In-stent restenosis is associated with proliferative skin healing and specific immune and endothelial cell profiles: results from the RACHEL trial.

Introduction: In-stent restenosis (ISR) is a major challenge in interventional cardiology. Both ISR and excessive skin healing are aberrant hyperplasic responses, which may be functionally related. However, the cellular component underlying ISR remains unclear, especially regarding vascular homeostasis. Recent evidence suggest that novel immune cell populations may be involved in vascular repair and damage, but their role in ISR has not been explored. The aims of this study is to analyze (i) the association between ISR and skin healing outcomes, and (ii) the alterations in vascular homeostasis mediators in ISR in univariate and integrative analyses. Methods: 30 patients with ≥1 previous stent implantation with restenosis and 30 patients with ≥1 stent without restenosis both confirmed in a second angiogram were recruited. Cellular mediators were quantified in peripheral blood by flow cytometry. Skin healing outcomes were analyzed after two consecutive biopsies. Results: Hypertrophic skin healing was more frequent in ISR patients (36.7%) compared to those ISR-free (16.7%). Patients with ISR were more likely to develop hypertrophic skin healing patterns (OR 4.334 [95% CI 1.044-18.073], p=0.033), even after correcting for confounders. ISR was associated with decreased circulating angiogenic T-cells (p=0.005) and endothelial progenitor cells (p<0.001), whereas CD4+CD28null and detached endothelial cells counts were higher (p<0.0001 and p=0.006, respectively) compared to their ISR-free counterparts. No differences in the frequency of monocyte subsets were found, although Angiotensin-Converting Enzyme expression was increased (non-classical: p<0.001; and intermediate: p<0.0001) in ISR. Despite no differences were noted in Low-Density Granulocytes, a relative increase in the CD16- compartment was observed in ISR (p=0.004). An unsupervised cluster analysis revealed the presence of three profiles with different clinical severity, unrelated to stent types or traditional risk factors. Conclusion: ISR is linked to excessive skin healing and profound alterations in cellular populations related to vascular repair and endothelial damage. Distinct cellular profiles can be distinguished within ISR, suggesting that different alterations may uncover different ISR clinical phenotypes.

Driving role of head and neck cancer cell secretome on the invasion of stromal fibroblasts: Mechanistic insights by phosphoproteomics.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are major players in tumor-stroma communication, and participate in several cancer hallmarks to drive tumor progression and metastatic dissemination. This study investigates the driving effects of tumor-secreted factors on CAF biology, with the ultimate goal of identifying effective therapeutic targets/strategies for head and neck squamous cell carcinomas (HNSCC). METHODS: Functionally, conditioned media (CM) from different HNSCC-derived cell lines and normal keratinocytes (Kc) were tested on the growth and invasion of populations of primary CAFs and normal fibroblasts (NFs) using 3D invasion assays in collagen matrices. The changes in MMPs expression were evaluated by RT-qPCR and kinase enrichment was analyzed using mass spectrometry phosphoproteomics. RESULTS: Our results consistently demonstrate that HNSCC-secreted factors (but not Kc CM) specifically and robustly promoted pro-invasive properties in both CAFs and NFs, thereby reflecting the plasticity of fibroblast subtypes. Concomitantly, HNSCC-secreted factors massively increased metalloproteinases levels in CAFs and NFs. By contrast, HNSCC CM and Kc CM exhibited comparable growth-promoting effects on stromal fibroblasts. Mechanistically, phosphoproteomic analysis predominantly revealed phosphorylation changes in fibroblasts upon treatment with HNSCC CM, and various promising kinases were identified: MKK7, MKK4, ASK1, RAF1, BRAF, ARAF, COT, PDK1, RSK2 and AKT1. Interestingly, pharmacologic inhibition of RAF1/BRAF using sorafenib emerged as the most effective drug to block tumor-promoted fibroblast invasion without affecting fibroblast viability CONCLUSIONS: Our findings demonstrate that HNSCC-secreted factors specifically fine tune the invasive potential of stromal fibroblasts, thereby generating tumor-driven pro-invasive niches, which in turn to ultimately facilitate cancer cell dissemination. Furthermore, the RAF/BRAF inhibitor sorafenib was identified as a promising candidate to effectively target the onset of pro-invasive clusters of stromal fibroblasts in the HNSCC microenvironment.

Improved Tool for Predicting Skin Irritation on Reconstructed Human Epidermis Models Based on Electrochemical Impedance Spectroscopy.

The rabbit skin irritation test has been the standard for evaluating the irritation potential of chemicals; however, alternative methods that do not use animal testing are actively encouraged. Reconstructed human epidermis (RhE) models mimic the biochemical and physiological properties of the human epidermis and can be used as an alternative method. On RhE methods, the metabolic activity of RhE models is used to predict skin irritation, with a reduction in metabolic activity indicating a reduced number of viable cells and linking cell death to skin irritation processes. However, new challenges have emerged as the use of RhE models increases, including the need for non-invasive and marker-free methodologies to assess cellular states. Electrochemical impedance spectroscopy (EIS) is one such methodology that can meet these requirements. In this study, our results showed that EIS can differentiate between irritant and non-irritant chemicals, with a significant increase in the capacitance values observed in the irritant samples. A ROC curve analysis showed that the prediction method based on EIS met OECD TG 439 requirements at all time points and had 95% within-laboratory reproducibility. Comparison with the MTT viability assay showed that prediction using EIS achieved higher sensitivity, specificity, and accuracy. These results suggest that EIS could potentially replace animal testing in the evaluation of irritation potential and could be a valuable addition to in vitro testing strategies.

Polymerizable Skin Hydrogel for Full Thickness Wound Healing.

The skin is the largest organ in the human body, comprising the main barrier against the environment. When the skin loses its integrity, it is critical to replace it to prevent water loss and the proliferation of opportunistic infections. For more than 40 years, tissue-engineered skin grafts have been based on the in vitro culture of keratinocytes over different scaffolds, requiring between 3 to 4 weeks of tissue culture before being used clinically. In this study, we describe the development of a polymerizable skin hydrogel consisting of keratinocytes and fibroblast entrapped within a fibrin scaffold. We histologically characterized the construct and evaluated its use on an in vivo wound healing model of skin damage. Our results indicate that the proposed methodology can be used to effectively regenerate skin wounds, avoiding the secondary in vitro culture steps and thus, shortening the time needed until transplantation in comparison with other bilayer skin models. This is achievable due to the instant polymerization of the keratinocytes and fibroblast combination that allows a direct application on the wound. We suggest that the polymerizable skin hydrogel is an inexpensive, easy and rapid treatment that could be transferred into clinical practice in order to improve the treatment of skin wounds.

DNA Repair and Immune Response Pathways Are Deregulated in Melanocyte-Keratinocyte Co-cultures Derived From the Healthy Skin of Familial Melanoma Patients.

Familial melanoma accounts for 10% of cases, being CDKN2A the main high-risk gene. However, the mechanisms underlying melanomagenesis in these cases remain poorly understood. Our aim was to analyze the transcriptome of melanocyte-keratinocyte co-cultures derived from healthy skin from familial melanoma patients vs. controls, to unveil pathways involved in melanoma development in at-risk individuals. Accordingly, primary melanocyte-keratinocyte co-cultures were established from the healthy skin biopsies of 16 unrelated familial melanoma patients (8 CDKN2A mutant, 8 CDKN2A wild-type) and 7 healthy controls. Whole transcriptome was captured using the SurePrint G3 Human Microarray. Transcriptome analyses included: differential gene expression, functional enrichment, and protein-protein interaction (PPI) networks. We identified a gene profile associated with familial melanoma independently of CDKN2A germline status. Functional enrichment analysis of this profile showed a downregulation of pathways related to DNA repair and immune response in familial melanoma (P < 0.05). In addition, the PPI network analysis revealed a network that consisted of double-stranded DNA repair genes (including BRCA1, BRCA2, BRIP1, and FANCA), immune response genes, and regulation of chromosome segregation. The hub gene was BRCA1. In conclusion, the constitutive deregulation of BRCA1 pathway genes and the immune response in healthy skin could be a mechanism related to melanoma risk.

Efficient CRISPR-Cas9-Mediated Gene Ablation in Human Keratinocytes to Recapitulate Genodermatoses: Modeling of Netherton Syndrome.

Current efforts to find specific genodermatoses treatments and define precise pathogenesis mechanisms require appropriate surrogate models with human cells. Although transgenic and gene knockout mouse models for several of these disorders exist, they often fail to faithfully replicate the clinical and histopathological features of the human skin condition. We have established a highly efficient method for precise deletion of critical gene sequences in primary human keratinocytes, based on CRISPR-Cas9-mediated gene editing. Using this methodology, in the present study we generated a model of Netherton syndrome by disruption of SPINK5. Gene-edited cells showed absence of LEKTI expression and were able to recapitulate a hyperkeratotic phenotype with most of the molecular hallmarks of Netherton syndrome, after grafting to immunodeficient mice and in organotypic cultures. To validate the model as a platform for therapeutic intervention, we tested an ex vivo gene therapy approach using a lentiviral vector expressing SPINK5. Re-expression of SPINK5 in an immortalized clone of SPINK5-knockout keratinocytes was capable of reverting from Netherton syndrome to a normal skin phenotype in vivo and in vitro. Our results demonstrate the feasibility of modeling genodermatoses, such as Netherton syndrome, by efficiently disrupting the causative gene to better understand its pathogenesis and to develop novel therapeutic approaches.

Development of an in-house reconstructed human epidermis model as an alternative method in skin corrosion assessment.

Defining the corrosive properties of chemical products generally involves the use of animal models for human health safety assessment. However, a few alternatives to animal experimentation are currently internationally accepted in order to reduce animal suffering. One of these alternatives makes use of in vitro reconstructed human epidermis (RhE) models and predicts corrosive potential based on the evaluation of cell viability after topical exposure. These models rely on its similarity to human skin, both functional and histological, and are currently worldwide marketed by a few private companies. In this manuscript, we describe the fundamentals of the production of a Do It Yourself (DIY) RhE model, and the operating procedures for the assessment of skin corrosion based on the guidelines proposed for the development of new alternative methods for skin corrosion. Our results indicate that the DIY-RhE model resembles the anatomy of the normal human epidermis as seen by immunohistochemical analysis. Moreover, barrier properties of DIY-RhE were assessed by the measure of Transepithelial Electrical Resistance. Applicability of DIY-RhE for the assessment of skin corrosion was evaluated by measuring cell viability after topical exposure of twelve reference chemicals for 3 and 60 min. Predictive performance resulted in 100% sensitivity, 100% specificity and 100% accuracy matching current requirements for new RhE models proposed for the discrimination of corrosives and non-corrosives.

Transcriptomic Analysis of a Diabetic Skin-Humanized Mouse Model Dissects Molecular Pathways Underlying the Delayed Wound Healing Response.

Defective healing leading to cutaneous ulcer formation is one of the most feared complications of diabetes due to its consequences on patients' quality of life and on the healthcare system. A more in-depth analysis of the underlying molecular pathophysiology is required to develop effective healing-promoting therapies for those patients. Major architectural and functional differences with human epidermis limit extrapolation of results coming from rodents and other small mammal-healing models. Therefore, the search for reliable humanized models has become mandatory. Previously, we developed a diabetes-induced delayed humanized wound healing model that faithfully recapitulated the major histological features of such skin repair-deficient condition. Herein, we present the results of a transcriptomic and functional enrichment analysis followed by a mechanistic analysis performed in such humanized wound healing model. The deregulation of genes implicated in functions such as angiogenesis, apoptosis, and inflammatory signaling processes were evidenced, confirming published data in diabetic patients that in fact might also underlie some of the histological features previously reported in the delayed skin-humanized healing model. Altogether, these molecular findings support the utility of such preclinical model as a valuable tool to gain insight into the molecular basis of the delayed diabetic healing with potential impact in the translational medicine field.

Human plasma gels: Their preparation and rheological characterization for cell culture applications in tissue engineering.

Tissue engineering is one of the fields of clinical medicine that has forged ahead in recent years, especially because of its role as a potential alternative to organ transplantation. The main aim of this study has been the development of biocompatible materials to form extracellular matrix (ECM) structures in order to provide the necessary conditions for the settlement, proliferation and differentiation of dermal cells such as fibroblasts. To this end, human plasma gels were synthesized with the addition of increasing concentrations of transglutaminase (TGase), which catalyses the formation of covalent bonds between Lys and Glu residues. These materials were structurally characterized using rheology and texturometry and were found to have good structural resistance and elasticity for fibroblast culture. A remarkable improvement in the mechanical properties of the human plasma gels was detected when the two highest TGase concentrations were tested, which may be interpreted as an increase in the number of covalent and non-covalent bonds formed between the plasma protein chains. Furthermore, a human fibroblast primary culture was seeded on human plasma scaffolds and satisfactorily proliferated at 37 °C. This was verified in the images obtained by optical microscopy (OM) and by scanning electron microscopy (SEM), which confirmed that the structure of this type of material is suitable for the growth and proliferation of dermal fibroblasts.

Deletion of a Pathogenic Mutation-Containing Exon of COL7A1 Allows Clonal Gene Editing Correction of RDEB Patient Epidermal Stem Cells.

Recessive dystrophic epidermolysis bullosa is a severe skin fragility disease caused by loss of functional type VII collagen at the dermal-epidermal junction. A frameshift mutation in exon 80 of COL7A1 gene, c.6527insC, is highly prevalent in the Spanish patient population. We have implemented gene-editing strategies for COL7A1 frame restoration by NHEJ-induced indels in epidermal stem cells from patients carrying this mutation. TALEN nucleases designed to cut within the COL7A1 exon 80 sequence were delivered to primary patient keratinocyte cultures by non-integrating viral vectors. After genotyping a large collection of vector-transduced patient keratinocyte clones with high proliferative potential, we identified a significant percentage of clones with COL7A1 reading frame recovery and Collagen VII protein expression. Skin equivalents generated with cells from a clone lacking exon 80 entirely were able to regenerate phenotypically normal human skin upon their grafting onto immunodeficient mice. These patient-derived human skin grafts showed Collagen VII deposition at the basement membrane zone, formation of anchoring fibrils, and structural integrity when analyzed 12 weeks after grafting. Our data provide a proof-of-principle for recessive dystrophic epidermolysis bullosa treatment through ex vivo gene editing based on removal of pathogenic mutation-containing, functionally expendable COL7A1 exons in patient epidermal stem cells.

Ectopic bone formation during tissue-engineered cartilage repair using autologous chondrocytes and novel plasma-derived albumin scaffolds.

PURPOSE: The aims of this study were twofold: first, to evaluate the production of cartilaginous tissue in vitro and in vivo using a novel plasma-derived scaffold, and second, to test the repair of experimental defects made on ears of New Zealand rabbits (NZr) using this approach. MATERIALS AND METHODS: Scaffolds were seeded with chondrocytes and cultured in vitro for 3 months to check in vitro cartilage production. To evaluate in vivo cartilage production, a chondrocyte-seeded scaffold was transplanted subcutaneously to a nude mouse. To check in vivo repair, experimental defects made in the ears of five New Zealand rabbits (NZr) were filled with chondrocyte-seeded scaffolds. RESULTS: In vitro culture produced mature chondrocytes with no extracellular matrix (ECM). Histological examination of redifferentiated in vitro cultures showed differentiated chondrocytes adhered to scaffold pores. Subcutaneous transplantation of these constructs to a nude mouse produced cartilage, confirmed by histological study. Experimental cartilage repair in five NZr showed cartilaginous tissue repairing the defects, mixed with calcified areas of bone formation. CONCLUSION: It is possible to produce cartilaginous tissue in vivo and to repair experimental auricular defects by means of chondrocyte cultures and the novel plasma-derived scaffold. Further studies are needed to determine the significance of bone formation in the samples.

Identification of two rare and novel large deletions in ITGB4 gene causing epidermolysis bullosa with pyloric atresia.

Epidermolysis bullosa with pyloric atresia (EB-PA) is a rare autosomal recessive hereditary disease with a variable prognosis from lethal to very mild. EB-PA is classified into Simplex form (EBS-PA: OMIM #612138) and Junctional form (JEB-PA: OMIM #226730), and it is caused by mutations in ITGA6, ITGB4 and PLEC genes. We report the analysis of six patients with EB-PA, including two dizygotic twins. Skin immunofluorescence epitope mapping was performed followed by PCR and direct sequencing of the ITGB4 gene. Two of the patients presented with non-lethal EB-PA associated with missense ITGB4 gene mutations. For the other four, early postnatal demise was associated with complete lack of ß4 integrin due to a variety of ITGB4 novel mutations (2 large deletions, 1 splice-site mutation and 3 missense mutations). One of the deletions spanned 278 bp, being one of the largest reported to date for this gene. Remarkably, we also found for the first time a founder effect for one novel mutation in the ITGB4 gene. We have identified 6 novel mutations in the ITGB4 gene to be added to the mutation database. Our results reveal genotype-phenotype correlations that contribute to the molecular understanding of this heterogeneous disease, a pivotal issue for prognosis and for the development of novel evidence-based therapeutic options for EB management.

Feeder Layer Cell Actions and Applications.

Cultures of growth-arrested feeder cells have been used for years to promote cell proliferation, particularly with low-density inocula. Basically, feeder cells consist in a layer of cells unable to divide, which provides extracellular secretions to help another cell to proliferate. It differs from a coculture system because only one cell type is capable to proliferate. It is known that feeder cells support the growth of target cells by releasing growth factors to the culture media, but this is not the only way that feeder cells promote the growth of target cells. In this work, we discuss the different mechanisms of action of feeder cells, tackling questions as to why for some cell cultures the presence of feeder cell layers is mandatory, while in some other cases, the growth of target cells can be achieved with just a conditioned medium. Different treatments to avoid feeder cells to proliferate are revised, not only the classical treatments as mitomycin or γ-irradiation but also the not so common treatments as electric pulses or chemical fixation. Regenerative medicine has been gaining importance in recent years as a discipline that moves biomedical technology from the laboratory to the patients. In this context, human stem and pluripotent cells play an important role, but the presence of feeder cells is necessary for these progenitor cells to grow and differentiate. This review addresses recent specific applications, including those associated to the growth of embryonic and induced pluripotent stem cells. In addition, we have also dealt with safety issues, including feeder cell sources, as major factors of concern for clinical applications.

Tissue-engineered oral mucosa grafts for intraoral lining reconstruction of the maxilla and mandible with a fibula flap.

PURPOSE: Many types of soft tissue grafts have been used for grafting or prelaminating bone flaps for intraoral lining reconstruction. The best results are achieved when prelaminating free flaps with mucosal grafts. We suggest a new approach to obtain keratinized mucosa over a fibula flap using full-thickness, engineered, autologous oral mucosa. PATIENTS AND METHODS: We report on a pilot study for grafting fibula flaps for mandibular and maxilla reconstruction with full-thickness tissue-engineered autologous oral mucosa. We describe 2 different techniques: prelaminating the fibula flap and second-stage grafting of the fibula after mandibular reconstruction. Preparation of the full-thickness tissue-engineered oral mucosa is also described. RESULTS: The clinical outcome of the tissue-engineered intraoral lining reconstruction and response after implant placement are reported. A peri-implant granulation tissue response was not observed when prelaminating the fibula, and little response was observed when intraoral grafting was performed. CONCLUSION: Tissue engineering represents an alternative method by which to obtain sufficient autologous tissue for reconstructing mucosal oral defects. The full-thickness engineered autologous oral mucosa offers definite advantages in terms of reconstruction planning, donor site morbidity, and quality of the intraoral soft tissue reconstruction, thereby restoring native tissue and avoiding peri-implant tissue complications.

Tissue-engineered oral mucosa for mucosal reconstruction in a pediatric patient with hemifacial microsomia and ankyloglossia.

Many types of soft tissue grafts have been used for the reconstruction of oral mucosal defects. The best results are achieved with mucosal grafts; however, when large areas must be grafted, sufficient donor tissue is not available. Tissue engineering represents an alternative method to obtain sufficient autologous tissue for reconstructing oral wounds. Herein we present a pediatric patient with hemifacial microsomia and congenital ankyloglossia requiring multiple surgical interventions, and in which an autologous full-thickness tissue-engineered oral mucosa was used for successful oral reconstruction. Our study demonstrates that even under challenging conditions, robust tissue-engineered products, such as the fibrin-based oral mucosa described here, can achieve successful tissue regeneration.

Keratinocyte cell lines derived from severe generalized recessive epidermolysis bullosa patients carrying a highly recurrent COL7A1 homozygous mutation: models to assess cell and gene therapies in vitro and in vivo.

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by deficiency of type VII collagen due to COL7A1 mutations such as c.6527insC, recurrently found in the Spanish RDEB population. Assessment of clonal correction-based therapeutic approaches for RDEB requires large expansions of cells, exceeding the replication capacity of human primary keratinocytes. Thus, immortalized RDEB cells with enhanced proliferative abilities would be valuable. Using either the SV40 large T antigen or papillomavirus HPV16-derived E6-E7 proteins, we immortalized and cloned RDEB keratinocytes carrying the c.6527insC mutation. Clones exhibited high proliferative and colony-forming features. Cytogenetic analysis revealed important differences between T antigen-driven and E6-E7-driven immortalization. Immortalized cells responded to differentiation stimuli and were competent for epidermal regeneration and recapitulation of the blistering RDEB phenotype in vivo. These features make these cell lines useful to test novel therapeutic approaches including those aimed at editing mutant COL7A1.

The regenerative potential of fibroblasts in a new diabetes-induced delayed humanised wound healing model.

Cutaneous diabetic wounds greatly affect the quality of life of patients, causing a substantial economic impact on the healthcare system. The limited clinical success of conventional treatments is mainly attributed to the lack of knowledge of the pathogenic mechanisms related to chronic ulceration. Therefore, management of diabetic ulcers remains a challenging clinical issue. Within this context, reliable animal models that recapitulate situations of impaired wound healing have become essential. In this study, we established a new in vivo humanised model of delayed wound healing in a diabetic context that reproduces the main features of the human disease. Diabetes was induced by multiple low doses of streptozotocin in bioengineered human-skin-engrafted immunodeficient mice. The significant delay in wound closure exhibited in diabetic wounds was mainly attributed to alterations in the granulation tissue formation and resolution, involving defects in wound bed maturation, vascularisation, inflammatory response and collagen deposition. In the new model, a cell-based wound therapy consisting of the application of plasma-derived fibrin dermal scaffolds containing fibroblasts consistently improved the healing response by triggering granulation tissue maturation and further providing a suitable matrix for migrating keratinocytes during wound re-epithelialisation. The present preclinical wound healing model was able to shed light on the biological processes responsible for the improvement achieved, and these findings can be extended for designing new therapeutic approaches with clinical relevance.

Clinical outcomes after the use of complete autologous oral mucosa equivalents: preliminary cases.

OBJECTIVE: Previously, we reported how to obtain complete autologous oral mucosa equivalents (CAOMEs) composed of an autologous plasma scaffold and fibroblasts together with immature keratinocytes able to build an oral epithelium with a structure similar to that of the oral mucosa. In this study, we present the clinical outcomes after applying our CAOMEs as grafts. STUDY DESIGN: Four patients who needed a CAOME to restore a defect of oral mucosa were selected. Two of the patients suffered from ankyloglossia, and the other 2 required a restoration of the keratinized gum of the alveolar rim. To assess the outcomes, the scale designed by Ewers et al. was used. RESULTS: Clinical and functional improvements were achieved in the patients with ankyloglossia. In cases of gum restoration, the mucosa was regenerated and a prosthetic restoration with implants was achieved. CONCLUSIONS: The results obtained points to the potential use of CAOME in intraoral lining.