Back to the future: from appendage development toward future human hair follicle neogenesis

Hair disorders such as alopecia and hirsutism often impact the social and psychological well-being of an individual. This also holds true for patients with severe burns who have lost their hair follicles (HFs). HFs stimulate proper wound healing and prevent scar formation; thus, HF research can benefit numerous patients. Although hair development and hair disorders are intensively studied, human HF development has not been fully elucidated. Research on human fetal material is often subject to restrictions, and thus development, disease, and wound healing studies remain largely dependent on time-consuming and costly animal studies. Although animal experiments have yielded considerable and useful information, it is increasingly recognized that significant differences exist between animal and human skin and that it is important to obtain meaningful human models. Human disease specific models could therefore play a key role in future therapy. To this end, hair organoids or hair-bearing skin-on-chip created from the patient's own cells can be used. To create such a complex 3D structure, knowledge of hair genesis, i.e., the early developmental process, is indispensable. Thus, uncovering the mechanisms underlying how HF progenitor cells within human fetal skin form hair buds and subsequently HFs is of interest. Organoid studies have shown that nearly all organs can be recapitulated as mini-organs by mimicking embryonic conditions and utilizing the relevant morphogens and extracellular matrix (ECM) proteins. Therefore, knowledge of the cellular and ECM proteins in the skin of human fetuses is critical to understand the evolution of epithelial tissues, including skin appendages. This review aims to provide an overview of our current understanding of the cellular changes occurring during human skin and HF development. We further discuss the potential implementation of this knowledge in establishing a human in vitro model of a full skin substitute containing hair follicles and the subsequent translation to clinical use.Thrombosis and Hemostasi

Resultado de las quemaduras tratadas mediante cultivo de células epidérmicas autólogas:reglamentos e implicaciones para ensayos clínicos internacionales con Productos de Terapia Avanzada

Introducción. Los Productos de Terapia Avanzada (ATMPs) son considerados el siguiente paso para mejorar la evolución de las quemaduras. Sin embargo, existen serios obstáculos para su aplicabilidad con respecto al reglamento internacional vigente. Aparte de las exigencias reglamentarias para los ensayos clínicos, la medición de los resultados ha llegado a ser importante para la investigación clínica. Objetivo. Investigamos un producto ATMP que contiene células epidérmicas autólogas para la mejora de la cicatrización de heridas en grandes quemados. Método. Se realizó un estudio prospectivo, aleatorizado y multicéntrico en 40 pacientes adultos con lesiones agudas por quemaduras de espesor total. El tratamiento experimental consistió en injertos de piel de espesor pacial (SSG) en combinación con células epidérmicas autólogas cultivadas, sembradas en un medio de colágeno, que es un producto ATMP. Esto se comparó con el tratamiento estándar, que era SSG solamente. El objetivo primario obtenido fue el cierre de la herida después de 5-7 días. Los objetivos secundarios fueron los aspectos de seguridad y calidad de la cicatriz medida por la captación del injerto en el lecho de la herida, la puntuación de la cicatriz (POSAS), la colorimetría de la piel (Dermatoespectrómetro) y la elasticidad (Cutómetro). Resultados. Cumplimos con las normativas existentes: una sala limpia para el cultivo, el permiso de Inspección de Sanidad, sistemas de calidad, el dictamen ético del Comité Central Holandés etc. La epitelización de la herida después de 5-7 días fue significativamente mejor con el tratamiento ATMP (71%) en comparación con el tratamiento estándar (67%) (p = 0,034, Wilcoxon), mientras que las tasas de captación de los injertos fueron similares. La calidad de la cicatriz según la evaluación de POSAS, a los 3 y 12 meses después de la quemadura, mostró mejores resultados para las heridas tratadas con células epidérmicas en cuanto al enrojecimiento, la pigmentación, el grosor, el alivio y la flexibilidad, con diferencias entre el 12 y el 23% (p≤0.010, Wilcoxon ). Esto fue confirmado por los datos obtenidos de las mediciones de color y elasticidad. Discusión. La relevancia de las células autólogos cultivadas en el resultado de la cicatriz tras sufrir quemaduras extensas está claramente apoyado por nuestros actuales resultados. Encontramos obstáculos importantes en la aplicabilidad de la reglamentación y del derecho internacional, que plantean complicaciones y falta de uniformidad internacional en relación con el estado de los productos ATMP

SPS-neutralization in tissue samples for efficacy testing of antimicrobial peptides

Background: Accurate determination of the efficacy of antimicrobial agents requires neutralization of residual antimicrobial activity in the samples before microbiological assessment of the number of surviving bacteria. Sodium polyanethol sulfonate (SPS) is a known neutralizer for the antimicrobial activity of aminoglycosides and polymyxins. In this study, we evaluated the ability of SPS to neutralize residual antimicrobial activity of antimicrobial peptides [SAAP-148 and pexiganan; 1% (wt/v) in PBS], antibiotics [mupirocin (Bactroban) and fusidic acid (Fucidin) in ointments; 2% (wt/wt))] and disinfectants [2% (wt/wt) silver sulfadiazine cream (SSD) and 0.5% (v/v) chlorhexidine in 70% alcohol].Methods: Homogenates of human skin models that had been exposed to various antimicrobial agents for 1 h were pipetted on top of Methicillin-resistant Staphylococcus aureus (MRSA) on agar plates to determine whether the antimicrobial agents display residual activity.To determine the optimal concentration of SPS for neutralization, antimicrobial agents were mixed with PBS or increasing doses of SPS in PBS (0.05-1% wt/v) and then 105 colony forming units (CFU)/mL MRSA were added. After 30min incubation, the number of viable bacteria was assessed. Next, the in vitro efficacy of SAAP-148 against various gram-positive and gram-negative bacteria was determined using PBS or 0.05% (wt/v) SPS immediately after 30 min incubation of the mixture. Additionally, ex vivo excision wound models were inoculated with 105 CFU MRSA for 1 h and exposed to SAAP-148, pexiganan, chlorhexidine or PBS for 1 h. Subsequently, samples were homogenized in PBS or 0.05% (wt/v) SPS and the number of viable bacteria was assessed.Results: All tested antimicrobials displayed residual activity in tissue samples, resulting in a lower recovery of surviving bacteria on agar. SPS concentrations at >= 0.05% (wt/v) were able to neutralize the antimicrobial activity of SAAP-148, pexiganan and chlorhexidine, but not of SSD, Bactroban and Fucidin. Finally, SPS-neutralization in in vitro and ex vivo efficacy tests of SAAP-148, pexiganan and chlorhexidine against gram-positive and gram-negative bacteria resulted in significantly higher numbers of CFU compared to control samples without SPS-neutralization.Conclusions: SPS was successfully used to neutralize residual activity of SAAP-148, pexiganan and chlorhexidine and this prevented an overestimation of their efficacy.Immunogenetics and cellular immunology of bacterial infectious disease

Extraction of osteocalcin from fossil bones and teeth

Osteocalcin (also called ‘bone Gla-protein’) was detected in fossil bovid bones ranging from 12 000 years to 13 million years old and in rodent teeth 30 million years old. Both the antigenic activity and the protein-bound Gla-residues have remained intact. The protein is indistinguishable from recent bovine osteocalcin when analyzed by HPLC using ion exchange and size exclusion columns. If sufficient amounts can be extracted and an adequate purification procedure is established, this would be the first time that amino acid sequences in a protein from fossil bones may be determined. Such sequence data could offer a new aproach to the phylogenetic study of extinct taxa

The number of immune cells is lower in healthy oral mucosa compared to skin and does not increase after scarring

Objective Depending on the location of injury, wounds can heal with different outcomes. In addition foetal wounds heal fast without scar formation, while scars are a common feature of regular skin repair. Since inflammation is very limited in these wounds reduced numbers or even absence of immune cells might be responsible for scarless foetal wound healing. It is thought that various immune cells, such as macrophages, neutrophils and T-cells, play a role in aberrant wound healing and the fibrotic process seen in scar formation in the adult skin. Similar to the foetus, oral wounds show comparable healing properties by means of accelerated reepithelialization and negligible scar formation. It is possible that reduced inflammatory reaction as a result of lower numbers of immune cells are present in oral wounds compared to skin wounds. Design Here we investigated the presence of various immune cells in human skin and oral mucosa, with or without scars. The presence or absence of these cells may play a role in the different modes of healing observed between the two types of tissue. Mast cells, neutrophils, M1/M2 macrophages, T-cells and blood vessels were localized in healthy and scarred skin and oral mucosa (scars >1 year old). Results Oral mucosa had significantly fewer neutrophils, macrophages, mannose receptor-positive M2 macrophages, but more blood vessels. Scars contained similar numbers of immune cells compared to healthy tissues. Conclusions Less immune cells in the healthy oral mucosa may induce a diminished immune reaction when wounding occurs, and could explain the better healing capacity of the oral mucosa