Search

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., (Copyright © 2013 John Wiley & Sons, Ltd.)

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.

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.

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.

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. [ABSTRACT FROM AUTHOR]

Autologous split-thickness skin grafts (STSGs) are the standard of care for closure of deep and large burns. However, perforation and extensive fishnet-like expansion of the grafts to achieve greater area wound coverage can lead to treatment failures or esthetically poor healing outcomes and scarring. The purpose of this study was to validate an autologous advanced therapy medicinal product (ATMP)-compliant skin cell suspension and evaluate its efficacy to promote epithelialization. Cells isolated from a piece of STSG according to ATMP classification requirements were sprayed onto 20 patients during a single operation in a validation study. Comparative evaluation of treatment efficacy was carried out using side-by-side skin graft donor site wounds that were standardized in depth. Firstly, we characterized wound healing transcriptomes at 14 and 21 days from serial wound biopsies in seven patients. Then, side-by-side wounds in four patients were treated with or without the skin cells. The wounds were photographed, clinical outcomes assessed, and the treatment and control wound transcriptomes at 14 days were compared to the untreated wounds' healing transcriptomes. The average cell yield after isolation from the STSG was 2.4 × 106 cells/cm2 with 96 % viability. The product contained mainly keratinocytes and their precursors but also other skin cells such as fibroblasts were present. As compared to vehicle-treated donor site wounds, the wounds treated with cells demonstrated improved epithelialization by both direct comparison and machine learning analysis of the transcriptomes. We showed that rapid and scalable ATMP-classified processing of skin cells is feasible, and application of the skin cells effectively promotes healing and epithelization of donor site wounds. • This study introduces a scalable and GMP-compliant method of rapid cell isolation from a STSG and its spray-on application on injury site. • Production of this advanced therapy medicinal product (ATMP)-classified cell spray is fast allowing application during the same operation. • Standardized Split-thickness skin graft (STSG) donor sites were utilized as study wounds since they enable harvesting of small biopsies over time for molecular level analysis • This study reports the validation and clinical use of this product for the treatment of skin wounds. • The results show that this treatment promotes wound healing as evaluated both clinically and on gene expression level. [ABSTRACT FROM AUTHOR]