high-resolution respirometer (Seahorse XFe24 Analyzer). RESULTS: There was no difference in age between BH and UH (34 ± 6 and 41 ± 4 years, respectively, P = 0.49). While passage 2 ASCs had lower ASC marker expression than subsequent passages, there were no significant differences in the expression between BH and UH ASCs. Similarly, no differences in mitochondrial abundance or membrane potential were found amongst passages or groups. Two-way ANOVA showed a significant effect (P < 0.01) of passaging on mitochondrial ROS production, with increased ROS in BH ASCs at later passages. Oxidative phosphorylation capacities (leak and maximal respiration) increased significantly in BH ASCs (P = 0.035) but not UH ASCs. On the contrary, basal glycolysis significantly decreased in BH ASCs (P = 0.011) with subsequent passaging, but not UH ASCs. CONCLUSIONS: In conclusion, ASCs from burned individuals become increasingly oxidative and less glycolytic upon passaging when compared to ASCs from unburned patients. This increase in oxidative capacities was associated with ROS production in later passages. While the autologous expansion of ASCs holds great promise for treating burned patients with limited donor sites, the potential negative consequences of using them require further investigation.

Accelerated Wound Closure of Deep Partial Thickness Burns with Acellular Fish Skin Graft.

Thermal injuries are caused by exposure to a variety of sources, and split thickness skin grafts are the gold standard treatment for severe burns; however, they may be impossible when there is no donor skin available. Large total body surface area burns leave patients with limited donor site availability and create a need for treatments capable of achieving early and complete coverage that can also retain normal skin function. In this preclinical trial, two cellular and tissue based products (CTPs) are evaluated on twenty-four 5 × 5 deep partial thickness (DPT) burn wounds. Using appropriate pain control methods, DPT burn wounds were created on six anesthetized Yorkshire pigs. Wounds were excised one day post-burn and the bleeding wound beds were subsequently treated with omega-3-rich acellular fish skin graft (FSG) or fetal bovine dermis (FBD). FSG was reapplied after 7 days and wounds healed via secondary intentions. Digital images, non-invasive measurements, and punch biopsies were acquired during rechecks performed on days 7, 14, 21, 28, 45, and 60. Multiple qualitative measurements were also employed, including re-epithelialization, contraction rates, hydration, laser speckle, and trans-epidermal water loss (TEWL). Each treatment produced granulated tissue (GT) that would be receptive to skin grafts, if desired; however, the FSG induced GT 7 days earlier. FSG treatment resulted in faster re-epithelialization and reduced wound size at day 14 compared to FBD (50.2% vs. 23.5% and 93.1% vs. 106.7%, p < 0.005, respectively). No differences in TEWL measurements were observed. The FSG integrated into the wound bed quicker as evidenced by lower hydration values at day 21 (309.7 vs. 2500.4 µS, p < 0.05) and higher blood flow at day 14 (4.9 vs. 3.1 fold change increase over normal skin, p < 0.005). Here we show that FSG integrated faster without increased contraction, resulting in quicker wound closure without skin graft application which suggests FSG improved burn wound healing over FBD.

Enzymatic Debridement of Porcine Burn Wounds via a Novel Protease, SN514.

Necrotic tissue generated by a thermal injury is typically removed via surgical debridement. However, this procedure is commonly associated with blood loss and the removal of viable healthy tissue. For some patients and contexts such as extended care on the battlefield, it would be preferable to remove devitalized tissue with a nonsurgical debridement agent. In this paper, a proprietary debridement gel (SN514) was evaluated for the ability to debride both deep-partial thickness (DPT) and full-thickness burn wounds using an established porcine thermal injury model. Burn wounds were treated daily for 4 days and visualized with both digital imaging and laser speckle imaging. Strip biopsies were taken at the end of the procedure. Histological analyses confirmed a greater debridement of the porcine burn wounds by SN514 than the vehicle-treated controls. Laser speckle imaging detected significant increases in the perfusion status after 4 days of SN514 treatment on DPT wounds. Importantly, histological analyses and clinical observations suggest that SN514 gel treatment did not damage uninjured tissue as no edema, erythema, or inflammation was observed on intact skin surrounding the treated wounds. A blinded evaluation of the digital images by a burn surgeon indicated that SN514 debrided more necrotic tissue than the control groups after 1, 2, and 3 days of treatment. Additionally, SN514 gel was evaluated using an in vitro burn model that used human discarded skin. Treatment of human burned tissue with SN514 gel resulted in greater than 80% weight reduction compared with untreated samples. Together, these data demonstrate that SN514 gel is capable of debriding necrotic tissue and suggest that SN514 gel could be a useful option for austere conditions, such as military multi-domain operations and prolonged field care scenarios.

PEGylated Platelet-Free Blood Plasma-Based Hydrogels for Full-Thickness Wound Regeneration.

Objective: To develop a cost-effective and clinically usable therapy to treat full-thickness skin injuries. We accomplished this by preparing a viscoelastic hydrogel using polyethylene glycol (PEG)-modified platelet-free plasma (PEGylated PFP) combined with human adipose-derived stem cells (ASCs). Approach: PEGylated PFP hydrogels were prepared by polymerizing the liquid mixture of PEG and PFP±ASCs and gelled either by adding calcium chloride (CaCl2) or thrombin. Rheological and in vitro studies were performed to assess viscoelasticity and the ability of hydrogels to direct ASCs toward a vasculogenic phenotype, respectively. Finally, a pilot study evaluated the efficacy of hydrogels±ASCs using an athymic rat full-thickness skin wound model. Results: Hydrogels prepared within the range of 11 to 27 mM for CaCl2 or 5 to 12.5 U/mL for thrombin exhibited a storage modulus of ∼62 to 87 Pa and ∼47 to 92 Pa, respectively. The PEGylated PFP hydrogels directed ASCs to form network-like structures resembling vasculature, with a fourfold increase in perivascular specific genes that were confirmed by immunofluorescent staining. Hydrogels combined with ASCs exhibited an increase in blood vessel density when applied to excisional rat wounds compared with those treated with hydrogels (110.3 vs. 95.6 BV/mm2; p < 0.05). Furthermore, ASCs were identified in the perivascular region associated with newly forming blood vessels. Innovation: This study demonstrates that PFP modified with PEG along with ASCs can be used to prepare cost-effective stable hydrogels, at the bed-side, to treat extensive skin wounds. Conclusion: These results indicate that PEGylated plasma-based hydrogels combined with ASCs may be a potential regenerative therapy for full-thickness skin wounds.

Delivery of silver sulfadiazine and adipose derived stem cells using fibrin hydrogel improves infected burn wound regeneration.

Infection control is necessary for improved burn wound regeneration. In this study contact burn wounds were induced on the dorsum of the rats and were infected with Pseudomonas aeruginosa (107cfu/ml of saline) and left overnight (12-14 hours) to establish the infection. After 12 hours, the wounds were treated with PEGylated fibrin hydrogel containing 50 mgs of silver sulfadiazine (SSD) loaded chitosan microsphere (SSD-CSM-FPEG). On day 9, SSD-CSM-FPEG treated burn wounds further received adipose derived stem cell (5×104 ASCs cells/ml) embedded in PEGylated fibrin hydrogel. Wounds were assessed for the healing outcomes such as neovascularization, granulation tissue formation, wound closure and collagen maturation. Analysis of bacterial load in the burn wound biopsies, demonstrated that SSD-CSM-FPEG significantly reduced bacterial infection, while overt infection was still observed in the untreated groups on day 14. Sequential treatment of infected wounds with SSD-CSM-FPEG followed by ASC-FPEGs (SSD-CSM-ASC-FPEG) significantly reduced bacterial colonization (9 log reduction) and pro-inflammatory cytokine (TNF-α) expression. A significant increase in neovascularization markers; NG2 and vWF was also observed. Histological analysis indicated the wounds treated with SSD-CSM-ASC-FPEG increased amount of dermal collagen matrix deposition, a thicker granulation tissue on day 21 and more mature collagen on day 28. This work demonstrates that the sequential treatment of infected burn wounds with SSD-CSM-FPEG followed by ASC-FPEG reduces bacterial infection as well as promotes neo-vascularization with improved matrix remodeling.

Platelet rich plasma hydrogels promote in vitro and in vivo angiogenic potential of adipose-derived stem cells.

Despite great advances in skin wound care utilizing grafting techniques, the resulting severe scarring, deformity and ineffective vascularization remains a challenge. Alternatively, tissue engineering of new skin using patient-derived stem cells and scaffolding materials promises to greatly increase the functional and aesthetic outcome of skin wound healing. This work focused on the optimization of a polyethylene glycol modified (PEGylated) platelet-rich plasma (PRP) hydrogel for the protracted release of cytokines, growth factors, and signaling molecules and also the delivery of a provisional physical framework for stem cell angiogenesis. Freshly collected whole blood was utilized to synthesize PEGylated PRP hydrogels containing platelet concentrations ranging from 0 to 200,000 platelets/µl. Hydrogels were characterized using thromboelastography and impedance aggregometry for platelet function and were visualized using scanning electron microscopy. To assess the effects of PEGylated PRP hydrogels on cells, PRP solutions were seeded with human adipose-derived stem cells (ASCs) prior to gelation. Following 14 days of incubation in vitro, increased platelet concentrations resulted in higher ASC proliferation and vascular gene and protein expression (assessed via RT-PCR, ELISA, and immunochemistry). Using a rat skin excision model, wounds treated with PRP + ASC hydrogels increased the number of vessels in the wound by day 8 (80.2 vs. 62.6 vessels/mm2) compared to controls. In conclusion, the proposed PEGylated PRP hydrogel promoted both in vitro and transient in vivo angiogenesis of ASCs for improved wound healing. STATEMENT OF SIGNIFICANCE: Our findings support an innovative means of cellular therapy intervention to improve surgical wound healing in a normal wound model. ASCs seeded within PEGylated PRP could be an efficacious and completely autologous therapy for treating patients who have poorly healing wounds caused by vascular insufficiency, previous irradiation, or full-thickness burns. Because wound healing is a dynamic and complex process, the application of more than one growth factor with ASCs demonstrates an advantageous way of improving healing.

Plasma-Alginate Composite Material Provides Improved Mechanical Support for Stem Cell Growth and Delivery.

Blood plasma-based products have been recently utilized in different tissue engineering applications, ranging from soft tissue repair to bone regeneration. Plasma contains fibrinogen which can be converted to an insoluble fibrin-laden gel in the presence of activated thrombin. In tissue engineering, these plasma-based materials can serve either as a three-dimensional scaffold to deliver therapeutic cells in vivo or as a growth factor-rich supply for tissue regeneration. Unfortunately, plasma-based materials are often mechanically weak and easily deformed, thus limiting their usability in harsh clinical settings. Simpler methods to create sturdier plasma-based materials are therefore needed. To this end, we hypothesized that combining alginate with plasma can create a composite plasma material with improved mechanical properties. Incorporating alginate into plasma produced composite gels with increasing bulk stiffness, as measured by rheology. Specifically, the plasma-alginate composite (PAC) gels with an alginate concentration of 2.86 mg/mL were 10-fold stiffer than pure plasma gels (11 vs 112 Pa). Interestingly, gel lysis rates were unchanged despite increasing alginate concentration (lysis time approximately 50 min). Adipose-derived stem cells cultured in the stiffer PAC gels expressed stemness markers (THY1, ENG, NT5E) at levels comparable to those in the pure plasma gels. Similarly, proangiogenic factor secretion was also constant across all gel conditions. In sum, we envision this PAC gel system will extend the use of plasma gel-based therapies into more rigorous clinical applications.

Peroxisome proliferator-activated receptor-α agonist and all-trans retinoic acid induce epithelial differentiation of subcutaneous adipose-derived stem cells from debrided burn skin.

This study demonstrates that adipose-derived stem cells from debrided skin (dsASCs) of burn patients can be isolated in sufficient quantities and differentiated into cytokeratin-expressing cells by treating them with all-trans retinoic acid (ATRA) and the peroxisome proliferator-activated receptor-α (PPARα) specific activator fenofibrate. Differentiation of dsASCs with ATRA and a combination of growth factors induced expression of simple epithelial markers (KRT7, KRT8, KRT18, and KRT19), along with low levels of stratified epithelial markers (KRT5, KRT10, KRT13, and KRT14). We have optimized a condition to induce dsASCs differentiation to epithelial cells by treatment with ATRA and fenofibrate alone. Real-time polymerase chain reaction analysis showed a significant increase in transcript levels (>75-fold) for basal (KRT5 and KRT14), suprabasal (KRT10), and cornified envelope markers (involucrin [IVL] and Loricrin [LOR]) with this treatment. Expression of the proteins encoded by these transcripts was confirmed by immunocytochemical analysis. Further, we show that dsASCs differentiated to a skin epithelial cell phenotype through activation of nuclear hormone receptors PPARα and RXRγ. Collectively this study shows that dsASCs can be differentiated to skin epithelial cells, without the requirement for exogenous growth factors. This differentiation protocol using dsASCs in combination with an appropriate biocompatible scaffold can be adapted to develop epithelial skin substitute for burn wound treatment.

PEG-Plasma Hydrogels Increase Epithelialization Using a Human Ex Vivo Skin Model.

In vitro cell culture methods are used extensively to study cellular migration, proliferation, and differentiation, which play major roles in wound healing but the results often do not translate to the in vivo environment. One alternative would be to establish an ex vivo model utilizing human discarded skin to evaluate therapies in a more natural setting. The purpose of this study was to institute such a model by creating 'wounds' in the center of a piece of discarded skin and treating them with three different biomaterials: collagen, polyethylene glycol (PEG)-fibrin, or PEG-platelet free plasma (PFP). Explants were cultured for 14 days with supernatant and microscopy images collected every 3 days to assess cytotoxicity and epithelialization. After 14 days, the explants were fixed, sectioned, and stained for cytokeratin-10 (CK-10), alpha-smooth muscle actin (α-SMA), and wheat germ (WG). Compared to controls, similar levels of cytotoxicity were detected for 12 days which decreased slightly at day 14. The PEG-PFP hydrogel-treated wounds epithelialized faster than other treatments at days 6 to 14. A 6-8 cell layer thick CK-10+ stratified epidermis had developed over the PEG-PFP hydrogel and cells co-stained by WG and α-SMA were observed within the hydrogel. An ex vivo model was established that can be used practically to screen different therapies exploring wound healing.

Advancements in Regenerative Strategies Through the Continuum of Burn Care.

Burns are caused by several mechanisms including flame, scald, chemical, electrical, and ionizing and non-ionizing radiation. Approximately half a million burn cases are registered annually, of which 40 thousand patients are hospitalized and receive definitive treatment. Burn care is very resource intensive as the treatment regimens and length of hospitalization are substantial. Burn wounds are classified based on depth as superficial (first degree), partial-thickness (second degree), or full-thickness (third degree), which determines the treatment necessary for successful healing. The goal of burn wound care is to fully restore the barrier function of the tissue as quickly as possible while minimizing infection, scarring, and contracture. The aim of this review is to highlight how tissue engineering and regenerative medicine strategies are being used to address the unique challenges of burn wound healing and define the current gaps in care for both partial- and full-thickness burn injuries. This review will present the current standard of care (SOC) and provide information on various treatment options that have been tested pre-clinically or are currently in clinical trials. Due to the complexity of burn wound healing compared to other skin injuries, burn specific treatment regimens must be developed. Recently, tissue engineering and regenerative medicine strategies have been developed to improve skin regeneration that can restore normal skin physiology and limit adverse outcomes, such as infection, delayed re-epithelialization, and scarring. Our emphasis will be centered on how current clinical and pre-clinical research of pharmacological agents, biomaterials, and cellular-based therapies can be applied throughout the continuum of burn care by targeting the stages of wound healing: hemostasis, inflammation, cell proliferation, and matrix remodeling.

Assessment of Ablative Fractional CO2 Laser and Er:YAG Laser to Treat Hypertrophic Scars in a Red Duroc Pig Model.

Hypertrophic scarring is a fibroproliferative process that occurs following a third-degree dermal burn injury, producing significant morbidity due to persistent pain, itching, cosmetic disfigurement, and loss of function due to contractures. Ablative fractional lasers have emerged clinically as a fundamental or standard therapeutic modality for hypertrophic burn scars. Yet the examination of their histopathological and biochemical mechanisms of tissue remodeling and comparison among different laser types has been lacking. In addition, deficiency of a relevant animal model limits our ability to gain a better understanding of hypertrophic scar pathophysiology. To evaluate the effect of ablative fractional lasers on hypertrophic third-degree burn scars, we have developed an in vivo Red Duroc porcine model. Third-degree burn wounds were created on the backs of animals, and burn scars were allowed to develop for 70 days before treatment. Scars received treatment with either CO2 or erbium: yttrium aluminum garnet (YAG) ablative fractional lasers. Here, we describe the effect of both lasers on hypertrophic third-degree burn scars in Red Duroc pigs. In this report, we found that Er:YAG has improved outcomes versus fractional CO2. Molecular changes noted in the areas of dermal remodeling indicated that matrix metalloproteinase 2, matrix metalloproteinase 9, and Decorin may play a role in this dermal remodeling and account for the enhanced effect of the Er:YAG laser. We have demonstrated that ablative fractional laser treatment of burn scars can lead to favorable clinical, histological, and molecular changes. This study provides support that hypertrophic third-degree burn scars can be modified by fractional laser treatment.