Kristo Nuutila

Human skin transcriptome during superficial cutaneous wound healing.

Healing of the epidermis is a crucial process for maintaining the skins defense integrity and its resistance to environmental threats. Compromised wound healing renders the individual readily vulnerable to infections and loss of body homeostasis. To clarify the human response of reepithelialization, we biopsied split‐thickness skin graft donor site wounds immediately before and after harvesting, as well as during the healing process 3 and 7 days thereafter. In all, 25 biopsies from eight patients qualified for the study. All samples were analyzed by genome‐wide microarrays. Here, we identified the genes associated with normal skin reepithelialization over time and organized them by similarities according to their induction or suppression patterns during wound healing. Our results provide the first elaborate insight into the transcriptome during normal human epidermal wound healing. The data not only reveal novel genes associated with epidermal wound healing but also provide a fundamental basis for the translational interpretation of data acquired from experimental models.

NOD-like receptor signaling and inflammasome-related pathways are highlighted in psoriatic epidermis

Psoriatic skin differs distinctly from normal skin by its thickened epidermis. Most gene expression comparisons utilize full-thickness biopsies, with substantial amount of dermis. We assayed the transcriptomes of normal, lesional, and non-lesional psoriatic epidermis, sampled as split-thickness skin grafts, with 5′-end RNA sequencing. We found that psoriatic epidermis contains more mRNA per total RNA than controls, and took this into account in the bioinformatic analysis. The approach highlighted innate immunity-related pathways in psoriasis, including NOD-like receptor (NLR) signaling and inflammasome activation. We demonstrated that the NLR signaling genes NOD2, PYCARD, CARD6, and IFI16 are upregulated in psoriatic epidermis, and strengthened these findings by protein expression. Interestingly, PYCARD, the key component of the inflammasome, showed an altered expression pattern in the lesional epidermis. The profiling of non-lesional skin highlighted PSORS4 and mitochondrially encoded transcripts, suggesting that their gene expression is altered already before the development of lesions. Our data suggest that all components needed for the active inflammasome are present in the keratinocytes of psoriatic skin. The characterization of inflammasome pathways provides further opportunities for therapy. Complementing previous transcriptome studies, our approach gives deeper insight into the gene regulation in psoriatic epidermis.

Gene expression profiling of negative-pressure-treated skin graft donor site wounds

Negative-pressure wound therapy (NPWT) is widely used to improve skin wound healing. Although NPWT has been studied as a treatment for wound closure and healing, the molecular mechanisms explaining its therapeutic effects remain unclear. To investigate the effect of NPWT on gene expression, and to discover the genes most dominantly responding to this treatment during skin wound healing, we applied negative pressure on split-thickness skin graft donor sites from the first postoperative day (POD) to the seventh POD. Biopsies were collected from 4 NPWT-treated and 2 control patients. Two biopsy samples were taken from each patient: one from intact skin before graft harvesting, and one on the seventh POD from the donor site wound. Genome-wide microarrays were performed on all samples. Gene expression changes on the seventh POD were compared between NPWT and control patients, and were analyzed for statistical significance. In addition, we analyzed wound exudates for volume, and for concentrations of leukocytes, erythrocytes, and haemoglobin. NPWT induced major changes in gene expression during healing. These changes ranged from 10-fold induction to 27-fold suppression. The genes most induced were associated with cell proliferation and inflammation, and the most down-regulated genes were linked to epidermal differentiation. Our results provide the first insight into the molecular mechanisms behind NPWT, and suggest that NPWT enhances specific inflammatory gene expression at the acute phase associated with epithelial migration and wound healing. However, its continued use may inhibit epithelial differentiation.

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.

Challenging the Conventional Therapy: Emerging Skin Graft Techniques for Wound Healing.

Background: Split-thickness skin grafting is the current gold standard for treatment of major traumatic skin loss. However, split-thickness skin grafting is limited by donor-skin availability, especially in large burns. In addition, the donor-site wound is associated with pain and scarring. Multiple techniques have been developed in the past to overcome these limitations but have been unable to achieve clinical relevance. In this study, the authors examine the novel emerging skin grafting techniques, aiming to improve the utility of split-thickness skin grafting. Methods: An extensive literature review was conducted on PubMed, MEDLINE, and Google Scholar to look for new skin grafting techniques. Special focus was given to techniques with potential for large expansion ratio and decreased donor-site pain. Results: The new modalities of modified skin grafting technique, discussed in this article, include (1) Xpansion Micrografting System, (2) fractional skin harvesting, (3) epidermal suction blister grafting, and (4) ReCell technology. These techniques are able to achieve significantly increased expansion ratios compared with conventional split-thickness skin grafting and also have decreased donor-site morbidity. Conclusions: These techniques can be used separately or in conjunction with split-thickness skin grafting to overcome the associated pitfalls. Further studies and clinical trials are needed to define the utility of these procedures and where they fit into routine clinical practice.

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 .

Pixel Grafting: An Evolution of Mincing for Transplantation of Full-Thickness Wounds.

Background: Split-thickness skin grafting is the gold standard for treatment of major skin loss. This technique is limited by donor-site availability in large burn injuries. With micrografting, a technique where split-thickness skin graft is minced into 0.8 × 0.8-mm pieces, the authors have demonstrated an expansion ratio of 1:100 and healing comparable to that achieved with split-thickness skin grafting. Methods: In this study, the authors explore the regenerative potential of a skin graft by cutting split-thickness skin grafts to pixel size (0.3 × 0.3 mm) grafts. Wound healing was studied in full-thickness wounds in a porcine model by creating an incubator-like microenvironment using polyurethane wound chambers. Multiple wound healing parameters were used to study the outcome of pixel grafting and compare it to micrografting and nontransplanted wounds. Results: The authors’ results show that 0.3 × 0.3-mm pixel grafts remain viable and contribute to skin regeneration. The pixel graft–transplanted wounds demonstrated a faster reepithelialization rate, decreased wound contraction, and increased mechanical stability compared with nontransplanted wounds. The reepithelialization rates of the wounds were significantly increased with pixel grafting at day 6 after wounding compared with micrografting. Among the other wound healing parameters, there were no significant differences between wounds transplanted with pixel grafts and micrografts. Conclusions: Pixel grafting technique would address the most commonly encountered limitations of the split-thickness skin graft with the possibility of an even larger expansion ratio than micrografting. This technique is simple and fast and can be conducted in the operating room or in the clinic.

Drug delivery systems and materials for wound healing applications

Chronic, non-healing wounds place a significant burden on patients and healthcare systems, resulting in impaired mobility, limb amputation, or even death. Chronic wounds result from a disruption in the highly orchestrated cascade of events involved in wound closure. Significant advances in our understanding of the pathophysiology of chronic wounds have resulted in the development of drugs designed to target different aspects of the impaired processes. However, the hostility of the wound environment rich in degradative enzymes and its elevated pH, combined with differences in the time scales of different physiological processes involved in tissue regeneration require the use of effective drug delivery systems. In this review, we will first discuss the pathophysiology of chronic wounds and then the materials used for engineering drug delivery systems. Different passive and active drug delivery systems used in wound care will be reviewed. In addition, the architecture of the delivery platform and its ability to modulate drug delivery are discussed. Emerging technologies and the opportunities for engineering more effective wound care devices are also highlighted.

Evolution of skin grafting for treatment of burns: Reverdin pinch grafting to Tanner mesh grafting and beyond

BACKGROUND Skin grafting is the current standard care in the treatment of full thickness burns. It was first described around 1500 BC but the vast majority of advancements have been achieved over the past 200 years. METHODS An extensive literature review was conducted on Pubmed, Medline and Google Scholar researching the evolution of skin grafting techniques. The authors concentrated on the major landmarks of skin grafting and also provide an overview of ongoing research efforts in this field. RESULTS The major innovations of skin grafting include Reverdin pinch grafting, Ollier grafting, Thiersch grafting, Wolfe grafting, Padgett dermatome and modifications, Meek-wall microdermatome and Tanner mesh grafting. A brief description of the usage, advantages and limitations of each technique is included in the manuscript. CONCLUSIONS Skin grafting technique have evolved significantly over past 200 years from Reverdin pinch grafting to modern day meshed skin grafts using powered dermatome. Increasing the expansion ratio and improving the cosmetic and functional outcome are the main focus of ongoing skin grafting research and emerging techniques (such as Integra®, Recell®, Xpansion®) are showing promise.

Development of a precise experimental burn model

BACKGROUND Porcine wounds closely mimic human wounds and are often used experimentally in burn studies. Multiple burn devices have been reported but they rarely described precise amount of heat transfer and the burn devices generally have low and varying heat capacity resulting in significant and varying temperature drop. METHODS The authors developed a customized aluminum burn device with cork insulation and high heat capacity. A thermistor probe was embedded in the device to accurately measure the temperature of the aluminum. The burn injury was inflicted by preheating the burn device to 100°C and pressing on the dorsum of pig skin for different time points ranging from 5 to 30s using standardized force of 10N on the device. With the knowledge of the heat capacity of the aluminum block and the temperature drop, the amount of heat transferred can be calculated. RESULT The temperature drop was 0°C, 1°C, 2°C, 3°C and 5°C for a wound-device contact time of 5, 10, 15, 20 and 30s, respectively. The depths of injury at 72h after burn were 0.46mm, 0.82mm, 1.21mm, 1.61mm and 1.91mm at 5, 10, 15, 20 and 30s respectively. 3.1mm represented a full thickness burn. The depth of the burn wounds significantly correlated with the heat transferred per cm2 (correlation coefficient=0.96, p-value=0.03). CONCLUSION The authors describe a simple, standardized and reproducible animal burn model using a customized burn device. The high heat capacity ensures minimal temperature drop which minimizes the variability of heat transferred with a large temperature drop. The correlation between the heat transfer and the depth of injury can facilitate standardization of burn depths in future studies.