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.

A Novel In Vitro Pathological Model for Studying Neural Invasion in Non-Melanoma Skin Cancer.

Neural Invasion (NI) is a key pathological feature of cancer in the colonization of distant tissues, and its underlying biological mechanisms are still scarcely known. The complex interactions between nerve and tumor cells, along with the stroma, make it difficult to reproduce this pathology in effective study models, which in turn has limited the understanding of NI pathogenesis. In this study, we have designed a three-dimensional model of NI squamous cell carcinoma combining human epidermoid carcinoma cells (hECCs) with a complete peripheral nerve segment encapsulated in a fibrine-agarose hydrogel. We recreated two vital processes of NI: a pre-invasive NI model in which hECCs were seeded on the top of the nerve-enriched stroma, and an invasive NI model in which cancer cells were immersed with the nerve in the hydrogel. Histological, histochemical and immunohistochemical analyses were performed to validate the model. Results showed that the integration of fibrin-agarose advanced hydrogel with a complete nerve structure and hECCs successfully generated an environment in which tumor cells and nerve components coexisted. Moreover, this model correctly preserved components of the neural extracellular matrix as well as allowing the proliferation and migration of cells embedded in hydrogel. All these results suggest the suitability of the model for the study of the mechanisms underlaying NI.

A novel 3D biofabrication strategy to improve cell proliferation and differentiation of human Wharton's jelly mesenchymal stromal cells for cell therapy and tissue engineering.

Purpose: Obtaining sufficient numbers of cells in a short time is a major goal of cell culturing in cell therapy and tissue engineering. However, current bidimensional (2D) culture methods are associated to several limitations, including low efficiency and the loss of key cell differentiation markers on cultured cells. Methods: In the present work, we have designed a novel biofabrication method based on a three-dimensional (3D) culture system (FIBRIAGAR-3D). Human Wharton's jelly mesenchymal stromal cells (HWJSC) were cultured in 3D using 100%, 75%, 50%, and 25% concentrations of fibrin-agarose biomaterials (FA100, FA75, FA50 and FA25 group) and compared with control cells cultured using classical 2D systems (CTR-2D). Results: Our results showed a significant increase in the number of cells generated after 7 days of culture, with cells displaying numerous expansions towards the biomaterial, and a significant overexpression of the cell proliferation marker KI67 was found for the FA75 and FA100 groups. TUNEL and qRT-PCR analyses demonstrated that the use of FIBRIAGAR-3D was not associated with an induction of apoptosis by cultured cells. Instead, the 3D system retained the expression of typical phenotypic markers of HWJSC, including CD73, CD90, CD105, NANOG and OCT4, and biosynthesis markers such as types-I and IV collagens, with significant increase of some of these markers, especially in the FA100 group. Finally, our analysis of 8 cell signaling molecules revealed a significant decrease of GM-CSF, IFN-g, IL2, IL4, IL6, IL8, and TNFα, suggesting that the 3D culture system did not induce the expression of pro-inflammatory molecules. Conclusion: These results confirm the usefulness of FIBRIAGAR-3D culture systems to increase cell proliferation without altering cell phenotype of immunogenicity and opens the door to the possibility of using this novel biofabrication method in cell therapy and tissue engineering of the human cornea, oral mucosa, skin, urethra, among other structures.

Expression of Basement Membrane Molecules by Wharton Jelly Stem Cells (WJSC) in Full-Term Human Umbilical Cords, Cell Cultures and Microtissues.

Wharton's jelly stem cells (WJSC) from the human umbilical cord (UC) are one of the most promising mesenchymal stem cells (MSC) in tissue engineering (TE) and advanced therapies. The cell niche is a key element for both, MSC and fully differentiated tissues, to preserve their unique features. The basement membrane (BM) is an essential structure during embryonic development and in adult tissues. Epithelial BMs are well-known, but similar structures are present in other histological structures, such as in peripheral nerve fibers, myocytes or chondrocytes. Previous studies suggest the expression of some BM molecules within the Wharton's Jelly (WJ) of UC, but the distribution pattern and full expression profile of these molecules have not been yet elucidated. In this sense, the aim of this histological study was to evaluate the expression of main BM molecules within the WJ, cultured WJSC and during WJSC microtissue (WJSC-MT) formation process. Results confirmed the presence of a pericellular matrix composed by the main BM molecules-collagens (IV, VII), HSPG2, agrin, laminin and nidogen-around the WJSC within UC. Additionally, ex vivo studies demonstrated the synthesis of these BM molecules, except agrin, especially during WJSC-MT formation process. The WJSC capability to synthesize main BM molecules could offer new alternatives for the generation of biomimetic-engineered substitutes where these molecules are particularly needed.

Development of stromal differentiation patterns in heterotypical models of artificial corneas generated by tissue engineering.

Purpose: We carried out a histological characterization analysis of the stromal layer of human heterotypic cornea substitutes generated with extra-corneal cells to determine their putative usefulness in tissue engineering. Methods: Human bioartificial corneas were generated using nanostructured fibrin-agarose biomaterials with corneal stromal cells immersed within. To generate heterotypical corneas, umbilical cord Wharton's jelly stem cells (HWJSC) were cultured on the surface of the stromal substitutes to obtain an epithelial-like layer. These bioartificial corneas were compared with control native human corneas and with orthotypical corneas generated with human corneal epithelial cells on top of the stromal substitute. Both the corneal stroma and the basement membrane were analyzed using histological, histochemical and immunohistochemical methods in samples kept in culture and grafted in vivo for 12 months in the rabbit cornea. Results: Our results showed that the stroma of the bioartificial corneas kept ex vivo showed very low levels of fibrillar and non-fibrillar components of the tissue extracellular matrix. However, in vivo implantation resulted in a significant increase of the contents of collagen, proteoglycans, decorin, keratocan and lumican in the corneal stroma, showing higher levels of maturation and spatial organization of these components. Heterotypical corneas grafted in vivo for 12 months showed significantly higher contents of collagen fibers, proteoglycans and keratocan. When the basement membrane was analyzed, we found that all corneas grafted in vivo showed intense PAS signal and higher contents of nidogen-1, although the levels found in human native corneas was not reached, and a rudimentary basement membrane was observed using transmission electron microscopy. At the epithelial level, HWJSC used to generate an epithelial-like layer in ex vivo corneas were mostly negative for p63, whereas orthotypical corneas and heterotypical corneas grafted in vivo were positive. Conclusion: These results support the possibility of generating bioengineered artificial corneas using non-corneal HWJSC. Although heterotypical corneas were not completely biomimetic to the native human corneas, especially ex vivo, in vivo grafted corneas demonstrated to be highly biocompatible, and the animal cornea became properly differentiated at the stroma and basement membrane compartments. These findings open the door to the future clinical use of these bioartificial corneas.

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.

Usefulness of a Nanostructured Fibrin-Agarose Bone Substitute in a Model of Severely Critical Mandible Bone Defect.

Critical defects of the mandibular bone are very difficult to manage with currently available materials and technology. In the present work, we generated acellular and cellular substitutes for human bone by tissue engineering using nanostructured fibrin-agarose biomaterials, with and without adipose-tissue-derived mesenchymal stem cells differentiated to the osteogenic lineage using inductive media. Then, these substitutes were evaluated in an immunodeficient animal model of severely critical mandibular bone damage in order to assess the potential of the bioartificial tissues to enable bone regeneration. The results showed that the use of a cellular bone substitute was associated with a morpho-functional improvement of maxillofacial structures as compared to negative controls. Analysis of the defect site showed that none of the study groups fully succeeded in generating dense bone tissue at the regeneration area. However, the use of a cellular substitute was able to improve the density of the regenerated tissue (as determined via CT radiodensity) and form isolated islands of bone and cartilage. Histologically, the regenerated bone islands were comparable to control bone for alizarin red and versican staining, and superior to control bone for toluidine blue and osteocalcin in animals grafted with the cellular substitute. Although these results are preliminary, cellular fibrin-agarose bone substitutes show preliminary signs of usefulness in this animal model of severely critical mandibular bone defect.

Injectable Magnetic-Responsive Short-Peptide Supramolecular Hydrogels: Ex Vivo and In Vivo Evaluation.

The inclusion of magnetic nanoparticles (MNP) in a hydrogel matrix to produce magnetic hydrogels has broadened the scope of these materials in biomedical research. Embedded MNP offer the possibility to modulate the physical properties of the hydrogel remotely and on demand by applying an external magnetic field. Moreover, they enable permanent changes in the mechanical properties of the hydrogel, as well as alterations in the micro- and macroporosity of its three-dimensional (3D) structure, with the associated potential to induce anisotropy. In this work, the behavior of biocompatible and biodegradable hydrogels made with Fmoc-diphenylalanine (Fmoc-FF) (Fmoc = fluorenylmethoxycarbonyl) and Fmoc-arginine-glycine-aspartic acid (Fmoc-RGD) short peptides to which MNP were incorporated was studied in detail with physicochemical, mechanical, and biological methods. The resulting hybrid hydrogels showed enhance mechanical properties and withstood injection without phase disruption. In mice, the hydrogels showed faster and improved self-healing properties compared to their nonmagnetic counterparts. Thanks to these superior physical properties and stability during culture, they can be used as 3D scaffolds for cell growth. Additionally, magnetic short-peptide hydrogels showed good biocompatibility and the absence of toxicity, which together with their enhanced mechanical stability and excellent injectability make them ideal biomaterials for in vivo biomedical applications with minimally invasive surgery. This study presents a new approach to improving the physical and mechanical properties of supramolecular hydrogels by incorporating MNP, which confer structural reinforcement and stability, remote actuation by magnetic fields, and better injectability. Our approach is a potential catalyst for expanding the biomedical applications of supramolecular short-peptide hydrogels.

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.

Efecto del uso de secretoma de células madre mesenquimales sobre heridas cutáneas. Análisis comparativo in vitro e in vivo de tres tipos de secretoma / Effects of mesenchymal stem cell secretome on skin wounds. In vitro and in vivo comparative analysis of three types of secretome

Introducción y objetivo: Los avances en el campo de la Ingeniería Tisular han promovido el desarrollo de sustitutos de piel y su aplicación en el campo de la Cirugía Plástica. Uno de los principales inconvenientes de la bioingeniería de la piel es la necesidad de obtener una gran cantidad de células viables en periodos cortos de tiempo, lo que conlleva que el proceso de biofabricación sea largo y complejo. En este estudio se pretende valorar los efectos de la aplicación de tres tipos diferentes de secretoma derivados de células madre mesenquimales humanas en cultivos de fibroblastos con el objetivo de favorecer la escalabilidad de los procesos de fabricación de piel artificial. Material y método: Se evaluaron los efectos a las 24, 48 y 72 horas sobre la viabilidad, la proliferación y la migración celular de fibroblastos humanos tras la aplicación de dos concentraciones (C1 y C2) de tres tipos diferentes de secretoma derivado de células madre mesenquimales humanas. Los resultados in vitro fueron contrastados en un modelo in vivo. Resultados: El uso de secretoma derivado de células madre mesenquimales mejoró los protocolos de cultivo de fibroblastos actualmente disponibles. El uso de secretoma se asoció a un aumento de la proliferación y migración celular manteniendo cifras altas de viabilidad. Los datos fueron especialmente positivos para el secretoma de células madre mesenquimales de pulpa dental y de tejido adiposo. Conclusiones: El efecto de la aplicación de secretoma procedentes de células madre mesenquimales permite mantener cifras de viabilidad celular elevadas, además de incrementar el ritmo de proliferación de fibroblastos. Los estudios in vivo e in vitro no evidenciaron efectos adversos a corto plazo. (AU)

Background and objective: Advances in Tissue Engineering promoted the development of skin substitutes. One of the main drawbacks of skin bioengineering is the requirement of a considerable quantity of viable cells in short periods of time, which leads to a challenging biofabrication process. This article aims to analyse the effects of the application of three different types of human mesenchymal stem cell-derived secretome in fibroblast cultures. The final goal is to achieve new strategies to promote the scalability of artificial skin manufacturing processes. Methods: The effects of the three different types of human mesenchymal stem cell-derived secretome were analyzed at 24, 48 and 72 hours. To study cell viability, cell proliferation and cell migration we exposed human fibroblasts to different secretome concentrations (C1 and C2). An in vivo study was proposed to corroborate in vitro results. Results: The use of mesenchymal stem cell-derived secretome improved the currently available fibroblast culture protocols. The use of secretome was associated with increased cell proliferation and cell migration while maintaining high viability values. Data were especially positive when secretome from dental pulp mesenchymal stem cells and adipose tissue mesenchymal stem cells were applied. Conclusions: The application of mesenchymal stem cell-derived secretome maintained high cell viability data and increased fibroblast proliferation. In vivo and in vitro studies showed no short-term adverse effects. (AU)

Implementación de un modelo de aula invertida para el autoaprendizaje de la ingeniería tisular en el grado de Farmacia / Implementation of a flipped learning classroom model for self-learning of tissue engineering in the Pharmacy degree

Introducción: El presente trabajo describe y evalúa la implementación de un modelo de aula invertida en la materia de Histología para el aprendizaje de ingeniería tisular en el grado de Farmacia con el objetivo de incorporar dichos conocimientos ante su escasa presencia en dicho grado. Sujetos y métodos: El modelo consiste en intercalar en el curso ordinario de Histología del grado de Farmacia un módulo de autoaprendizaje inverso. Participan 110 alumnos que responden a un cuestionario sobre ámbitos conceptuales, procedimentales y actitudinales al comienzo y al final del proceso. Los resultados se analizan estadísticamente. Resultados: Los resultados muestran valores superiores en la evaluación final con respecto a la evaluación inicial. Esta diferencia fue estadísticamente significativa (p < 0,05) tanto en mujeres y hombres como en el total de estudiantes. Los valores obtenidos fueron decrecientes desde el componente actitudinal hasta el componente conceptual. En el componente procedimental, se obtienen valores intermedios. Conclusión: Los valores más elevados observados en los componentes actitudinal y procedimental, tras la implementación del modelo propuesto, ponen de relieve la necesidad de incrementar el componente conceptual en la formación de la ingeniería tisular en el currículo de farmacia.(AU)

Introduction: This paper describes and evaluates the implementation of a flipped learning model in the discipline of histology for learning tissue engineering contents in the Pharmacy degree, with the aim of incorporating this knowledge for the scarce presence of such matter in that degree. Subjects and methods: The model consists of inserting in the ordinary course of Histology of the pharmacy degree an inverse self-learning module. A questionnaire on conceptual, procedural and attitudinal fields was answered by the 110 students involved in the study at the beginning and end of the process. Results were statistically analysed. Results: The results after the implementation of the flipped learning model show statistically significant higher values (p < 0.05) in the final evaluation both in female and male and in all the students with decreasing values from those reached for the attitudinal component to those reached by the conceptual component. In the procedural component, intermediate values are obtained. Conclusion: The highest values observed in the attitudinal and procedural components, after the implementation of the proposed model, highlight the need to increase the conceptual component in the formation of tissue engineering in the pharmacy curriculum.(AU)