Histological characterization of the human masticatory oral mucosa. A histochemical and immunohistochemical study.

BACKGROUND: Histology of human oral mucosa is closely related with its function and anatomical location, and a proper characterization of the human masticatory oral mucosa could be very useful in periodontal pathology. OBJECTIVE: In the present work, we have carried out a comprehensive study in order to determine the main histological features of parakeratinized (POM) and orthokeratinized (OOM) masticatory human oral mucosa using light and electron microscopy. METHODS: To perform this, we have used several histological, histochemical and immunohistochemical methods to detect key markets at the epithelial, basement membrane and connective tissue levels. RESULTS: Our results demonstrated that POM and OOM share many histological similarities, as expected. However, important differences were observed at the epithelial layer of POM, that was significantly thicker than the epithelial layer found in OOM, especially due to a higher number of cells at the stratum spinosum. The expression pattern of CK10 and filaggrin revealed intense signal expression in OOM as compared to POM. Collagen and proteoglycans were more abundant in OOM stroma than in POM. No differences were found for blood vessels and basement membrane. CONCLUSION: These results may contribute to a better understanding of the pathological conditions affecting the human masticatory oral mucosa. In addition, these findings could be useful for the generation of different types of oral mucosa by tissue engineering techniques. RESEARCH HIGHLIGHTS: Microscopical features of parakeratinized and orthokeratinized masticatory human oral mucosa showed important differences at both, epithelial and stromal levels. Parakeratinized masticatory human oral mucosa exert thicker epithelial layer, especially, at the stratum spinosum in comparison to orthokeratinized human oral mucosa. Cytokeratin 10 and filaggrin human epithelial markers were intensively expressed in orthokeratinized masticatory human oral mucosa in comparison to parakeratinized masticatory human oral mucosa. At the stromal level, orthokeratinized masticatory human oral mucosa exhibit higher levels of collagen and proteoglycans than parakeratinized masticatory oral mucosa. The deep knowledge of histological features of masticatory oral mucosa could lead to a better understanding of oral mucosa pathology and advanced treatments.

Histological Profiling of the Human Umbilical Cord: A Potential Alternative Cell Source in Tissue Engineering.

The embryonic development of the human umbilical cord (hUC) is complex, and different regions can be identified in this structure. The aim of this work is to characterize the hUC at in situ and ex vivo levels to stablish their potential use in vascular regeneration. Human umbilical cords were obtained and histologically prepared for in the situ analysis of four hUC regions (intervascular-IV, perivascular-PV, subaminoblastic-SAM, and Wharton's jelly-WH), and primary cell cultures of mesenchymal stem cells (hUC-MSC) isolated from each region were obtained. The results confirmed the heterogeneity of the hUC, with the IV and PV zones tending to show the higher in situ expression of several components of the extracellular matrix (collagens, proteoglycans, and glycosaminoglycans), vimentin, and MSC markers (especially CD73), although isolation and ex vivo culture resulted in a homogeneous cell profile. Three vascular markers were positive in situ, especially vWF, followed by CD34 and CD31, and isolation and culture revealed that the region associated with the highest expression of vascular markers was IV, followed by PV. These results confirm the heterogeneity of the hUC and the need for selecting cells from specific regions of the hUC for particular applications in tissue engineering.

Generation of a novel model of bioengineered human oral mucosa with increased vascularization potential.

OBJECTIVE: The aim of this study was to generate novel models of bioartificial human oral mucosa with increased vascularization potential for future use as an advanced therapies medicinal product, by using different vascular and mesenchymal stem cell sources. BACKGROUND: Oral mucosa substitutes could contribute to the clinical treatment of complex diseases affecting the oral cavity. Although several models of artificial oral mucosa have been described, biointegration is a major issue that could be favored by the generation of novel substitutes with increased vascularization potential once grafted in vivo. METHODS: Three types of mesenchymal stem cells (MSCs) were obtained from adipose tissue, bone marrow, and dental pulp, and their in vitro potential was evaluated by inducing differentiation to the endothelial lineage using conditioning media. Then, 3D models of human artificial oral mucosa were generated using biocompatible fibrin-agarose biomaterials combined with human oral mucosa fibroblasts and each type of MSC before and after induction to the endothelial lineage, using human umbilical vein endothelial cells (HUVEC) as controls. The vascularization potential of each oral mucosa substitute was assessed in vitro and in vivo in nude mice. RESULTS: In vitro induction of MSCs kept in culture was able to increase the expression of VEGF, CD31, and vWF endothelial markers, especially in bone marrow and dental pulp-MSCs, and numerous proteins with a role in vasculogenesis become overexpressed. Then, in vivo grafting resulted in a significant increase in blood vessels formation at the interface area between the graft and the host tissues, with significantly positive expression of VEGF, CD31, vWF, and CD34 as compared to negative controls, especially when pre-differentiated MSCs derived from bone marrow and dental pulp were used. In addition, a significantly higher number of cells committed to the endothelial lineage expressing the same endothelial markers were found within the bioartificial tissue. CONCLUSION: Our results suggest that the use of pre-differentiated MSCs could contribute to a rapid generation of a vascular network that may favor in vivo biointegration of bioengineered human oral mucosa substitutes.

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, tight-junction 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.

Erratum: Blanco-Elices, C. et al. In Vitro Generation of Novel Functionalized Biomaterials for Use in Oral and Dental Regenerative Medicine Applications. Running Title: Fibrin-Agarose Functionalized Scaffolds. Materials 2020, 13, 1692.

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In Vitro Generation of Novel Functionalized Biomaterials for Use in Oral and Dental Regenerative Medicine Applications. Running Title: Fibrin-Agarose Functionalized Scaffolds.

Recent advances in tissue engineering offer innovative clinical alternatives in dentistry and regenerative medicine. Tissue engineering combines human cells with compatible biomaterials to induce tissue regeneration. Shortening the fabrication time of biomaterials used in tissue engineering will contribute to treatment improvement, and biomaterial functionalization can be exploited to enhance scaffold properties. In this work, we have tested an alternative biofabrication method by directly including human oral mucosa tissue explants within the biomaterial for the generation of human bioengineered mouth and dental tissues for use in tissue engineering. To achieve this, acellular fibrin-agarose scaffolds (AFAS), non-functionalized fibrin-agarose oral mucosa stroma substitutes (n-FAOM), and novel functionalized fibrin-agarose oral mucosa stroma substitutes (F-FAOM) were developed and analyzed after 1, 2, and 3 weeks of in vitro development to determine extracellular matrix components as compared to native oral mucosa controls by using histochemistry and immunohistochemistry. Results demonstrate that functionalization speeds up the biofabrication method and contributes to improve the biomimetic characteristics of the scaffold in terms of extracellular matrix components and reduce the time required for in vitro tissue development.

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

Blindness 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.

Qualitative and Quantitative Evaluation of a Novel Detergent-Based Method for Decellularization of Peripheral Nerves.

Tissue engineering is an emerging strategy for the development of nerve substitutes for peripheral nerve repair. Especially decellularized peripheral nerve allografts are interesting alternatives to replace the gold standard autografts. In this study, a novel decellularization protocol was qualitatively and quantitatively evaluated by histological, biochemical, ultrastructural and mechanical methods and compared to the protocol described by Sondell et al. and a modified version of the protocol described by Hudson et al. Decellularization by the method described by Sondell et al. resulted in a reduction of the cell content, but was accompanied by a loss of essential extracellular matrix (ECM) molecules such as laminin and glycosaminoglycans. This decellularization also caused disruption of the endoneurial tubes and an increased stiffness of the nerves. Decellularization by the adapted method of Hudson et al. did not alter the ECM composition of the nerves, but an efficient cell removal could not be obtained. Finally, decellularization by the method developed in our lab by Roosens et al. led to a successful removal of nuclear material, while maintaining the nerve ultrastructure and ECM composition. In addition, the resulting ECM scaffold was found to be cytocompatible, allowing attachment and proliferation of adipose-derived stem cells. These results show that our decellularization combining Triton X-100, DNase, RNase and trypsin created a promising scaffold for peripheral nerve regeneration.

Evaluation of freeze-drying and cryopreservation protocols for long-term storage of biomaterials based on decellularized intestine.

We evaluated the efficiency of several protocols to preserve the main components of decellularized tissue scaffolds for delayed use. Decellularized rat intestine scaffolds were generated by using SDS and triton X-100 and preserved for 3 months subjected to eight freeze-drying (F1 to F8) and 14 cryopreservation protocols (C1 to C14). Morphological analysis showed that cryopreservation tended to preserve the tissue morphostructure more efficiently than freeze-drying. Histological analysis showed that the content of proteoglycans and glycoproteins was efficiently preserved by most methods. The protocols that most efficiently preserved collagen fibers were those using trehalose and saccharose for freeze-drying (F2, F3, and F7 protocols) and DMSO, albumin, and saccharose (C3, C5, C6, C12) for cryopreservation. Most freeze-drying protocols and cryopreservation protocols with DMSO, albumin, and maltose (C6, C7, C13, and C14) efficiently preserved reticular fibers. For the elastic fibers, freeze-drying methods with trehalose and maltose (F2, F4, F6, and F8) properly preserved these fibers, with the results of most cryopreservation methods comparable to controls. These results suggest that freeze-drying using 0.1M trehalose and cryopreservation in the presence of 8% DMSO and 4.6% albumin are more efficient than other protocols in preserving the scaffold morphostructure and histological composition. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 488-500, 2018.

Bone mineral density aspects in the femoral neck of hip fracture patients.

Elderly people, due to neurological conditions and muscular atrophy, present a greater propensity to falls and thus are very susceptible to hip fractures. Other variables, such as osteoporosis, may also be related to the etiopathogenesis of hip fractures, although osteoporosis is in fact a concurrent disease, and merely a coadjutant cause. Nonetheless, osteoporosis can make fracture patterns more severe and interfere with osteosynthesis. Osteoporosis is the radiological image of osteopenia, a pathological concept meaning a smaller quantity of bone per unit of volume. The radiological expression of osteopenia is therefore that of bone tissue with a lower radiological density than normal. In the context of hip fractures, bone mineral density and bone architecture of the femoral neck together with protein expression profiles and cross-links of this anatomical area are of special interest which is reviewed in the current paper. Spatial variations in bone mineral density in the femoral neck were found in the literature with increased porosity from the periosteal to the endosteal region and also from the distal to the proximal part of the femoral neck. Furthermore, increased crystal size, increased cortical porosity, reduced osteocyte lacunar density and an increased Ca/P ratio associated with higher concentrations of Ca and P were described in hip fracture patients compared to control patients. Osteocalcin/collagen type 1 expression ratio and enzymatic cross-link content in high-density bone was found to be significantly lower in hip fractures compared to controls. In conclusion, further research in bone mineral density and associated parameters are of interest to deepen the understanding of osteoporotic hip fractures.

An early and late cytotoxicity evaluation of lidocaine on human oral mucosa fibroblasts.

Local anesthetic drugs are extensively used in dentistry. However, the cytotoxic effects of these pharmaceutical compounds remain unclear. In this work, we have evaluated the cell viability and cell function of human oral mucosa fibroblasts exposed to different concentrations of lidocaine for increasing incubation times, using a global screening methods including structural, metabolic and microanalytical analyses. Our results demonstrate that lidocaine is able to alter cell viability and function even at low concentrations and times, although the effect of lidocaine concentration was more important than the incubation time. First, the structural analysis methods revealed that ≥5% concentrations of lidocaine are able to significantly reduce cell viability. Then, the metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and water-soluble tetrazolium salt (WST-1) assays suggest that concentrations starting from 1% were able to significantly hinder cell physiology. Finally, electron-probe X-ray microanalysis confirmed the deleterious effects of lidocaine and allowed us to demonstrate that these effects are associated to an apoptosis process of cell death. Therefore, care should be taken when lidocaine is clinically used, and the lowest efficient concentrations should always be used. Furthermore, these results suggest that the comprehensive evaluation method used in this work is accurate and efficient for screening of local anesthetics.

Wharton's jelly stem cells: a novel cell source for oral mucosa and skin epithelia regeneration.

Perinatal stem cells such as human umbilical cord Wharton's jelly stem cells (HWJSCs) are excellent candidates for tissue engineering because of their proliferation and differentiation capabilities. However, their differentiation potential into epithelial cells at in vitro and in vivo levels has not yet been reported. In this work we have studied the capability of HWJSCs to differentiate in vitro and in vivo to oral mucosa and skin epithelial cells using a bioactive three-dimensional model that mimics the native epithelial-mesenchymal interaction. To achieve this, primary cell cultures of HWJSCs, oral mucosa, and skin fibroblasts were obtained in order to generate a three-dimensional heterotypical model of artificial oral mucosa and skin based on fibrin-agarose biomaterials. Our results showed that the cells were unable to fully differentiate to epithelial cells in vitro. Nevertheless, in vivo grafting of the bioactive three-dimensional models demonstrated that HWJSCs were able to stratify and to express typical markers of epithelial differentiation, such as cytokeratins 1, 4, 8, and 13, plakoglobin, filaggrin, and involucrin, showing specific surface patterns. Electron microscopy analysis confirmed the presence of epithelial cell-like layers and well-formed cell-cell junctions. These results suggest that HWJSCs have the potential to differentiate to oral mucosa and skin epithelial cells in vivo and could be an appropriate novel cell source for the development of human oral mucosa and skin in tissue engineering protocols.

A combined approach for the assessment of cell viability and cell functionality of human fibrochondrocytes for use in tissue engineering.

Temporo-mandibular joint disc disorders are highly prevalent in adult populations. Autologous chondrocyte implantation is a well-established method for the treatment of several chondral defects. However, very few studies have been carried out using human fibrous chondrocytes from the temporo-mandibular joint (TMJ). One of the main drawbacks associated to chondrocyte cell culture is the possibility that chondrocyte cells kept in culture tend to de-differentiate and to lose cell viability under in in-vitro conditions. In this work, we have isolated human temporo-mandibular joint fibrochondrocytes (TMJF) from human disc and we have used a highly-sensitive technique to determine cell viability, cell proliferation and gene expression of nine consecutive cell passages to determine the most appropriate cell passage for use in tissue engineering and future clinical use. Our results revealed that the most potentially viable and functional cell passages were P5-P6, in which an adequate equilibrium between cell viability and the capability to synthesize all major extracellular matrix components exists. The combined action of pro-apoptotic (TRAF5, PHLDA1) and anti-apoptotic genes (SON, HTT, FAIM2) may explain the differential cell viability levels that we found in this study. These results suggest that TMJF should be used at P5-P6 for cell therapy protocols.

Histología., embriología e ingeniería tisular bucodental

Histología, embriología e ingeniería tisular bucodental. Métodos y técnicas de estudio en histología e ingeniería tisular bucodental. Embriología general humana. Histología general humana. Embriología especial bucomaxilofacial. Embriología dental (odontogénesis). Cavidad bucal. Glándulas salivales. Complejo articular temporomandibular (CATM). Complejo dentino-pulpar I: pulpa dental. Complejo dentino-pulpar II: dentina. Esmalte. Periodoncio de protección: encía y unión dentogingival. Periodoncio de inserción: cemento, ligamento periodontal y hueso alveolar. Erupción dentaria. Dientes primarios

Histología., embriología e ingeniería tisular bucodental

Histología, embriología e ingeniería tisular bucodental. Métodos y técnicas de estudio en histología e ingeniería tisular bucodental. Embriología general humana. Histología general humana. Embriología especial bucomaxilofacial. Embriología dental (odontogénesis). Cavidad bucal. Glándulas salivales. Complejo articular temporomandibular (CATM). Complejo dentino-pulpar I: pulpa dental. Complejo dentino-pulpar II: dentina. Esmalte. Periodoncio de protección: encía y unión dentogingival. Periodoncio de inserción: cemento, ligamento periodontal y hueso alveolar. Erupción dentaria. Dientes primarios

El mapa conceptual. Un instrumento educativo polivalente para las ciencias de la salud. Su aplicación en histología / Concept maps as a polyvalent educational tool for health sciences. Their application to histology

En el presente artículo se analiza el mapa conceptual como instrumento de estrategia educativa aplicada a las ciencias de la salud y, especialmente, al ámbito de la histología. Tras considerar los elementos constitutivos y los tipos de mapas conceptuales y el fundamento epistemológico de los mismos para estimular el aprendizaje significativo, se examina la aplicación de los mapas al desarrollo curricular, la evaluación, el diseño pedagógico por parte del profesor y el autoaprendizaje por parte del alumno en el contexto del proceso de Bolonia. En el ámbito de la histología se analiza la utilización de los distintos tipos de mapas para la definición y jerarquización de sus contenidos, su relación con el resto de las disciplinas y su nuevo paradigma vinculado a la Ingeniería tisular (AU)

This article analyzes the concept map as a tool for educational strategy applied to health sciences, particularly in the area of histology. After considering the elements that make up these maps, the different types of concept maps and the epistemological basis of maps as instruments to enhance significant learning, the article examines how maps can be used for curriculum development, evaluation, pedagogic design by teachers, and self-learning by students within the context of the Bologna process. With reference to histology, we analyze how different types of maps are used to define and rank concepts, examine the relationship between disciplines and to understand the new paradigm of histology related to tissue engineering (AU)

Histología y embriología bucodental

Prólogo a la segunda edición. Prólogo a la primera edición. Introducción al estudio de la histología y la embriología bucodental. Embriología general humana. Embriología especial bucomaxilofacial. Embriología dentaria (odontogénesis). Cavidad bucal. Glándulas salivales. Complejo articular temporomandibular (CATM). Complejo dentino-pulpar: pulpa dental. Complejo dentino-pulpar: dentina. Esmalte. Periodoncio de protección: encía y unión dentogingival. Periodoncio de inserción: cemento, ligamento periodontal y hueso alveolar. Erupción dentaria. Dientes primarios. Respuestas a las situaciones problemáticas

Histología y embriología bucodental

Prólogo a la segunda edición. Prólogo a la primera edición. Introducción al estudio de la histología y la embriología bucodental. Embriología general humana. Embriología especial bucomaxilofacial. Embriología dentaria (odontogénesis). Cavidad bucal. Glándulas salivales. Complejo articular temporomandibular (CATM). Complejo dentino-pulpar: pulpa dental. Complejo dentino-pulpar: dentina. Esmalte. Periodoncio de protección: encía y unión dentogingival. Periodoncio de inserción: cemento, ligamento periodontal y hueso alveolar. Erupción dentaria. Dientes primarios. Respuestas a las situaciones problemáticas