Microscopía electrónica con electrones secundarios y retrodispersos en el esmalte del diente fluorótico

Introducción: El objetivo del presente trabajo consistió en definir los patrones morfológicos del esmalte en dientes fluoróticos mediante la utilización de la Microscopía Electrónica de Barrido con electrones secundarios y retrodispersos. Material y Métodos: Se estudiaron 20 piezas dentarias permanentes de pacientes con fluorosis dental y 5 dientes controles. Todas las muestras fueron procesadas para su estudio morfológico. Resultados: Se distinguen tres zonas en dientes fluoróticos: zona externa, zona subsuperficial o lesión fluorótica y zona interna. La zona subsuperficial presenta un triple patrón morfológico característico. Discusión: Este estudio permite sistematizar patrones estructurales que en un futuro pueden constituir una base para el desarrollo de futuras estrategias reparativas en dientes fluoróticos.Introduction: The aim of the present study was to establish histological patterns of fluorotic teeth by scanning electron microscopy (SEM) using secondary and retrodispersive electrons. Material and Methods: We studied 20 permanent teeth belonging to pacients whith dental fluorosis and 5 control teeth. All samples were processed for morphological study and scanning electron microscopy (SEM). Results: We distinguished three areas in fluorotic teeth: external area, subsuperficial area (or fluorotic lesion) and internal area. Subsuperficial area showed three different types of morphological patterns, which were characteristic of fluorotic lesions. Discussion: This study provides systematic structural patterns which in the future can provide a basis for developing future strategies reparative in fluorotic teeth

Mineralization of human premolar occlusal fissures: a quantitative histochemical microanalysis

The mechanisms of cariogenesis in occlusal fissures remain elusive because of limited information about fissure structure and wall mineralization. The purpose of the present study was to determine the correlation between morphological patterns in occlusal fissures in human premolars and quantitative histochemical patterns of mineralization in the walls of these formations. We used scanning electron microscopy and quantitative X-ray microanalysis with the peak-tolocal background ratio method and microcrystalline calcium salts as standards. We distinguished three morphological patterns of fissures in scanning electron microscopic images. The wall of the fissures was less mineralized than the control enamel in all three types of fissures. Because the fissure walls are hypomineralized, we suggest that practicing dentists should take into account the degree of mineralization when they are preparing the fissures for the application of sealant.This work was partially supported by the Ministerio de Educación y Cultura (PB97-0840) and the Agencia Española de Cooperación Internacional (AECI)

Biofabrication of a Tubular Model of Human Urothelial Mucosa Using Human Wharton Jelly Mesenchymal Stromal Cells

Several models of bioartificial human urothelial mucosa (UM) have been described recently. In this study, we generated novel tubularized UM substitutes using alternative sources of cells. Nanostructured fibrin–agarose biomaterials containing fibroblasts isolated from the human ureter were used as stroma substitutes. Then, human Wharton jelly mesenchymal stromal cells (HWJSC) were used to generate an epithelial-like layer on top. Three differentiation media were used for 7 and 14 days. Results showed that the biofabrication methods used here succeeded in generating a tubular structure consisting of a stromal substitute with a stratified epithelial-like layer on top, especially using a medium containing epithelial growth and differentiation factors (EM), although differentiation was not complete. At the functional level, UM substitutes were able to synthesize collagen fibers, proteoglycans and glycosaminoglycans, although the levels of control UM were not reached ex vivo. Epithelial differentiation was partially achieved, especially with EM after 14 days of development, with expression of keratins 7, 8, and 13 and pancytokeratin, desmoplakin, tightjunction protein-1, and uroplakin 2, although at lower levels than controls. These results confirm the partial urothelial differentiative potential of HWJSC and suggest that the biofabrication methods explored here were able to generate a potential substitute of the human UM for future clinical use.CTS-115 Tissue Engineering Group and by the Spanish Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica, Ministry of Science and Innovation, Instituto de Salud Carlos III, grant FIS PI21/0981 (cofinanced by FEDER funds, European Union)

Development of a diagnostic algorithm in periodontal disease and identification of genetic expression patterns: A preliminary report

AbstractBackground/purposeTo identify genetic expression patterns that can be used to define an appropriate diagnostic algorithm of clinical use in periodontal disease.Materials and methodsTotal RNA was extracted from 13 samples corresponding to normal human gingiva (NHG) and human gingiva affected by periodontal disease (PDHG). A comprehensive gene expression analysis was carried out by microarray analysis using Affymetrix Human Genome U133 plus 2.0 oligonucleotide arrays.ResultsSixty-six probe sets (genes and expressed sequence tags – EST) overexpressed in all samples of one of the comparison groups, were used for the diagnostic algorithm. All samples, including an independent test sample, were correctly classified as normal or periodontally affected using the diagnostic algorithm. In addition, 2596 genes/EST were upregulated and 1542 genes/EST were downregulated in PDHG, with numerous gene functions impaired in PDHG, especially those related to the immune response, cell-cell junctions, and extracellular matrix remodeling.ConclusionOur study reveals differential gene expression profiles in NHG and PDHG. The proposed diagnostic algorithm could have clinical usefulness for differential diagnosis in periodontal disease

Histological and histochemical evaluation of human oral mucosa constructs developed by tissue engineering

Reconstruction of large oral mucosa defects is often challenging, since the shortage of healthy oral mucosa to replace the excised tissues is very common. In this context, tissue engineering techniques may provide a source of autologous tissues available for transplant in these patients. In this work, we developed a new model of artificial oral mucosa generated by tissue engineering using a fibrin-agarose scaffold. For that purpose, we generated primary cultures of human oral mucosa fibroblasts and keratinocytes from small biopsies of normal oral mucosa using enzymatic treatments. Then we determined the viability of the cultured cells by electron probe quantitative X-ray microanalysis, and we demonstrated that most of the cells in the primary cultures were alive and had high K/Na ratios. Once cell viability was determined, we used the cultured fibroblasts and keratinocytes to develop an artificial oral mucosa construct by using a fibrin-agarose extracellular matrix and a sequential culture technique using porous culture inserts. Histological analysis of the artificial tissues showed high similarities with normal oral mucosa controls. The epithelium of the oral substitutes had several layers, with desmosomes and apical microvilli and microplicae. Both the controls and the oral mucosa substitutes showed high suprabasal expression of cytokeratin 13 and low expression of cytokeratin 10. All these results suggest that our model of oral mucosa using fibrin-agarose scaffolds show several similarities with native human oral mucosa.This work was supported by the grants FIS 03/0141 and FIS 04/1306 from the Spanish National Ministry of Health (Instituto de Salud Carlos III) and by CM 2005/011 from Junta de Andalucía

Applications of Tissue Engineering in reparation of abdominal wall defects

En este trabajo se ha realizado una revisión bibliográfica de más de treinta artículos relevantes que han sido publicados en revistas científicas de impacto durante los últimos años con el objetivo de conocer la efectividad de las nuevas opciones terapéuticas en la reparación de defectos de la pared abdominal. La Ingeniería Tisular permite el desarrollo de nuevos sustitutos biológicos consistentes en matrices dérmicas acelulares sobre las que se cultivan células de diverso origen. Estas nuevas mallas biológicas no presentan las complicaciones habituales de las mallas artificiales que a día de hoy se utilizan en la clínica quirúrgica de forma rutinaria. Además de reducir la incidencia de complicaciones como son la infección con necesidad de retirada de la misma, falta de estímulo de la cicatrización o formación de seromas, estas nuevas opciones terapéuticas aportan beneficios adicionales como favorecer la angiogénesis, biocompatibilidad, y por tanto disminución del riesgo de infección. Existen varios tipos de matrices acelulares (humana, porcina, bovina,…) sin evidenciarse diferencias significativas entre ellas, siendo la más utilizada la humana. El cultivo de células sobre estas matrices ha demostrado mejores resultados que cuando se utiliza una matriz acelular. La colocación quirúrgica de la malla parece influir en el porcentaje de recidivas, habiéndose mostrado la colocación pre-peritoneal y la técnica de separación anatómica por componentes como las mejores. El principal problema que existe es que los estudios tienen un período de seguimiento corto, por lo que aún no se conocen los resultados a largo plazo de las mismas. A esto se añade el elevado coste de los biomateriales empleados, aún inviable en la práctica quirúrgica diaria. Por todo ello es fundamental una mayor investigación de los nuevos biomateriales para poder trasladar este tipo de opciones terapéuticas a la clínica, así como una adecuada selección del caso y el paciente cuando se decida utilizarlas.In this work we have reviewed more than thirty relevant issues recently published in scientific journal with impact factor in order to estimate the effectiveness of new therapeutic approaches in reparation of abdominal wall defects. Tissue Engineering can be used for the development of new biological substitutes consisting on acellular dermal matrix seeded with different types of cells. These new biological meshes do not present some complications that are relatively common when artificial polymeric meshes are used in actual surgical practice. Besides the decrease of incidences such as infection and need to removal, lack of healing signals, or seromas, these new therapeutic approaches lead to some additional benefits such as to induce angiogenesis, biocompatibility and, thus, decrease of infection risk. There are several types of acellular dermal matrix (human, porcine, bovine …) with no significant evidence among them, although the most used is humanderived matrix. Cell culture over these biomaterials has reported better results than using acellular matrix. Matrix localization can also modify the relapse rate. In this sense, pre-peritoneal localization and anatomical separation of components technique have been reported as the best techniques. The main concern about the included studies is a short-term following period after treatment, and thus, scarce data about long-term effectiveness of these therapeutic approaches. Furthermore, due to the high price of the used biomaterials, these therapies are unfeasible nowadays in daily surgical practice. In summary, it is necessary advanced research about these new biomaterials in order to translate them to daily practice, as well as an adequate diagnosis and selection of the patient to be treated

Effective use of mesenchymal stem cells in human skin substitutes generated by tissue engineering

Mesenchymal stem cells (MSCs) can differentiate toward epithelial cells and may be used as an alternative source for generation of heterotypical artificial human skin substitutes, thus, enhancing their development and translation potential to the clinic. The present study aimed at comparing four types of heterotypical human bioengineered skin generated using MSCs as an alternative epithelial cell source. Adipose-tissue-derived stem cells (ADSCs), dental pulp stem cells (DPSCs), Wharton’s jelly stem cells (WJSCs) and bone marrow stem cells (BMSCs) were used for epidermal regeneration on top of dermal skin substitutes. Heterotypic human skin substitutes were evaluated before and after implantation in immune-deficient athymic mice for 30 d. Histological and genetic studies were performed to evaluate extracellular matrix synthesis, epidermal differentiation and human leukocyte antigen (HLA) molecule expression. The four cell types differentiated into keratinocytes, as shown by the expression of cytokeratin 10 and filaggrin 30 d post-grafting; also, they induced dermal fibroblasts responsible for the synthesis of extracellular fibrillar and non-fibrillar components, in a similar way among each other. WJSCs and BMSCs showed higher expression of cytokeratin 10 and filaggrin, suggesting these cells were more prone to epidermal regeneration. The absence of HLA molecules, even when the epithelial layer was differentiated, supports the future clinical use of these substitutes – especially ADSCs, DPSCs and WJSCs – with low rejection risk. MSCs allowed the generation of bioengineered human skin substitutes with potential clinical usefulness. According to their epidermal differentiation potential and lack of HLA antigens, WJSCs should preferentially be used.Spanish Ministry of Economy and Competitiveness (Instituto de Salud Carlos III), FIS PI15/2048 (co-financed by ERDF-FEDER, European Union), award number AC17/00013 (NanoGSkin) by ISCIII thorough AES 2017 and within the EuroNanoMed framework and PE-0393-20

Successful development and clinical translation of a novel anterior lamellar artificial cornea

We thank the Andalusian Public Foundation Progress and Health, through the Andalusian Initiative for Advanced Therapies, for assuming the roles and responsibilities of sponsoring this clinical trial. We thank Dr. Manuel de la Rosa and Dr. Salvador Arias Santiago for providing insight and expertise that assisted the research.The datasets generated and/or analyzed during the current study are available in the Gene Expression Omnibus (GEO) public repository, ref. GSE86584 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE86584Blindness due to corneal diseases is a common pathology affecting up to 23 million individuals worldwide. The tissue‐engineered anterior human cornea, which is currently being tested in a Phase I/II clinical trial to treat severe corneal trophic ulcers with preliminary good feasibility and safety results. This bioartificial cornea is based on a nanostructured fibrin–agarose biomaterial containing human allogeneic stromal keratocytes and cornea epithelial cells, mimicking the human native anterior cornea in terms of optical, mechanical, and biological behavior. This product is manufactured as a clinical‐grade tissue engineering product, fulfilling European requirements and regulations. The clinical translation process included several phases: an initial in vitro and in vivo preclinical research plan, including preclinical advice from the Spanish Medicines Agency followed by additional preclinical development, the adaptation of the biofabrication protocols to a good manufacturing practice manufacturing process, including all quality controls required, and the design of an advanced therapy clinical trial. The experimental development and successful translation of advanced therapy medicinal products for clinical application has to overcome many obstacles, especially when undertaken by academia or SMEs. We expect that our experience and research strategy may help future researchers to efficiently transfer their preclinical results into the clinical settings.This study was supported by the Spanish National Plan for Scientific and Technical Research and Innovation (I + D + I) from the Spanish Ministry of Economy and Competitiveness (Carlos III Institute of Health), grants FIS PI14/0955 and FIS PI17/0391 (both cofinanced by ERDF‐FEDER, European Union); by the Spanish Ministry of Health, Social Policy and Equity, grant EC10‐285; and by preclinical research funds from the Regional Ministry of Health through the Andalusian Initiative for Advanced Therapies