Recombinant human collagen III gel for transplantation of autologous skin cells in porcine full-thickness wounds.

Complex skin wounds, such as chronic ulcers and deep burns, require lengthy treatments and cause extensive burdens on healthcare and the economy. Use of biomaterials and cell transplantation may improve traditional treatments and promote the healing of difficult-to-treat wounds. In this study, we investigated the use of recombinant human collagen III (rhCol-III) gel as a delivery vehicle for cultured autologous skin cells (keratinocytes only or keratinocyte-fibroblast mixtures). We examined its effect on the healing of full-thickness wounds in a porcine wound-healing model. Two Landrace pigs were used for the study. Fourteen deep dermal wounds were created on the back of each pig with an 8 mm biopsy punch. Syringes containing acellular rhCol-III gel (n = 8) or rhCol-III gel with autologous keratinocytes (n = 8) or rhCol-III gel with autologous keratinocytes and fibroblasts (n = 8) were applied into wounds. Untreated wounds were used as controls for the treatment groups (n = 4). We used rhCol-III gel to manufacture a cell-delivery syringe containing autologous skin cells. In a full-thickness wound-healing model, we observed that rhCol-III gel enhances early granulation tissue formation. Interestingly, we found cell type-dependent differences in the stability of rhCol-III in vivo. Fibroblast-containing gel was effectively removed from the wound, whereas gels without cells or with keratinocytes only remained intact. Our results demonstrate that the properties of rhCol-III gel for skin cell transplantation can be significantly altered in a cell type-dependent manner.

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.

Human skin transcriptome during superficial cutaneous wound healing.

Healing of the epidermis is a crucial process for maintaining the skin's 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.

Improved skin wound epithelialization by topical delivery of soluble factors from fibroblast aggregates.

INTRODUCTION: Timely coverage of an excised burn wound with a split-thickness skin graft, and efficient epithelialization at the donor site wound are key components in the treatment of burn patients. Prompt healing is dependent on paracrine support from underlying dermal connective tissue fibroblasts. STUDY AIM: Using the skin graft donor site in pig as a model for epithelialization, our aim was to evaluate if dermal signals, derived from cultured dermal fibroblast aggregates (Finectra), can promote epidermal regeneration. MATERIALS AND METHODS: Partial-thickness skin wounds were made with a dermatome on the backs of three domestic pigs. After randomization, topical treatment was initiated by application of Finectra (n=6) or factors from standard fibroblast monolayer cultures (n=6) trapped in a slow-clotting fibrin matrix. Saline was applied to contralateral wounds to serve as corresponding untreated controls (n=12). After 3 days, full-thickness skin samples representing the whole wound area were obtained. Histological sections of these samples were analyzed for epithelialization, cell migration from lateral wound edges and hair follicles, as well as for formation of granulation tissue. RESULTS: In response to topical delivery of Finectra, a significant acceleration of epithelialization (p<0.001) across the wound surface as well as from the wound edges was evident. Marked increase in thickness of granulation tissue (p<0.001) was noted in wounds treated with Finectra. Epihelialization originated from adnexal structures in which epithelial islets showed positive staining for cytokeratin-14 and PCNA. CONCLUSION: These data show that the fibroblast aggregate-derived paracrine mediators, Finectra, stimulate epidermal regeneration in vivo.

Paracrine factors from fibroblast aggregates in a fibrin-matrix carrier enhance keratinocyte viability and migration.

Efficient re-epithelialization of skin lesions is dependent on paracrine support from connective tissue fibroblasts. In deep skin defects, the supporting growth factor incentive is lacking. Current methods of keratinocyte transplantation with compromised attachment, spread, and cell proliferation warrant improvement and refinement. We describe here how human keratinocytes can be stimulated by matrix-embedded factors from a novel process of fibroblast activation: nemosis. Interestingly, the unique set of mediators released in this process also plays a key role in normal wound healing. To develop a system for targeted delivery of nemosis-derived paracrine effectors, herein designated as Finectra, we combined them with fibrin to establish a controlled-release gel. Keratinocytes seeded to cover this active matrix showed better adherence, outgrowth, and viability than did cells on control matrix. The matrix incorporating Finectra supported viability of both primary keratinocytes and green fluorescent protein (GFP)-labeled HaCaT cells, as evaluated by MTT assay and persistence of GFP-fluorescence. The fibrin-Finectra matrix promoted migration of keratinocytes to cover a larger area on the matrix, suggesting better wound coverage on transplantation. An inhibitor of EGFR/c-Met receptor tyrosine kinases abolished keratinocyte responses to fibrin-Finectra matrix. This matrix can thus deliver biologically relevant synergistic stimuli to keratinocytes and hasten re-epithelialization.

Bone marrow mesenchymal stem cells undergo nemosis and induce keratinocyte wound healing utilizing the HGF/c-Met/PI3K pathway.

We previously showed cell-cell contacts of human dermal fibroblasts to induce expression of the hepatocyte growth factor/scatter factor (HGF) in a process designated as nemosis. Now we report on nemosis initiation in bone marrow mesenchymal stem cells (BMSCs). Because BMSCs are being used increasingly in cell transplantation therapy we aimed to demonstrate a functional effect and benefit of BMSC nemosis for wound healing. Nemotic and monolayer cells were used to stimulate HaCaT keratinocyte migration in a scratch-wound healing assay. Both indicators of nemosis, HGF production and cyclooxygenase-2 expression, were induced in BMSC spheroids. When compared with a similar amount of cells as monolayer, nemotic cells induced keratinocyte in vitro scratch-wound healing in a concentration-dependent manner. The HGF receptor, c-Met, was rapidly phosphorylated in the nemosis-stimulated keratinocytes. Nemosis-induced in vitro scratch-wound healing was inhibited by an HGF-neutralizing antibody as well as the small molecule c-Met inhibitor, SU11274. HGF-induced in vitro scratch-wound healing was inhibited by PI3K inhibitors, wortmannin and LY294002, while LY303511, an inactive structural analogue of LY294002, had no effect. Inhibitors of the mitogen-activated protein kinases MEK/ERK1/2 (PD98059 and U0126), and p38 (SB203580) attenuated HGF-induced keratinocyte in vitro scratch-wound healing. We conclude that nemosis of BMSCs can induce keratinocyte in vitro scratch-wound healing, and that in this effect signaling via HGF/c-Met is involved.