Topical Noneuphoric Phytocannabinoid Elixir 14 Reduces Inflammation and Mitigates Burn Progression.

INTRODUCTION: Thermal injuries are caused by exposure to a wide variety of agents including heat, electricity, radiation, chemicals, and friction. Early intervention can decrease injury severity by preventing excess inflammation and mitigating burn wound progression for improved healing outcomes. Previous studies have demonstrated that cannabinoids can trigger anti-inflammatory responses and promote wound closure. Therefore, the purpose of this study was to investigate whether a topical application of Noneuphoric Phytocannabinoid Elixir 14 (NEPE14) containing a full complement of phytocannabinoids (< 0.3% delta-9-tetrahydrocannabinol or cannabidiol) and other phytochemicals would mitigate burn wound progression in the treatment of deep partial-thickness burn wounds. METHODS: Deep partial-thickness burns were created on the dorsum of four anesthetized pigs and treated with NEPE14, Vehicle control, Silverlon, or gauze. The burns were assessed on postburn days 4, 7, and 14. Assessments consisted of digital photographs, Laser-Speckle imagery (blood perfusion), MolecuLight imagery (qualitative bacterial load), and biopsies for histology and immunohistochemistry (interleukin six and tumor necrosis factor-α). RESULTS: Topical treatment with NEPE14 significantly (P < 0.001) decreased inflammation (interleukin six and tumor necrosis factor-α) in comparison to control groups. It was also demonstrated that the reduction in inflammation led to mitigation of burn wound progression. In terms of wound healing and presence of bacteria, no statistically significant differences were observed. CONCLUSIONS: Topical treatment of deep partial-thickness burns with NEPE14 decreased wound inflammation and mitigated burn wound progression in comparison to control treatments.

Topical Synthetic Platelets Loaded With Gentamicin Decrease Bacteria in Deep Partial-Thickness Burns.

INTRODUCTION: Prolonged inflammation and infection in burns may cause inadequate healing. Platelet granules contain anti-inflammatory mediators that impact wound healing. Synthetic platelets (SPs) avoid portability and storage difficulties of natural platelets and can be loaded with bioactive agents. We evaluated wound healing outcomes in deep partial-thickness (DPT) burns treated topically with SP loaded with antibiotics. MATERIALS AND METHODS: Thirty DPT burns were created on the dorsum of two Red Duroc hybrid pigs. Six wounds were randomized into five groups: SP alone, SP loaded with gentamicin vesicles, SP with gentamicin mixture, vehicle control (saline), or dry gauze. Wounds were assessed from postburn days 3-90. Primary outcome was re-epithelialization percentage at postburn day 28. Secondary outcomes included wound contraction percentage, superficial blood flow relative to normal skin controls, and bacterial load score. RESULTS: Results showed that re-epithelialization with the standard of care (SOC) was 98%, SP alone measured 100%, SP loaded with gentamicin vesicles was 100%, and SP with gentamicin mixture was 100%. Wound contraction was 5.7% in the SOC and was ∼10% in both the SP loaded with gentamicin vesicles and SP with gentamicin mixture groups. Superficial blood flow in the SOC was 102.5%, SP alone was 170%, the SP loaded was 155%, and gentamicin mixture 162.5%. Bacterial load score in the SOC was 2.2/5.0 and was significantly less at 0.8/5.0 in SP loaded with gentamicin vesicles (P > 0.05). SP and gentamicin mixture scored 2.7 and 2.3/5.0. CONCLUSIONS: Topical SP treatment did not significantly improve outcomes. However, SP loaded with gentamicin-infused vesicles decreased bacterial load.

Full-thickness skin columns: A method to reduce healing time and donor site morbidity in deep partial-thickness burns.

The current standard of care for the coverage of large wounds often involves split thickness skin grafts (STSGs) which have numerous limitations. One promising technique that has gained traction is fractional autologous skin grafting using full-thickness skin columns (FTSC). Harvesting occurs orthogonally by taking numerous individual skin columns containing the epidermis down through the dermis and transferring them to the wound bed. The purpose of this porcine study was to investigate the efficacy of implanting FTSCs directly into deep partial-thickness burn wounds, as well as examining donor site healing at the maximal harvest density. It was hypothesised that by utilising FTSCs, the rate of healing in deep partial thickness burns can be improved without incurring the donor morbidity seen in other methods of skin grafting. Deep partial-thickness burns were created on the dorsum of female red duroc swine, debrided 3 days later and FTSCs were implanted at varying expansion ratios directly into the burn wounds. At day 14, 1:50 expansion ratio showed significantly faster re-epithelialisation compared to the debrided burn control and 1:200. Donor sites (at 7%-10% harvest density) were 100% re-epithelialised by day 7. Additionally, the maximal harvest density was determined to be 28% in an ex vivo model, which then five donor sites were harvested at 28% density on a red duroc swine and compared to five STSG donor sites. At maximal harvest density, FTSC donor sites were significantly less hypopigmented compared to STSGs, but no significant differences were observed in re-epithelialisation, contraction, blood flow or dermal thickness. In conclusion, implantation directly into deep partial-thickness burns is a viable option for the application of FTSCs, favouring lower expansion ratios like 1:50 or lower. Little difference in donor site morbidity was observed between FTSC at a maximal harvest density of 28% and STSGs, exceeding the optimal harvest density.

Delivery of topical gentamicin cream via platform wound device to reduce wound infection-A prospective, controlled, randomised, clinical study.

The platform wound device (PWD) is a wound coverage system that is designed to decrease wound infection rates by allowing for direct delivery of topical antibiotics and antimicrobials while creating a sealed, protective barrier around the area of injury. This study evaluated the safety and efficacy of the PWD as a protective dressing and a delivery system for topical antibiotics compared to the current standard of care (SoC). This was a multi-center, prospective, randomised, controlled clinical trial. The wounds were treated with the PWD with gentamicin cream or SoC dressings. The wounds were evaluated before the start of treatment and after 48-96 hours via clinical assessment, photographs, and qualitative bacterial swabs for bacterial analysis. The delivery of gentamicin via the PWD was safe and did not cause any adverse effects. The treatment decreased both inflammation and bacterial growth during the study period. No significant differences in the SoC were observed. The PWD is a transparent and impermeable polyurethane chamber that encloses and protects the injured area. The delivery of topical gentamicin via the PWD was safe and effective. Clinical assessment for infection found the PWD to be non-inferior to the current SoC treatment options.

Analysis of the Utility of CO2 and Pulse-Dye Lasers Together and Separately in the Treatment of Hypertrophic Burn Scars.

INTRODUCTION: Hypertrophic burn scars (HTBSs) remain a significant source of morbidity. Contemporary treatment has evolved to use CO2 lasers and/or pulse-dye lasers (PDLs) to reduce scar thickness (ST) and erythema. This study seeks to compare treatment efficacy with CO2 or PDL individually and in combination. METHODS: Patients undergoing laser treatments for HTBSs were enrolled. Three 3 × 3 cm squares of HTBSs were randomized to receive treatment with CO2 laser, PDL or CO2 + PDL. Patients underwent 3 treatments, 4 to 6 weeks apart and were followed up over 3 to 6 months. Scar assessments occurred at each visit before treatment and consisted of photographs, ultrasound, colorimetry, and the Patient and Observer Scar Assessment Score. RESULTS: Twenty-five patients were enrolled. Twenty completed 2 treatments (80%) and 11 completed all 3 treatments (44%). Median initial ST was 0.3 cm. Median time since injury was 8 months. Hypertrophic burn scars treated with CO2 or PDL showed a significant decrease in Patient and Observer Scar Assessment Scale score from visit 1 to 3 (P = 0.01 and 0.01, respectively). When separated by ST, thick scars (≥0.3 cm) showed a significant decrease in thickness between visit 1 and 2 using all laser modalities (CO2 + PDL, P = 0.01; CO2, P = 0.02; PDL, P = 0.03). Thin scars (<0.3 cm) showed a reduction in thickness by visit 3 after CO2 + PDL or PDL alone (P = 0.01 and 0.04, respectively). Separating scars by age, younger scars (<9 months) showed a significant reduction in thickness between visit 1 and 2 for CO2 treatment (P = 0.04), and between visit 2 and 3 for CO2 + PDL treatment (P = 0.04). Hypertrophic burn scars treated with PDL did not demonstrate a significant reduction in thickness until visit 3 (P = 0.002). Older scars (≥9 months) showed a significant reduction in thickness between visit 1 and 2 only after CO2 + PDL (P = 0.01). CONCLUSIONS: Hypertrophic burn scars of varying ages, etiologies, and thicknesses were examined in this study with greater degree of early reduction seen in thicker scars using all laser modalities of CO2, PDL or in combination. However, there was no clinically meaningful benefit found with combination as compared with individual treatment. These data support the use of laser to improve HTBS but does not support one modality or combination of modalities over another.

Loss of ARNT in skeletal muscle limits muscle regeneration in aging.

The ability of skeletal muscle to regenerate declines significantly with aging. The expression of aryl hydrocarbon receptor nuclear translocator (ARNT), a critical component of the hypoxia signaling pathway, was less abundant in skeletal muscle of old (23-25 months old) mice. This loss of ARNT was associated with decreased levels of Notch1 intracellular domain (N1ICD) and impaired regenerative response to injury in comparison to young (2-3 months old) mice. Knockdown of ARNT in a primary muscle cell line impaired differentiation in vitro. Skeletal muscle-specific ARNT deletion in young mice resulted in decreased levels of whole muscle N1ICD and limited muscle regeneration. Administration of a systemic hypoxia pathway activator (ML228), which simulates the actions of ARNT, rescued skeletal muscle regeneration in both old and ARNT-deleted mice. These results suggest that the loss of ARNT in skeletal muscle is partially responsible for diminished myogenic potential in aging and activation of hypoxia signaling holds promise for rescuing regenerative activity in old muscle.

A porous collagen-GAG scaffold promotes muscle regeneration following volumetric muscle loss injury.

Volumetric muscle loss (VML) is a segmental loss of skeletal muscle which commonly heals with fibrosis, minimal muscle regeneration, and loss of muscle strength. Treatment options for these wounds which promote functional recovery are currently lacking. This study was designed to investigate whether the collagen-GAG scaffold (CGS) promotes functional muscle recovery following VML. A total of 66 C57/Bl6 mice were used in a three-stage experiment. First, 24 animals were split into three groups which underwent sham injury or unilateral quadriceps VML injury with or without CGS implantation. Two weeks post-surgery, muscle was harvested for histological and gene expression analysis. In the second stage, 18 mice underwent bilateral quadriceps VML injury, followed by weekly functional testing using a treadmill. In the third stage, 24 mice underwent sham or bilateral quadriceps VML injury with or without CGS implantation, with tissue harvested six weeks post-surgery for histological and gene expression analysis. VML mice treated with CGS demonstrated increased remnant fiber hypertrophy versus both the VML with no CGS and uninjured groups. Both VML groups showed greater muscle fiber hypertrophy than non-injured muscle. This phenomenon was still evident in the longer-term experiment. The gene array indicated that the CGS promoted upregulation of factors involved in promoting wound healing and regeneration. In terms of functional improvement, the VML mice treated with CGS ran at higher maximum speeds than VML without CGS. A CGS was shown to enhance muscle hypertrophy in response to VML injury with a resultant improvement in functional performance. A gene array highlighted increased gene expression of multiple growth factors following CGS implantation. This suggests that implantation of a CGS could be a promising treatment for VML wounds.

Novel negative pressure wound therapy device without foam or gauze is effective at -50 mmHg.

Negative pressure wound therapy (NPWT) promotes healing in acute or chronic wounds. Conventional NPWT devices consist of a filler (such as foam or gauze) that covers the wound and of a permeable membrane and tubing that connects the space under the membrane to a suction pump. The permeable membrane increases airflow and thus increases the required pump capacity that can cause patient discomfort or even ischemia in wounds with compromised vascularity. In addition, foam or gauze may fragment and become colonized with bacteria over time. To mitigate these, negative aspects, we have developed a new impermeable single layer component membrane dressing to deliver NPWT that does not need a foam or gauze to function. Therefore, the purpose of this study was to introduce this novel NPWT system (platform wound device, PWD) and evaluate its usability and effectiveness in the treatment of porcine full-thickness burns. A total of 48 burn wounds were created across four Yorkshire pigs on the dorsum. Wounds were created on day 0 and continuous NPWT with -50 mmHg and - 80 mmHg was initiated immediately. Subsequently, the burns were debrided on day 3 and animals were euthanized on day 7. The efficacy of the PWD on wound healing and reduction of bacterial burden was measured and compared to wounds that did not receive NPWT. The results showed that PWD promoted wound healing by outperforming the wounds that did not receive NPWT and that PWD was efficient at reducing bacteria from the burn eschar and from the wound bed. In conclusion, this study demonstrated that PWD promoted wound healing with a negative pressure as low as -50 mmHg, which likely benefits healing and avoids potential safety issues.

Intracellular signalling pathways and cytoskeletal functions converge on the psoriasis candidate gene CCHCR1 expressed at P-bodies and centrosomes.

BACKGROUND: CCHCR1 (Coiled-Coil α-Helical Rod protein 1) is a putative psoriasis candidate gene with the risk alleles CCHCR1*WWCC and *Iso3, the latter inhibiting the translation of isoform 1. CCHCR1 was recently shown to be a centrosomal protein, as well as a component of cytoplasmic processing bodies (P-bodies) that regulate mRNA turnover. The function of CCHCR1 has remained unsettled, partly because of the inconsistent findings; it has been shown to play a wide variety of roles in divergent processes, e.g., cell proliferation and steroidogenesis. Here we utilized RNA sequencing (RNAseq) using HEK293 cells overexpressing isoforms 1 or 3 (Iso1, Iso3 cells), in combination with the coding non-risk or risk (*WWCC) haplotype of CCHCR1. Our aim was to study the overall role of CCHCR1 and the effects of its variants. RESULTS: The overexpression of CCHCR1 variants in HEK293 cells resulted in cell line-specific expression profiles though several similarities were observable. Overall the Iso1 and Iso3 cells showed a clear isoform-specific clustering as two separate groups, and the Non-risk and Risk cells often exhibited opposite effects. The RNAseq supported a role for CCHCR1 in the centrosomes and P-bodies; the most highlighted pathways included regulation of cytoskeleton, adherens and tight junctions, mRNA surveillance and RNA transport. Interestingly, both the RNAseq and immunofluorescent localization revealed variant-specific differences for CCHCR1 within the P-bodies. CONCLUSIONS: CCHCR1 influenced a wide variety of signaling pathways, which could reflect its active role in the P-bodies and centrosomes that both are linked to the cytoskeleton; as a centrosomal P-body protein CCHCR1 may regulate diverse cytoskeleton-mediated functions, such as cell adhesion and -division. The present findings may explain the previous inconsistent observations about the functions of CCHCR1.

Evaluation of the efficacy of cell and micrograft transplantation for full-thickness wound healing.

BACKGROUND: Skin grafting is the current standard of care in the treatment of full-thickness burns and other wounds. It is sometimes associated with substantial problems, such as poor quality of the healed skin, scarring, and lack of donor-site skin in large burns. To overcome these problems, alternative techniques that could provide larger expansion of a skin graft have been introduced over the years. Particularly, different cell therapies and methods to further expand skin grafts to minimize the need for donor skin have been attempted. The purpose of this study was to objectively evaluate the efficacy of cell and micrograft transplantation in the healing of full-thickness wounds. MATERIALS AND METHODS: Allogeneic cultured keratinocytes and fibroblasts, separately and together, as well as autologous and allogeneic skin micrografts were transplanted to full-thickness rat wounds, and healing was studied over time. In addition, wound fluid was collected, and the level of various cytokines and growth factors in the wound after transplantation was measured. RESULTS: Our results showed that both autologous and allogeneic micrografts were efficient treatment modalities for full-thickness wound healing. Allogeneic skin cell transplantation did not result in wound closure, and no viable cells were found in the wound 10 d after transplantation. CONCLUSIONS: Our study demonstrated that allogeneic micrografting is a possible treatment modality for full-thickness wound healing. The allografts stayed viable in the wound and contributed to both re-epithelialization and formation of dermis, whereas allogeneic skin cell transplantation did not result in wound closure.

Induced Granulation Tissue but not Artificial Dermis Enhances Early Host-Graft Interactions in Full-Thickness Burn Wounds.

BACKGROUND: Cellular grafts used for skin repair require rapid integration with the host tissue to remain viable and especially to nourish the epidermal cells. Here, we evaluated the responses in the split-thickness skin grafts (STSGs) grafted on three differently treated wound beds: directly on excised wound bed (EX), on an artificial dermal template (DT) and on granulation tissue (GT) induced by cellulose sponge. METHODS: In ten burn patients, after excision, a test area was divided into three sections: One transplanted with STSG instantaneously and two sections had a pre-treatment for 2 weeks with either DT or a cellulose sponge inducing granulation tissue formation and thereafter grafted with STSGs. RESULTS: One week after grafting, the STSGs on GT demonstrated most endothelial CD31+ staining, largest average vessel diameters as well as most CD163+ staining of M2-like macrophages and most MIB1+ proliferating epidermal cells, suggesting an active regenerative environment. STSGs on DT had smallest vessel diameters and the least CD163+ macrophages. STSGs on EX had the least CD31+ cells and the least MIB1+ proliferating cells. After 3 months, this reactivity in STSGs had subsided, except increased dermal cell proliferation was observed in STSGs on EX. CONCLUSIONS: Results show that pre-treatment of wound bed and induction of granulation tissue formation can accelerate host-graft interaction by stimulating graft vasculature and inducing cell proliferation.

Use of a novel chitosan-based dressing on split-thickness skin graft donor sites: a pilot study.

OBJECTIVE: Split-thickness skin graft (STSG) donor site dressings can play an integral role in reducing donor site morbidity. This study tested a novel, chitosan-based wound dressing, Opticell Ag, as an STSG donor site dressing for wounds <10% total body surface area (TBSA). METHOD: Between January and December 2016, the chitosan-based dressing was placed on participating patients' donor sites immediately following graft harvest and covered with a transparent occlusive dressing. Pain was evaluated on postoperative day one, before dressing change between days 5-7, and before and after dressing removal between days 10-14 using the Visual Analog Scale (VAS). The extent of re-epithelialisation was determined between day 10-14 and at one month, and healing quality was also evaluated at one month post-operatively using the Vancouver Scar Scale (VSS). RESULTS: A total of 19 patients were recruited, of which 16 completed the study. Patients experienced mild-to-moderate pain in their donor sites when the chitosan-based dressing was used. Pain decreased significantly between postoperative day one and days 10-14, as well as between days 5-7 and 10-14. The mean percentage of re-epithelialisation on days 10-14 was 92% and by one month was 99%. The mean VSS at one month was 3.2±1.4. There were no statistically significant differences between patients' re-epithelialisation rates or VSS scores. There were unplanned dressing changes in four patients. No donor site infections or other adverse events were identified. CONCLUSION: The chitosan-based dressing tested in this study is safe, effective, and associated with reasonable pain control and acceptable healing quality. The results suggest that it is a promising STSG donor site dressing.

Gene expression profiling of skeletal muscle after volumetric muscle loss.

Volumetric muscle loss (VML), usually occurring following traumatic injury, results in a composite loss of muscle mass. These injuries manifest as decreased strength and functional impairments. Clinically, these injuries often heal with fibrosis, as opposed to skeletal muscle regeneration. This study examines the healing patterns of a skeletal muscle following VML in a murine model. Eight-week old male C57BL/6J mice used in the study underwent either bilateral VML injury or cryoinjury, a widely used model known to induce skeletal muscle regeneration. Skeletal muscle was harvested at 2 and 4 weeks following injury and subjected to histological analysis. H&E staining demonstrated skeletal muscle regeneration following cryoinjury, but not VML, at either timepoint post-injury. Additionally, samples were analyzed using a wound-healing PCR array to identify differentially regulated genes of interest in VML and cryoinjury, as compared to noninjured controls. The gene array data further demonstrated prolonged inflammation and increased pro-fibrotic activity in the VML injured muscles, as compared to cryoinjury. In addition, IGF1, a known myogenic factor, was significantly decreased following VML, as compared to cryoinjury, in both ELISA and PCR. This study offers an insight into the pathophysiology of VML injury and reveals a gene profile of a nonregenerating skeletal muscle.

The effect of pH on cell viability, cell migration, cell proliferation, wound closure, and wound reepithelialization: In vitro and in vivo study.

Wound microenvironment plays a major role in the process of wound healing. It contains various external and internal factors that participate in wound pathophysiology. The pH is an important factor that influences wound healing by changing throughout the healing process. Several previous studies have investigated the role of pH in relation to pathogens but studies concentrating on the effects of pH on wound healing itself are inconclusive. The purpose of this study was to comprehensively and in a controlled fashion investigate the effect of pH on wound healing by studying its effect on human primary keratinocyte and fibroblast function in vitro and on wound healing in vivo. In vitro, primary human keratinocytes and fibroblasts were cultured in different levels of pH (5.5-12.5) and the effect on cell viability, proliferation, and migration was studied. A rat full-thickness wound model was used to investigate the effect of pH (5.5-9.5) on wound healing in vivo. The effect of pH on inflammation was monitored by measuring IL-1 α concentrations from wounds and cell cultures exposed to different pH environments. Our results showed that both skin cell types tolerated wide range of pH very well. They further demonstrated that both acidic and alkaline environments decelerated cell migration in comparison to neutral environments and interestingly alkaline conditions significantly enhanced cell proliferation. Results from the in vivo experiments indicated that a prolonged, strongly acidic wound environment prevents both wound closure and reepithelialization while a prolonged alkaline environment did not have any negative impact on wound closure or reepithelialization. Separately, both in vitro and in vivo studies showed that prolonged acidic conditions significantly increased the expression of IL-1 α in fibroblast cultures and in wound fluid, whereas prolonged alkaline conditions did not result in elevated amounts of IL-1 α.

The effect of local hyperglycemia on skin cells in vitro and on wound healing in euglycemic rats.

BACKGROUND: Multiple previous studies have established that high systemic blood glucose concentration impairs skin wound healing. However, the effects of local hyperglycemia on wound healing are not well defined. Comprehensive animal studies and in vitro studies using both fibroblasts and keratinocytes are lacking. MATERIALS AND METHODS: Primary keratinocytes and fibroblasts were isolated from discarded human tissue, cultured under different concentrations of glucose, and the effect on cell function was examined. In addition, a rat full-thickness wound model was used to topically treat the wounds with different glucose concentrations and the effect on wound closure and re-epithelialization was investigated over time. RESULTS: The cell viability experiments indicated that both keratinocytes and fibroblasts endure high glucose well and concentrations under 26 mM did not have a remarkable effect on their viability over time. Moderate addition of glucose (10 mM) boosted fibroblast proliferation (6-fold) but did not have an effect on keratinocyte proliferation. In both keratinocytes and fibroblasts, glucose inhibited their migration and already the addition of 5.6-mM glucose had an inhibitory effect. In vivo experiments showed that full-thickness wounds treated with topical glucose had impaired wound closure and lower re-epithelialization rate in comparison to nontreated control wounds. The results also showed that higher glucose concentrations inhibited wound healing more efficiently. CONCLUSIONS: In conclusion, our study indicates that high glucose inhibits both keratinocyte and fibroblast migration as well as wound healing in vivo in a concentration dependent manner.

Endotoxin-induced inflammation in a rodent model up-regulates IL-1a expression and CD45+ leukocyte recruitment and increases the rate of reepithelialization and wound closure.

Wound healing is traditionally divided into inflammation, proliferation, and remodeling phases. Several inflammatory mediators and cells regulate the inflammation phase. The specific roles for different mediators have not been clearly defined. The effects of inflammation phase modulation on wound healing were evaluated in this study. Rat full-thickness wounds were divided into different experimental groups: (1) sterile hyper-inflammatory wounds/endotoxin (topical endotoxin), (2) sterile hypo-inflammatory/inhibitor group (cocktail of topical COX-1 plus COX-2 plus lipoxygenase inhibitors), and (3) control groups: topical saline or DMSO. After full-thickness wound creation, custom-made titanium chambers enclosed the wound, creating an isolated well-controlled environment. Wound healing was followed over time; tissue biopsies and wound fluid samples were collected on days 1, 4, and 8 postoperatively. The validity of the inflammation model was confirmed by increased IL-1a expression, increased CD45+ leukocytes recruitment in the hyper-inflamed group as compared to the inhibitor and control groups. The reepithelialization percentage was significantly increased in the endotoxin group as compared to the inhibitor group on day 4 (60.75 vs. 22.05, p-value <0.05) and both the inhibitor and the control group on day 8 (control group: 63.2%, inhibitor group: 28.9%, endotoxin group: 84.2%, p-value <0.05). Also, the macroscopic wound closure was increased in the endotoxin group as compared to the inhibitor group and control group both on day 4 (control group: 69.9%, inhibitor group: 62.9%, endotoxin group: 81.9%, p-value <0.05) and on day 8 (control group: 68.5%, inhibitor group: 69.1%, endotoxin group: 83.7%, p-value <0.05). Endotoxin-induced sterile inflammation up-regulates IL-1a expression and CD45+ leukocyte recruitment and results in faster rate of wound reepithelialization and wound closure in full-thickness rodent wounds. Conversely, the wound reepithelialization and wound closure can be significantly delayed on treatment with a combination of cyclooxygenase and lipoxygenase inhibitors.

Titanium wound chambers for wound healing research.

Standardized and reproducible animal models are crucial in medical research. Rodents are commonly used in wound healing studies since, they are easily available, affordable and simple to handle and house. However, the most significant limitation of rodent models is that the wounds heal by contraction while in humans the primary mechanisms of healing are reepithelialization and granulation tissue formation. The robust contraction results in faster wound closure that complicates the reproducibility of rodent studies in clinical trials. We have developed a titanium wound chamber for rodent wound healing research. The chamber is engineered from two pieces of titanium and is placed transcutaneously on the dorsum of a rodent. The chamber inhibits wound contraction and provides a means for controlled monitoring and sampling of the wound environment in vivo with minimal foreign body reaction. This technical report introduces two modalities utilizing the titanium chambers in rats: (1) Wound in a skin island model and, (2) Wound without skin model. Here, we demonstrate in rats how the "wound in a skin island model" slows down wound contraction and how the "wound without skin" model completely prevents the closure. The titanium wound chamber provides a reproducible standardized models for wound healing research in rodents.

Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control.

BACKGROUND: Keratinocytes (KCs) are the most frequent cells in the epidermis, and they are often isolated and cultured in vitro to study the molecular biology of the skin. Cultured primary cells and various immortalized cells have been frequently used as skin models but their comparability to intact skin has been questioned. Moreover, when analyzing KC transcriptomes, fluctuation of polyA+ RNA content during the KCs' lifecycle has been omitted. RESULTS: We performed STRT RNA sequencing on 10 ng samples of total RNA from three different sample types: i) epidermal tissue (split-thickness skin grafts), ii) cultured primary KCs, and iii) HaCaT cell line. We observed significant variation in cellular polyA+ RNA content between tissue and cell culture samples of KCs. The use of synthetic RNAs and SAMstrt in normalization enabled comparison of gene expression levels in the highly heterogenous samples and facilitated discovery of differences between the tissue samples and cultured cells. The transcriptome analysis sensitively revealed genes involved in KC differentiation in skin grafts and cell cycle regulation related genes in cultured KCs and emphasized the fluctuation of transcription factors and non-coding RNAs associated to sample types. CONCLUSIONS: The epidermal keratinocytes derived from tissue and cell culture samples showed highly different polyA+ RNA contents. The use of SAMstrt and synthetic RNA based normalization allowed the comparison between tissue and cell culture samples and thus proved to be valuable tools for RNA-seq analysis with translational approach. Transciptomics revealed clear difference both between tissue and cell culture samples and between primary KCs and immortalized HaCaT cells.

The external microenvironment of healing skin wounds.

The skin wound microenvironment can be divided into two main components that influence healing: the external wound microenvironment, which is outside the wound surface; and the internal wound microenvironment, underneath the surface, to which the cells within the wound are exposed. Treatment methods that directly alter the features of the external wound microenvironment indirectly affect the internal wound microenvironment due to the exchange between the two compartments. In this review, we focus on the effects of temperature, pressure (positive and negative), hydration, gases (oxygen and carbon dioxide), pH, and anti-microbial treatment on the wound. These factors are well described in the literature and can be modified with treatment methods available in the clinic. Understanding the roles of these factors in wound pathophysiology is of central importance in wound treatment.

Split-Thickness Skin and Dermal Pixel Grafts Can Be Expanded up to 500 Times to Re-Epithelialize a Full-Thickness Burn Wound.

Objective: Autologous skin transplantation is limited by donor site availability for patients with extensive burns. The objective of this study was to demonstrate the feasibility and efficacy of split-thickness skin (STS) and dermal pixel grafts (PG) in the treatment of burns. Approach: The study was divided into three arms of validation, expansion, and combination that all followed the same study design. Sixteen deep partial-thickness burns were created on the dorsum of anesthetized pigs. Three days postinjury the burns were debrided and grafted with STS and dermal PGs. The PGs were prepared by harvesting two skin grafts (split-thickness skin graft [STSG] and dermal graft) from the same donor site going down in depth. The grafts were minced to 0.3 × 0.3 × 0.3 mm PGs and suspended in a small volume of hydrogel. Healing was monitored for 6, 10, 14, or 28 days. In the validation study the PGs at 1:2 expansion ratio were transplanted and compared with STSG and untreated controls. The expansion study investigated the maximum expansion potential of the PGs and the combination of the benefits of transplanting STS and dermal PGs together. Results: The validation study showed that when STS and dermal PGs were transplanted in a 1:2 ratio they fully re-epithelialized the wounds in 14 days. The expansion study demonstrated that using expansion ratios up to 1:500 the wounds were re-epithelialized by day 28. The combination study showed that there was no additional benefit to use STS and dermal PGs together. Innovation: Pixel grafting provides expansion ratios greater than conventional STSG. The possibility to harvest both STS and dermal PGs from the same donor area further reduces the need for healthy skin. Conclusion: STSG and dermal grafts can be minced to PGs with preserved viability and expanded up to 500 times to re-epithelialize a wound.