Gene expression profiling of human skin donor site wound healing to guide novel regenerative therapies

Information on the molecular details of human skin wound healing has been limited due to several types of challenges faced when data from basic research are adapted for use in clinical studies. Comprehensive genomewide data on gene behaviour during the various phases of wound healing have not been available, although many techniques, such as gene expression microarrays and RNA sequencing would, have made it possible. The aim of my doctoral thesis was to examine human skin regeneration and to develop ways to enhance wound healing, especially in burn wound treatment. The main goal was to characterize the gene expression transcriptome of human donor site wounds over time. Skin biopsies were collected from patients before and after split-thickness skin graft harvesting. The first biopsy of intact skin was obtained immediately before the site was wounded by the graft harvest. Immediately thereafter, a second biopsy was performed to serve as a sample an of acute wound. Later, the next biopsies were obtained on the 3rd, 7th, 14th and 21st postoperative days. The skin graft donor site wound was chosen because it is controllably made in the operating room and represents a normal, uncomplicated, and consistently healing superficial skin wound. Negative pressure was applied to some of the donor sites to clarify the effects of this widely used therapy on gene expression in a healing wound. Moreover, this approach enabled the initial evaluation of the gene expression profiling principle for clinical research purposes. The tissue samples collected were homogenized, and ribonucleic acid (RNA) was isolated. Thereafter, genomewide microarrays were performed on all samples. The gene profiles at different time points were compared, and data were presented as fold-change alterations in gene expression. As a result, the data reveals various types of gene expression patterns relating to, loss of expression due to tissue removal, down-regulation or induction of expression as the wound-healing cascade advances or regain of expression according to tissue regeneration. The results also give molecular level information on the effects of negative-pressure wound therapy. These studies provide the first insights into the transcriptome during normal human superficial cutaneous wound healing. The data reveal novel genes associated with epidermal wound healing. Moreover, they provide a fundamental background for future studies that can be utilized in the clinical evaluation of the effects of therapies.Väitöskirjatutkimukseni tarkoituksena on tutkia ihmisen ihon uudistumisen mekanismeja ja ihon paranemisen tehostamista erityisesti palovammojen hoidossa. Tutkimukseni motivaationa on ollut se, että kliinisen haavatutkimuksen tekeminen potilailla on erittäin hankalaa, koska haavan paranemisen merkkigeenejä ei tunneta ja ihon regeneraation kliininen arviointi on subjektiivista. Lisäksi vaikka haavan parantumiseen liittyviä mekanismeja on tutkittu jo vuosikymmeniä, on suuri osa tiedoista kertynyt erilaisista eläinmalleista. Eläinmalleilla on tärkeä osa tutkimuksessa, mutta niistä kertyneen tiedon varaukseton käyttö ihmisen tauti- ja regeneraatiomekanismien selittämisessä on johtanut eriasteisten virhearviointien syntyyn esim. lääkekehityksessä. Väitöskirjani kolmessa ensimmäisessä osatyössä tutkin potilasaineistosta miten ihmisgenomin geenit ilmentyvät haavan parantumisen eri vaiheissa. Tutkimusta varten kerättiin kudosnäytteitä palovammapotilaiden ihonsiirteiden ottokohtahaavoista eri aikapisteissä. Näytteiden RNA:sta analysoitiin mikrosirumenetelmää käyttäen koko genomi, ja osa geenien ilmentymislöydöksistä varmennettiin polymeraasiketjureaktiomenetelmällä. Myös kokeellinen haavatutkimus on oleellinen osa väitöskirjatutkimustani. Tässä työssä käytän erilaisia soluviljely- ja koe-eläinmalleja. Eläinmallien avulla on kokeiltu solusiirtoja ja biomateriaaleja sekä näiden yhdistämistä erilaisten ihohaavojen hoitoon. Neljännessä osatyössä tutkin ihmisen rekombinantin kollageenin (rhCol-III) soveltuvuutta syvien ihohaavojen hoitoon sekä mahdollisuutta siirtää ihosoluja haavaan kollageenigeelin sisällä

The Immune and Regenerative Response to Burn Injury

Burn are diverse and complex injuries that not only have local effects but also serious systemic consequences through severe and prolonged inflammatory response. They are caused by heat, electricity, friction, chemicals, or radiation and are commonly divided into superficial, superficial partial-, deep partial- and full-thickness injuries. The severity of the burn depends mainly on the size and depth of the injury but also on location, age, and underlying systemic diseases. A prolonged and strong immune response makes major burns even worse by causing multiple systemic effects including damage to the heart, lungs, blood vessels, kidneys, and other organs. Burns that do not require surgical excision, superficial and superficial partial-thickness, follow the known progression of wound healing (inflammation, proliferation, remodeling), whilst deep partial- and full thickness injuries requiring excision and grafting do not. For these burns, intervention is required for optimal coverage, function, and cosmesis. Annually millions of people worldwide suffer from burns associated with high morbidity and mortality. Fortunately, over the past decades, burn care has significantly improved. The improvement in understanding the pathophysiology of burn injury and burn wound progression has led to developments in skin grafting, fluid resuscitation, infection control and nutrition This review article focuses on the immune and regenerative responses following burn injury. In the Introduction, we describe the epidemiology of burns and burn pathophysiology. The focus of the following chapter is on systemic responses to burn injury. Next, we define the immune response to burns introducing all the different cell types involved. Subsequently, we discuss the regenerative cell response to burns as well as some of the emerging novel treatments in the battle against burns

The Immune and Regenerative Response to Burn Injury

Burn are diverse and complex injuries that not only have local effects but also serious systemic consequences through severe and prolonged inflammatory response. They are caused by heat, electricity, friction, chemicals, or radiation and are commonly divided into superficial, superficial partial-, deep partial- and full-thickness injuries. The severity of the burn depends mainly on the size and depth of the injury but also on location, age, and underlying systemic diseases. A prolonged and strong immune response makes major burns even worse by causing multiple systemic effects including damage to the heart, lungs, blood vessels, kidneys, and other organs. Burns that do not require surgical excision, superficial and superficial partial-thickness, follow the known progression of wound healing (inflammation, proliferation, remodeling), whilst deep partial- and full thickness injuries requiring excision and grafting do not. For these burns, intervention is required for optimal coverage, function, and cosmesis. Annually millions of people worldwide suffer from burns associated with high morbidity and mortality. Fortunately, over the past decades, burn care has significantly improved. The improvement in understanding the pathophysiology of burn injury and burn wound progression has led to developments in skin grafting, fluid resuscitation, infection control and nutrition This review article focuses on the immune and regenerative responses following burn injury. In the Introduction, we describe the epidemiology of burns and burn pathophysiology. The focus of the following chapter is on systemic responses to burn injury. Next, we define the immune response to burns introducing all the different cell types involved. Subsequently, we discuss the regenerative cell response to burns as well as some of the emerging novel treatments in the battle against burns

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

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. 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. 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. 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.Peer reviewe

In vivo printing of growth factor-eluting adhesive scaffolds improves wound healing

Acute and chronic wounds affect millions of people around the world, imposing a growing financial burden on patients and hospitals. Despite the application of current wound management strategies, the physiological healing process is disrupted in many cases, resulting in impaired wound healing. Therefore, more efficient and easy-to-use treatment modalities are needed. In this study, we demonstrate the benefit of in vivo printed, growth factor-eluting adhesive scaffolds for the treatment of full-thickness wounds in a porcine model. A custom-made handheld printer is implemented to finely print gelatin-methacryloyl (GelMA) hydrogel containing vascular endothelial growth factor (VEGF) into the wounds. In vitro and in vivo results show that the in situ GelMA crosslinking induces a strong scaffold adhesion and enables printing on curved surfaces of wet tissues, without the need for any sutures. The scaffold is further shown to offer a sustained release of VEGF, enhancing the migration of endothelial cells in vitro. Histological analyses demonstrate that the administration of the VEGF-eluting GelMA scaffolds that remain adherent to the wound bed significantly improves the quality of healing in porcine wounds. The introduced in vivo printing strategy for wound healing applications is translational and convenient to use in any place, such as an operating room, and does not require expensive bioprinters or imaging modalities

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

Background: CCHCR1 (Coiled Coil alpha-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 centiosomal piotein, as well as a component of cytoplasmic piocessmg bodies (P-bodies) that regulate mRNA turnovel. 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 seveial 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 piesent findings may explain the previous inconsistent obseivations about the functions of CCHCR1.Peer reviewe

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.Peer reviewe

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.Peer reviewe

Topical Drug Delivery in the Treatment of Skin Wounds and Ocular Trauma Using the Platform Wound Device

Topical treatment of injuries such as skin wounds and ocular trauma is the favored route of administration. Local drug delivery systems can be applied directly to the injured area, and their properties for releasing therapeutics can be tailored. Topical treatment also reduces the risk of adverse systemic effects while providing very high therapeutic concentrations at the target site. This review article highlights the Platform Wound Device (PWD) (Applied Tissue Technologies LLC, Hingham, MA, USA) for topical drug delivery in the treatment of skin wounds and eye injuries. The PWD is a unique, single-component, impermeable, polyurethane dressing that can be applied immediately after injury to provide a protective dressing and a tool for precise topical delivery of drugs such as analgesics and antibiotics. The use of the PWD as a topical drug delivery platform has been extensively validated in the treatment of skin and eye injuries. The purpose of this article is to summarize the findings from these preclinical and clinical studies