Evaluating the QileX-RhE skin corrosion test for chemical subcategorization in accordance with OECD TG 431.

Skin corrosion testing is integral to evaluating the potential harm posed by chemicals, impacting regulatory decisions on safety, transportation, and labeling. Traditional animal testing methods are giving way to in vitro alternatives, such as reconstructed human epidermis (RhE) models, aligning with evolving ethical standards. This study evaluates the QileX-RhE test system's performance for chemical subcategorization within the OECD TG 431 framework. Results demonstrate its ability to differentiate subcategories, accurately predicting 83% of UN GHS Category 1A and 73% of UN GHS Category 1B/1C chemicals with 100% sensitivity in corrosive prediction. Additionally, this study provides a comprehensive assessment of the test method's performance by employing nuanced parameters such as positive predictive value (PPV), negative predictive value (NPV), post-test odds and likelihood rations, offering valuable insights into the applicability and effectiveness of the QileX-RhE test method.

Xeno-free approach for the expansion of human adipose derived mesenchymal stem cells for ocular therapies.

To restore corneal transparency and vision loss after an injury on the ocular surface, the use of human stem cells from different origins has been recently proposed. Mesenchymal stem cells (MSCs) seem to be an appropriate adult source of autologous stem cells due to their accessibility, high proliferation rate, and multipotent capacity. In this work, we developed a simple culture system to prepare a graft based on a fibrin membrane seeded with human MSCs. A commercial kit, PRGF Endoret®, was used to prepare both, the growth factors used as culture media supplement and the fibrin membrane grafts. Adipose-derived MSCs (Ad-MSCs) were expanded, characterised by flow cytometry and their multilineage differentiation potential confirmed by inducing adipogenesis, osteogenesis and chondrogenesis. Ad-MSCs seeded on the fibrin membranes were grafted onto athymic mice showing good biocompatibility with no adverse reactions observed during the follow up period. These findings support the assumption that a system in which all the biological components (cells, grow factors and carrier) are autologous, could potentially be used for future ex vivo expansion of Ad-MSCs to treat ocular conditions such as an inflammatory milieu, traumatic scars and loss of the regenerative capacity of the corneal epithelium that compromise the quality of vision.

Fibroblast activation and abnormal extracellular matrix remodelling as common hallmarks in three cancer-prone genodermatoses.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB), Kindler syndrome (KS) and xeroderma pigmentosum complementation group C (XPC) are three cancer-prone genodermatoses whose causal genetic mutations cannot fully explain, on their own, the array of associated phenotypic manifestations. Recent evidence highlights the role of the stromal microenvironment in the pathology of these disorders. OBJECTIVES: To investigate, by means of comparative gene expression analysis, the role played by dermal fibroblasts in the pathogenesis of RDEB, KS and XPC. METHODS: We conducted RNA-Seq analysis, which included a thorough examination of the differentially expressed genes, a functional enrichment analysis and a description of affected signalling circuits. Transcriptomic data were validated at the protein level in cell cultures, serum samples and skin biopsies. RESULTS: Interdisease comparisons against control fibroblasts revealed a unifying signature of 186 differentially expressed genes and four signalling pathways in the three genodermatoses. Remarkably, some of the uncovered expression changes suggest a synthetic fibroblast phenotype characterized by the aberrant expression of extracellular matrix (ECM) proteins. Western blot and immunofluorescence in situ analyses validated the RNA-Seq data. In addition, enzyme-linked immunosorbent assay revealed increased circulating levels of periostin in patients with RDEB. CONCLUSIONS: Our results suggest that the different causal genetic defects converge into common changes in gene expression, possibly due to injury-sensitive events. These, in turn, trigger a cascade of reactions involving abnormal ECM deposition and underexpression of antioxidant enzymes. The elucidated expression signature provides new potential biomarkers and common therapeutic targets in RDEB, XPC and KS. What's already known about this topic? Recessive dystrophic epidermolysis bullosa (RDEB), Kindler syndrome (KS) and xeroderma pigmentosum complementation group C (XPC) are three genodermatoses with high predisposition to cancer development. Although their causal genetic mutations mainly affect epithelia, the dermal microenvironment likely contributes to the physiopathology of these disorders. What does this study add? We disclose a large overlapping transcription profile between XPC, KS and RDEB fibroblasts that points towards an activated phenotype with high matrix-synthetic capacity. This common signature seems to be independent of the primary causal deficiency, but reflects an underlying derangement of the extracellular matrix via transforming growth factor-ß signalling activation and oxidative state imbalance. What is the translational message? This study broadens the current knowledge about the pathology of these diseases and highlights new targets and biomarkers for effective therapeutic intervention. It is suggested that high levels of circulating periostin could represent a potential biomarker in RDEB.

Two novel recessive mutations in KRT14 identified in a cohort of 21 Spanish families with epidermolysis bullosa simplex.

BACKGROUND: Basal epidermolysis bullosa simplex (EBS) is a group of blistering genodermatoses mostly caused by mutations in the keratin genes, KRT5 and KRT14. Recessive mutations represent about 5% of all EBS mutations, being common and specific in populations with high consanguinity, where affected patients show severe phenotypes. OBJECTIVES: To accomplish the first mutational analysis in patients of Spanish origin with EBS and to delineate a comprehensive genotype-phenotype correlation. METHODS: Twenty-one EBS families were analysed. Immunofluorescence mapping at the dermoepidermal junction level was performed on skin biopsies from patients. Mutation screening of the entire coding sequences of KRT5 and KRT14 in genomic DNA was assessed by polymerase chain reaction and direct sequencing. RESULTS: KRT5 or KRT14 causative mutations were identified in 18 of the 21 EBS families. A total of 14 different mutations were disclosed, of which 12 were dominant missense mutations and two truncating recessive mutations. Five of the 14 mutations were novel including three dominant in KRT5 (p.V186E, p.T321P and p.A428T) and two recessive in KRT14 (p.K116X and p.K250RfsX8). The two patients with EBS carrying homozygous recessive mutations were affected by severe phenotypes and belonged to consanguineous families. All five families with the EBS Dowling-Meara subtype carried recurrent mutations affecting the highly conserved ends of the α-helical rod domain of K5 and K14. The seven mutations associated with the localized EBS subtype were widely distributed along the KRT5 and KRT14 genes. Two families with mottled pigmentation carried the P25L mutation in KRT5, commonly associated with this subtype. CONCLUSIONS: This study further confirms the genotype-phenotype correlation established for EBS in other ethnic groups, and is the first in a Mediterranean country (excluding Israel). This study adds two novel recessive mutations to the worldwide record to date, which includes a total of 14 mutations. As in previous reports, the recessive mutations resulted in a lack of keratin K14, giving rise to a generalized and severe presentation.

Use of an autologous bioengineered composite skin in extensive burns: Clinical and functional outcomes. A multicentric study.

OBJECTIVE: We report clinical and functional outcomes obtained after application of an autologous bioengineered composite skin (ABCS) produced in a single Spanish tissue-engineering unit. MATERIALS/METHODS: Twenty-five burned patients treated with ABCS from 1999 to 2007 in five burn centres were included in the study. Mean age was 29 years (SD 11), with mean total body surface area (TBSA) burned being 74% (SD 17) and mean full-thickness injury of 61% (SD 19) of TBSA. RESULTS: The mean area initially engrafted with ABCS was 24% (SD 13) of TBSA, with a final take of 49% (SD 30, range 0-100%). ABCS achieved permanent coverage of a mean of 11% (SD 8) of TBSA. In subset analyses, lack of pre- and post-application wound bed infection and lack of serious acute systemic complications at the time of engraftment were significantly associated with better ABCS take. CONCLUSIONS: Final take obtained with ABCS could be improved with the use of non-cytotoxic topical antibiotics following engraftment. The use of plasma to prepare ABCS reduces production costs: cost-effectiveness ratio is not a limitation for its use. In terms of patient satisfaction, cosmetic/functional outcomes (general appearance, texture, flexibility, sensitivity and colour) of ABCS and split-thickness autografts are not different statistically.

Spinal and peripheral analgesic effects of the CB2 cannabinoid receptor agonist AM1241 in two models of bone cancer-induced pain.

BACKGROUND AND PURPOSE: The activation of CB(2) receptors induces analgesia in experimental models of chronic pain. The present experiments were designed to study whether the activation of peripheral or spinal CB(2) receptors relieves thermal hyperalgesia and mechanical allodynia in two models of bone cancer pain. EXPERIMENTAL APPROACH: NCTC 2472 osteosarcoma or B16-F10 melanoma cells were intratibially inoculated to C3H/He and C57BL/6 mice. Thermal hyperalgesia was assessed by the unilateral hot plate test and mechanical allodynia by the von Frey test. AM1241 (CB(2) receptor agonist), AM251 (CB(1) receptor antagonist), SR144528 (CB(2) receptor antagonist) and naloxone were used. CB(2) receptor expression was measured by Western blot. KEY RESULTS: AM1241 (0.3-10 mg.kg(-1)) abolished thermal hyperalgesia and mechanical allodynia in both tumour models. The antihyperalgesic effect was antagonized by subcutaneous, intrathecal or peri-tumour administration of SR144528. In contrast, the antiallodynic effect was inhibited by systemic or intrathecal, but not peri-tumour, injection of SR144528. The effects of AM1241 were unchanged by AM251 but were prevented by naloxone. No change in CB(2) receptor expression was found in spinal cord or dorsal root ganglia. CONCLUSIONS AND IMPLICATIONS: Spinal CB(2) receptors are involved in the antiallodynic effect induced by AM1241 in two neoplastic models while peripheral and spinal receptors participate in the antihyperalgesic effects. Both effects were mediated by endogenous opiates. The use of drugs that activate CB(2) receptors could be a useful strategy to counteract bone cancer-induced pain symptoms.

The first COL7A1 mutation survey in a large Spanish dystrophic epidermolysis bullosa cohort: c.6527insC disclosed as an unusually recurrent mutation.

BACKGROUND: Dystrophic epidermolysis bullosa (DEB) is a genodermatosis caused by mutations in COL7A1. The clinical manifestations are highly variable from nail dystrophy to life-threatening blistering, making early molecular diagnosis and prognosis of utmost importance for the affected families. Mutation identification is mandatory for prenatal testing. OBJECTIVES: To conduct the first mutational analysis of COL7A1 in a Spanish cohort, to assess mutation consequences at protein/mRNA level and to establish genotype-phenotype correlations. METHODS: Forty-nine Spanish patients with DEB were studied. Antigen mapping was performed on patient skin biopsies. COL7A1 mutation screening in genomic DNA was performed by polymerase chain reaction (PCR) and direct sequencing. Mutation consequences were determined by reverse transcriptase-PCR. RESULTS: Eight patients belonged to three unrelated families with dominant DEB. Forty-one were affected with recessive DEB (RDEB). Specifically, 27 displayed the severe generalized subtype, eight the other generalized subtype and six a localized phenotype (two pretibial, three acral and one inversa). Thirty-five mutations were identified, 20 of which are novel. The pathogenic mutation c.6527insC accounted for 46.3% of Spanish RDEB alleles. A consistent genotype-phenotype correlation was established. CONCLUSIONS: Although the COL7A1 database indicates that most DEB mutations are family specific, the pathogenic mutation c.6527insC was highly recurrent in our cohort. This level of recurrence for a single genetic defect has never previously been reported for COL7A1. Our findings are essential to the clinicians caring for patients with DEB in Spain and in the large population of Spanish descendants in Latin America. They also provide geneticists a molecular clue for a priority mutation screening strategy.

Cancellous bone homograft storage with aluminium-polyethylene bags.

In order to transport and cryopreserve human tissues, it is essential to have an easy-to-use recipient where tissues can be kept in sterile conditions. Here we show the results obtained by using Macopharma's tissue freezing bags, an aluminium-polyethylene multilayer bag, in our tissue bank of the Centro Comunitario de Sangre y Tejidos de Asturias. Five hundred and twenty-seven cancellous bone homografts were obtained from hospitals located 120 km around our Bank. The homografts were submitted to bacteriological controls and sent to our bank in these bags. They were stored at -70 degrees C and sent in dry ice to about 50 hospitals, where the tissue was bacteriologically controlled and grafted. Furthermore, the behaviour of these bags at -140 degrees C (vapour nitrogen) or -196 degrees C (liquid nitrogen) was tested. Our results indicate that Macopharma aluminium-polyethylene bags are suitable for the transporting and cryopreserving of cancellous bone homografts. These bags could also be used for keeping tissues in nitrogen containers.

[In vitro three-dimensional reconstruction of human bladder mucosa]. / Reconstrucción tridimensional in vitro de mucosa vesical humana.

OBJECTIVE: The purpose of this study is to apply the in vitro keratinocyte culture techniques and the tissue engineering principles to human urothelium, to reconstruct an in vitro three-dimensional human bladder mucosa, suitable for grafting. MATERIAL AND METHODS: Biopsy specimens of human bladder mucosa were obtained from patients undergoing suprapubic prostatectomy, in vitro cultured and finally, an immunohistochemical study was made. RESULTS: A three-dimensional in vitro tissue was obtained, composed of a bio-artificial submucosa (fibrin gel and fibroblast) where the uroepithelial cells were seeding. We used a biodegradable polyglycolic acid mesh to facilitate the tissue manipulation and implantation. An immature epithelium was obtained with a weak immunostaining to cytokeratins. The immunohistochemical study could not demonstrate the development of basement membrane. CONCLUSIONS: In vitro keratinocyte culture techniques could be applied to other epithelial tissues like the urothelium. We obtained a three-dimensional in vitro tissue suitable for grafting in a relatively short time, which needs the matrix interactions in order to mature.

Reconstrucción tridimensional in vitro de mucosa vesical humana / In vitro three-dimensional reconstruction of human bladder mucosa

Objetivo: Aplicar las técnicas de cultivo in vitro de queratinocitos así como los principios de la ingeniería tisular al epitelio urinario humano, con el fin de reconstruir tridimensionalmente una mucosa vesical humana in vitro, apta para trasplantar. Material y Métodos: Se obtuvieron muestras de mucosa vesical de pacientes programados para cirugía abierta de próstata, previo consentimiento de los mismos, las cuales fueron cultivadas in vitro, para proceder posteriormente al estudio histomorfológico de los tejidos obtenidos. Resultados: Se obtuvo un tejido tridimensional compuesto por una submucosa bioartificial a base de un gel de fibrina y fibroblastos, sobre la que descansan las células uroepiteliales, pudiendo utilizar una malla de ácido poliglicólico, que facilite la manipulación de la mucosa y el posterior injerto de la misma. El tejido obtenido tenía el aspecto de un epitelio inmaduro con muy escasa reacción a citoqueratinas, sin poderse demostrar inmunohistoquímicamente el desarrollo de una membrana basal. Conclusiones: Las técnicas de cultivo in vitro de queratinocitos son aplicables a otros epitelios, entre ellos el urotelio humano. En un periodo de tiempo relativamente corto se puede obtener un tejido in vitro tridimensional apto para trasplantar, precisando posiblemente de las interacciones con el lecho receptor para poder madurar

Objetive: The purpose of this study is to apply the in vitro keratinocyte culture techniques and the tissue engineering principles to human urothelium, to reconstruct an in vitro three-dimensional human bladder mucosa, suitable for grafting. Material and Methods: Biopsy specimens of human bladder mucosa were obtained from patients undergoing suprapubic prostatectomy, in vitro cultured and finally, an immunohistochemical study was made. Results: A three-dimensional in vitro tissue was obtained, composed of a bio-artificial submucosa (fibrin gel and fibroblast) where the uroepithelial cells were seeding. We used a biodegradable polyglycolic acid mesh to facilitate the tissue manipulation and implantation. An immature epithelium was obtained with a weak immunostaining to cytokeratins. The immunohistochemical study could not demonstrate the development of basement membrane. Conclusions: In vitro keratinocyte culture techniques could be applied to other epithelial tissues like the urothelium. We obtained a three-dimensional in vitro tissue suitable for grafting in a relatively short time, which needs the matrix interactions in order to mature

Estado actual de la ingeniería de tejidos en urología. Revisión de la literatura / Current state of the tissue engineering in urology. Revision of the literature

En la década de los 80 surge un nuevo campo de la medicina que aplica los principios del cultivo celular a polímeros sintéticos biodegradables de soporte con el fin de crear sustitutos biológicos autólogos que puedan mejorar, mantener o restaurar la función de órganos o tejidos dañados. La Ingeniería Tisular constituye una nueva disciplina en plena fase de desarrollo, especialmente en USA, con múltiples potenciales aplicaciones en las diferentes especialidades médicas. Nuestra especialidad no puede permanecer ajena al interés y esperanzador futuro suscitado por esta nueva ciencia. En el presente trabajo realizamos una amplia revisión bibliográfica en Medline con objeto de conocer los antecedentes, estado actual y las posibles aplicaciones futuras de la Ingeniería de Tejidos en Urología

In the eighties a new field of the medicine appears wich applies the principles of cellular cultivation to synthetic biodegradable polymers scaffolds with the purpose of creating autologous biological substitutes that could improve, maintain or restore the function of organs or damaged tissues. The Tissue Engineering constitutes a new discipline in full phase of development especially in USA, with multiple potential applications in several medical specialities. Our speciality can’t remain indifferent to interest and encouraging future originated by this new science. In this work we have made a wide bibliographical revision in the Medline to know the antecedents, current state and the possible future applications of Tissue Engineering in Urology

[Current status of tissue engineering in urology. Review of the literature]. / Estado actual de la ingeniería de tejidos en urología. Revisión de la literatura.

In the eighties a new field of the medicine appears wich applies the principles of cellular cultivation to synthetic biodegradable polymers scaffolds with the purpose of creating autologous biological substitutes that could improve, maintain or restore the function of organs or damaged tissues. The Tissue Engineering constitutes a new discipline in full phase of development especially in USA, with multiple potential applications in several medical specialities. Our speciality can't remain indifferent to interest and encouraging future originated by this new science. In this work we have made a wide bibliographical revision in the Medline to know the antecedents, current state and the possible future applications of Tissue Engineering in Urology.

Tratamiento de úlceras vasculares crónicas con equivalentes cutáneos obtenidos mediante ingeniería tisular / Treatment of chronic vascular ulcers with skin equivalents obtained by tissue engineering

Objetivo. Valorar la efectividad de un equivalente cutáneo diseñado en un banco de tejidos, en el tratamiento de úlceras vasculares crónicas. Pacientes y métodos. Entre septiembre de 1999 y diciembre de 2001 se incluyó a 25 pacientes con úlceras vasculares de evolución tórpida (>4 meses) en quienes, tras ingreso hospitalario y tratamiento estándar, se objetivó un estancamiento en su cicatrización. Se constataron nueve úlceras venosas, dos arteriales, siete hipertensivas y siete mixtas (componente flebostático y arterial). Descartada la infección clínica y bacteriológica de la úlcera, los injertos se colocaron una vez a la semana hasta la cicatrización o suspensión del tratamiento por no respuesta. Se realizaron controles fotográficos digitales semanales para medir el área de las lesiones. A partir del segundo injerto, el tratamiento se realizó ambulatoriamente. El seguimiento medio fue de 18 meses (intervalo: 6-30 meses). Se valoró la ausencia de rechazo, la tasa de cicatrización, el tiempo de reducción del área al 50 y 75%, y la recidiva ulcerosa. Resultados. No se detectaron signos de rechazo. Tasa global de cicatrización del 80% (20/25); en hipertensivas y mixtas, tasa del 100% (14/14). Tiempo medio de cierre: 5,3 semanas (intervalo: 3-12 semanas). Número medio de injertos utilizados: 5,8 (intervalo: 3-13 injertos). La recidiva ulcerosa fue del 25% (cuatro venosas y una hipertensiva). De las cinco úlceras que no cicatrizaron (cuatro venosas y una arterial), ninguna empeoró: en tres no hubo respuesta y en dos se redujo el área más de un 85%. Conclusiones. Los equivalentes cutáneos podrían constituir una buena alternativa en el tratamiento de úlceras vasculares crónicas (AU)

Aims. To assess the effectiveness of a skin equivalent designed in a tissue bank in the treatment of chronic vascular ulcers. Patients and methods. Between September 1999 and December 2001, 25 patients with vascular ulcers that progressed in a torpid fashion (>4 months) were admitted to hospital and administered standard therapy, following which sluggish healing processes were observed. Nine venous, two arterial, seven hypertensive and seven mixed ulcers (the latter with a phlebostatic and arterial component) were found. After ruling out the possibility of the ulcer being affected by clinical and bacteriological infections, the grafts were placed once a week until healing had been achieved or the treatment was suspended due to lack of response. Weekly digital photographic monitoring was carried out to measure the extension of the lesions. From the second graft onwards, patients were treated in the outpatients department. Average follow-up time was 18 months (interval: 6-30 months). Values measured included the absence of rejection, healing rate, the time required to reduce the extension to 50 and 75%, and recurrence of ulcers. Results. No signs of rejection were detected. Overall healing rate was 80% (20/25); in hypertensive and mixed cases, the rate was 100% (14/14). Average closing time: 5.3 weeks (interval: 3-12 weeks). Average number of grafts used: 5.8 (interval: 3-13 grafts). Recurrence of ulcers was 25% (four venous and one hypertensive). Of the five ulcers that did not heal (four venous and one arterial), none of them got worse: there was no response in three cases and two of them showed a reduction of the extension that reached 85%. Conclusions. Skin equivalents could be a good alternative in the treatment of chronic vascular ulcers (AU)