Development of secretome-based strategies to improve cell culture protocols in tissue engineering.

Advances in skin tissue engineering have promoted the development of artificial skin substitutes to treat large burns and other major skin loss conditions. However, one of the main drawbacks to bioengineered skin is the need to obtain a large amount of viable epithelial cells in short periods of time, making the skin biofabrication process challenging and slow. Enhancing skin epithelial cell cultures by using mesenchymal stem cells secretome can favor the scalability of manufacturing processes for bioengineered skin. The effects of three different types of secretome derived from human mesenchymal stem cells, e.g. hADSC-s (adipose cells), hDPSC-s (dental pulp) and hWJSC-s (umbilical cord), were evaluated on cultured skin epithelial cells during 24, 48, 72 and 120 h to determine the potential of this product to enhance cell proliferation and improve biofabrication strategies for tissue engineering. Then, secretomes were applied in vivo in preliminary analyses carried out on Wistar rats. Results showed that the use of secretomes derived from mesenchymal stem cells enhanced currently available cell culture protocols. Secretome was associated with increased viability, proliferation and migration of human skin epithelial cells, with hDPSC-s and hWJSC-s yielding greater inductive effects than hADSC-s. Animals treated with hWJSC-s and especially, hDPSC-s tended to show enhanced wound healing in vivo with no detectable side effects. Mesenchymal stem cells derived secretomes could be considered as a promising approach to cell-free therapy able to improve skin wound healing and regeneration.

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.

Characterization of the human ridged and non-ridged skin: a comprehensive histological, histochemical and immunohistochemical analysis.

The structure of the human skin is directly dependent on its location and the mechanical forces to which it is subjected. In the present work, we have performed a comprehensive analysis of the human ridged and non-ridged skin to identify the differences and similarities between both skin types. For this purpose, human skin samples were obtained from dorsal hand skin (DHS), palmar hand skin (PHS), dorsal foot skin (DFS) and plantar foot skin (PFS) from the same cadaveric donors. Histological, histochemical and semiquantitative and quantitative immunohistochemical analyses were carried out to evaluate the epidermis, dermis and basement membrane. Results show that the epithelial layer of ridged skin had larger cell number and size than non-ridged skin for most strata. Melanocytes and Langerhans cells were more abundant in non-ridged skin, whereas Merkel cells were preferentially found in ridged skin. The expression pattern of CK5/6 was slightly differed between non-ridged and ridged skin. Involucrin expression was slightly more intense in non-ridged skin than in ridged skin. Collagen was more abundant in foot skin dermis than in hand skin, and in ridged skin as compared to non-ridged skin. Elastic fibers were more abundant in DHS. Biglycan was more abundant in foot skin than in hand skin. No differences were found for blood and lymphatic vessels. The basement membrane laminin was preferentially found in foot skin. These results revealed important differences at the epithelial, dermal and basement membrane levels that could contribute to a better knowledge of the human skin histology.

Chiral symmetry breaking yields the I-Au60 perfect golden shell of singular rigidity.

The combination of profound chirality and high symmetry on the nm-scale is unusual and would open exciting avenues, both fundamental and applied. Here we show how the unique electronic structure and bonding of quasi-2D gold makes this possible. We report a chiral symmetry breaking, i.e., the spontaneous formation of a chiral-icosahedral shell (I-Au60) from achiral (Ih) precursor forms, accompanied by a contraction in the Au-Au bonding and hence the radius of this perfect golden sphere, in which all 60 sites are chemically equivalent. This structure, which resembles the most complex of semi-regular (Archimedean) polyhedra (34.5*), may be viewed as an optimal solution to the topological problem: how to close a 60-vertex 2D (triangular) net in 3D. The singular rigidity of the I-Au60 manifests in uniquely discrete structural, vibrational, electronic, and optical signatures, which we report herein as a guide to its experimental detection and ultimately its isolation in material forms.

Bioactive injectable aggregates with nanofibrous microspheres and human dental pulp stem cells: A translational strategy in dental endodontics.

Regeneration of the pulp-dentin complex with stem cells is a potential alternative to conventional root canal treatments. Human dental pulp stem cells (hDPSCs) have been extensively studied because of their ability to proliferate and differentiate into mineralized dental and non-dental tissues. Here we combined hDPSCs with two types of injectable poly-l-lactic acid (PLLA) microsphere with a nanofibrous or smooth surface to form bioactive injectable aggregates, and examined their ability to promote pulp regeneration in the root canal in an in vivo model. We investigated the biocompatibility, biosafety and odontogenic potential of fibrous (F-BIM) and smooth bioactive injectable microspheres (S-BIM) in vitro and in vivo. Our results demonstrated that PLLA microspheres and hDPSCs were able to form bioactive injectable aggregates that promoted dentin regeneration in both in vitro and in vivo models. Our results suggest that F-BIM and S-BIM may induce dentinogenesis upon in vivo grafting, and propose that the potential usefulness of the microsphere-hDPSC aggregates described here should be evaluated in clinical settings. Copyright © 2017 John Wiley & Sons, Ltd.

Membranes derived from human umbilical cord Wharton's jelly stem cells as novel bioengineered tissue-like constructs.

Cell-derived matrices were recently described as novel biomaterials generated by human cells allowed to grow and synthetize their own extracellular matrix in culture. In the present work, we generated and evaluated a novel tissue-like substitute (WDM) consisting of a membrane derived from cultured human Wharton's jelly stem cells. WDM were evaluated ex vivo and in vivo by histochemistry and immunohistochemistry for several mesenchymal cell markers and fibrillar and non-fibrillar extracellular matrix components. Results show that WDM were heterogeneous and consisted of dense cell-poor areas surrounded by cell-rich zones with abundant HWJSC. Histological analyses demonstrated that cell-poor areas were very rich in fibrillar and non-fibrillar extracellular matrix components such as collagen and proteoglycans, and cells in the WDM were highly viable and mostly PCNA-positive. HWJSC in the WDM expressed all markers of this cell type, including CD90, CD105, pan cytokeratin and CK8. In vivo analysis showed that the WDM was highly biocompatible and grafting this membrane in the muscle of laboratory rats was not associated to increased inflammation, necrosis, tumorigenesis or other side effects, while cells properly integrated at the damage site and showed high proliferation index. These results suggest that the structure and composition of the extracellular matrix of these novel WDM could reproduce the situation of native human tissues and that WDM implanted in vivo are highly biocompatible and rapidly integrate in the host tissues. For these reasons, we hypothesize that WDM could be used in regenerative medicine protocols.

Development of a multilayered palate substitute in rabbits: a histochemical ex vivo and in vivo analysis.

Current tissue engineering technology focuses on developing simple tissues, whereas multilayered structures comprising several tissue types have rarely been described. We developed a highly biomimetic multilayered palate substitute with bone and oral mucosa tissues using rabbit cells and biomaterials subjected to nanotechnological techniques based on plastic compression. This novel palate substitute was autologously grafted in vivo, and histological and histochemical analyses were used to evaluate biointegration, cell function, and cell differentiation in the multilayered palate substitute. The three-dimensional structure of the multilayered palate substitute was histologically similar to control tissues, but the ex vivo level of cell and tissue differentiation were low as determined by the absence of epithelial differentiation although cytokeratins 4 and 13 were expressed. In vivo grafting was associated with greater cell differentiation, epithelial stratification, and maturation, but the expression of cytokeratins 4, 13, 5, and 19 at did not reach control tissue levels. Histochemical analysis of the oral mucosa stroma and bone detected weak signals for proteoglycans, elastic and collagen fibers, mineralization deposits and osteocalcin in the multilayered palate substitute cultured ex vivo. However, in vivo grafting was able to induce cell and tissue differentiation, although the expression levels of these components were always significantly lower than those found in controls, except for collagen in the bone layer. These results suggest that generation of a full-thickness multilayered palate substitute is achievable and that tissues become partially differentiated upon in vivo grafting.

Relevant aspects in the surface properties in titanium dental implants for the cellular viability.

Roughness and topographical features are the most relevant of the surface properties for a dental implant for its osseointegration. For that reason, we studied the four surfaces more used in titanium dental implants: machined, sandblasted, acid etching and sandblasted plus acid etching. The roughness and wettability (contact angle and surface free energy) was studied by means 3D-interferometric microscope and sessile drop method. Normal human gingival fibroblasts (HGF) were obtained from small oral mucosa biopsies and were used for cell cultures. To analyze cell integrity, we first quantified the total amount of DNA and LDH released from dead cells to the culture medium. Then, LIVE/DEAD assay was used as a combined method assessing cell integrity and metabolism. All experiments were carried out on each cell type cultured on each Ti material for 24h, 48h and 72h. To evaluate the in vivo cell adhesion capability of each Ti surface, the four types of discs were grafted subcutaneously in 5 Wistar rats. Sandblasted surfaces were significantly rougher than acid etching and machined. Wettability and surface free energy decrease when the roughness increases in sand blasted samples. This fact favors the protein adsorption. The DNA released by cells cultured on the four Ti surfaces did not differ from that of positive control cells (p>0.05). The number of cells per area was significantly lower (p<0.05) in the sand-blasted surface than in the machined and surface for both cell types (7±2 cells for HGF and 10±5 cells for SAOS-2). The surface of the machined-type discs grafted in vivo had a very small area occupied by cells and/or connective tissue (3.5%), whereas 36.6% of the sandblasted plus acid etching surface, 75.9% of sandblasted discs and 59.6% of acid etching discs was covered with cells and connective tissue. Cells cultured on rougher surfaces tended to exhibit attributes of more differentiated osteoblasts than cells cultured on smoother surfaces. These surface properties justify that the sandblasted implants is able to significantly increase bone contact and bone growth with very good osseointegration results in vivo.

Ex vivo and in vivo modulatory effects of umbilical cord Wharton's jelly stem cells on human oral mucosa stroma substitutes.

Novel oral mucosa substitutes have been developed in the laboratory using human umbilical cord Wharton's jelly stem cells -HWJSC- as an alternative cell source. In the present work, we have generated human oral mucosa substitutes with oral mucosa keratinocytes and HWJSC to determine the influence of these cell sources on stromal differentiation. First, acellular and cellular stroma substitutes and bilayered oral mucosa substitutes with an epithelial layer consisting of oral mucosa keratinocytes -OM samples- or HWJSC -hOM- were generated. Then, tissues were analyzed by light and electron microscopy, histochemistry and immunohistochemistry to quantify all major extracellular matrix components after 1, 2 and 3 weeks of ex vivo development, and OM and hOM were also analyzed after in vivo grafting. The results showed that bioengineered oral mucosa stromas displayed an adequate fibrillar mesh. Synthesis of abundant collagen fibers was detected in OM and hOM after 3 weeks, and in vivo grafting resulted in an increased collagen synthesis. No elastic or reticular fibers were found. Glycoprotein synthesis was found at the epithelial-stromal layer when samples were grafted in vivo. Finally, proteoglycans, decorin, versican and aggrecan were strongly dependent on the in vivo environment and the presence of a well-structured epithelium on top. The use of HWJSC was associated to an increased synthesis of versican. These results confirm the usefulness of fibrin-agarose biomaterials for the generation of an efficient human oral mucosa stroma substitute and the importance of the in vivo environment and the epithelial-mesenchymal interaction for the adequate differentiation of the bioengineered stroma.

Human Dental Pulp Stem Cells. A promising epithelial-like cell source.

Several models of tissue-engineered human skin based on three-dimensional (3D) co-culture techniques have been proposed to the date. However, normal skin biopsies are not always available, especially in patients with a high percentage of skin affected by deep burning, and the generation of large amounts of cultured keratinocytes may take very long time, with an associated risk for the patients' survival. For those reasons, the search of alternative cell sources for tissue reconstruction is a clinical need. In this context, Human Dental Pulp Stem Cells (HDPSC) have the potential to differentiate into multiple cell lineages by the appropriate differentiation conditions, but skin epidermis differentiation has not been demonstrated so far. Here, we hypothesize that HDPSC may have pluripotent differentiation capability, and may be able to differentiate into skin epithelial keratinocytes in culture using organotypic 3D models based on the interaction with the subjacent dermal fibroblasts. By using HDPSC, the problems associated to the donor site availability and the proliferation capability of the epithelial cells could be solved. The rapid accessibility to these cells could be translated to a more immediate generation of a bioengineered human skin substitute for the future clinical treatment.

Sequential keratinocytic differentiation and maturation in a three-dimensional model of human artificial oral mucosa.

BACKGROUND AND OBJECTIVE: Oral mucosa shortage may limit or condition some clinical approaches in maxillofacial, periodontal and implant treatment. The availability of a human oral mucosa model generated by tissue engineering could help clinicians to address the lack of oral mucosa. In this work, we carried out a sequential maturation and differentiation study of the epithelial cell layer of an artificial human oral mucosa substitute based on fibrin-agarose biomaterials with fibroblasts and keratinocytes. MATERIAL AND METHODS: Histological, immunohistochemical and gene expression analyses were carried out in artificial human oral mucosa models developed and cultured for 1, 2 and 3 wk. RESULTS: Artificial oral mucosa models showed expression of tight junction proteins and cytokeratins from the first week of in vitro development. Mature samples of 3 wk of development subjected to air-liquid conditions showed signs of epithelial differentiation and expressed specific RNAs and proteins corresponding to adherent and gap junctions and basement lamina. Moreover, these mature samples overexpressed some desmosomal and tight junction transcripts, with gap junction components being downregulated. CONCLUSION: These results suggest that bioengineered human oral mucosa substitutes form a well-developed epithelial layer that was very similar to human native tissues. In consequence, the epithelial layer could be fully functional in these oral mucosa substitutes, thus implying that these tissues may have clinical usefulness.

Expression of epithelial markers by human umbilical cord stem cells. A topographical analysis.

INTRODUCTION: Human umbilical cord stem cells have inherent differentiation capabilities and potential usefulness in regenerative medicine. However, the epithelial differentiation capability and the heterogeneity of these cells have not been fully explored to the date. METHODS: We analyzed the expression of several undifferentiation and epithelial markers in cells located in situ in different zones of the umbilical cord -in situ analysis- and in primary ex vivo cell cultures of Wharton's jelly stem cells by microarray and immunofluorescence. RESULTS: Our results demonstrated that umbilical cord cells were heterogeneous and had intrinsic capability to express in situ stem cell markers, CD90 and CD105 and the epithelial markers cytokeratins 3, 4, 7, 8, 12, 13, 19, desmoplakin and zonula occludens 1 as determined by microarray and immunofluorescence, and most of these markers remained expressed after transferring the cells from the in situ to the ex vivo cell culture conditions. However, important differences were detected among some cell types in the umbilical cord, with subvascular zone cells showing less expression of stem cell markers and cells in Wharton's jelly and the amnioblastic zones showing the highest expression of stem cells and epithelial markers. CONCLUSIONS: These results suggest that umbilical cord mesenchymal cells have intrinsic potential to express relevant epithelial markers, and support the idea that they could be used as alternative cell sources for epithelial tissue engineering.

Generación in vitro de un sustituto de pared vesical humana obtenido mediante ingeniería tisular / In vitro generation of a human bladder wall substitute by tissue engineering

Objetivos. Numerosas patologías que afectan a la vejiga, de origen congénito (extrofia) o adquirido (traumatismos, tumores), requieren la reconstrucción de la pared vesical utilizando intestino delgado, sigma o estómago, los cuales no están exentos de complicaciones. Por ese motivo, en el presente trabajo pretendemos desarrollar un nuevo modelo de pared vesical humana mediante ingeniería tisular que pudiese tener una utilidad clínica. Material y métodos. En primer lugar, se procedió a generar cultivos primarios de células epiteliales y estromales de la mucosa vesical a partir de pequeñas biopsias de la pared vesical humana, utilizando para ello técnicas de digestión enzimática mediante tripsina-EDTA y colagenasa. Posteriormente, se generó un sustituto tridimensional de la mucosa vesical utilizando como soporte biomateriales de fibrina-agarosa. El análisis de las muestras se realizó a los 14 días mediante examen histológico de muestras teñidas con hematoxilina-eosina. Resultados. La aplicación de los métodos de digestión enzimática permitió generar eficientemente cultivos primarios de células epiteliales y estromales de la mucosa vesical humana, comprobándose que la tasa de proliferación de las células estromales era superior a la de las células epiteliales. Una vez generados los sustitutos de la pared vesical, se comprobó el adecuado nivel de biocompatibilidad del biomaterial y las células estromales y epiteliales. La estructura histológica de los sustitutos de pared vesical presentaba una gran analogía con la mucosa vesical humana nativa. Conclusiones. El tejido vesical generado por ingeniería tisular muestra importantes similitudes estructurales e histológicas con el tejido vesical nativo. Estos resultados sugieren que los tejidos generados mediante ingeniería tisular podrían tener utilidad terapéutica en el futuro (AU)

Introduction. Certain urological congenital conditions, such as bladder exstrophy and acquired conditions such as trauma and tumors may require the use of different tissues like small bowel, sigmoid colon or stomach for bladder reconstruction. However, these tissues are often associated to important complications. The aim of this study is to develop a novel substitute of the human bladder wall by tissue engineering. Material and methods. We first generated primary cell cultures of epithelial and stromal bladder mucosa cells from small tissue biopsies of human bladder by using enzymatic methods based on trypsin-EDTA and collagenase I. Then, a three-dimensional substitute of the bladder mucosa was generated using fibrin-agarose biomaterials. The analysis of the tissue substitutes was carried out at day 14th of development by histological examination of samples stained with hematoxylin-eosin. Results. The use of enzymatic digestion methods allowed us to efficiently generate primary cell cultures of the human bladder epithelial and stromal cells. The proliferation rate was higher in stromal cells as compared to epithelial cells. Once the bladder mucosa substitutes were generated, a good biocompatibility of the stromal and epithelial cells into the biomaterial was found. The histological structure of the bladder wall substitutes was analogue to that of the native human bladder mucosa. Conclusions. The bladder mucosa substitute generated by tissue engineering showed structural and histological similarities with the native human bladder tissues and open the door to the future therapeutic use of these bioengineered tissues (AU)

The effects of fibrin and fibrin-agarose on the extracellular matrix profile of bioengineered oral mucosa.

Several studies have developed efficient oral mucosa constructs using different types of scaffold. However, the changes in the morphology and gene and protein expression profile that could occur in these artificial constructs remain unknown. This study compared the histology and expression of several extracellular matrix molecules in human artificial oral mucosa developed using two different types of scaffolds: fibrin and fibrin-agarose. To that end, bioengineered oral mucosa stromas were constructed from biopsy samples of human oral mucosa and the substitute generated was analyzed at different periods of time in culture. Histological analysis was carried out by light and transmission electron microscopy and the expression of collagen types I, III, and VI, the proteoglycans decorin and biglycan, and the different chains of laminin, were assessed by immunoperoxidase technique. This study found that fibrin scaffolds accelerated fibroblast growth and remodeling of the scaffold, thus enhancing collagen fibrillogenesis. In the fibrin-agarose scaffold, the morphology and organization of the fibroblasts did not change during the culture period. All extracellular matrix proteins analyzed were expressed in both scaffolds. However, in fibrin scaffolds, these proteins were widely distributed and replaced the scaffold during the follow-up period. These results show that the substitutes generated showed histological and molecular similarities with native human oral mucosa stroma. In addition, it was observed that the nature of the biomaterial influenced the behaviour of the oral stromal fibroblasts, thereby modulating their growth, protein synthesis, and collagen fibrillogenesis.

[In vitro generation of a human bladder wall substitute by tissue engineering]. / Generación in vitro de un sustituto de pared vesical humana obtenido mediante ingeniería tisular.

INTRODUCTION: Certain urological congenital conditions, such as bladder exstrophy and acquired conditions such as trauma and tumors may require the use of different tissues like small bowel, sigmoid colon or stomach for bladder reconstruction. However, these tissues are often associated to important complications. The aim of this study is to develop a novel substitute of the human bladder wall by tissue engineering. MATERIAL AND METHODS: We first generated primary cell cultures of epithelial and stromal bladder mucosa cells from small tissue biopsies of human bladder by using enzymatic methods based on trypsin-EDTA and collagenase I. Then, a three-dimensional substitute of the bladder mucosa was generated using fibrin-agarose biomaterials. The analysis of the tissue substitutes was carried out at day 14th of development by histological examination of samples stained with hematoxylin-eosin. RESULTS: The use of enzymatic digestion methods allowed us to efficiently generate primary cell cultures of the human bladder epithelial and stromal cells. The proliferation rate was higher in stromal cells as compared to epithelial cells. Once the bladder mucosa substitutes were generated, a good biocompatibility of the stromal and epithelial cells into the biomaterial was found. The histological structure of the bladder wall substitutes was analogue to that of the native human bladder mucosa. CONCLUSIONS: The bladder mucosa substitute generated by tissue engineering showed structural and histological similarities with the native human bladder tissues and open the door to the future therapeutic use of these bioengineered tissues.

Generación de un sustituto de mucosa oral humana y comprobación de su viabilidad mediante ingeniería tisular / Generation of a substitute for human oral mucosa and verification of its viability by tissue-engineering

La reconstrucción de grandes defectos de mucosa oral a menudo es desafiante, por la escasez de mucosa oral sana para reemplazar tejidos dañados. De esta forma, las técnicas de ingeniería tisular pueden suponer una fuente de tejidos autólogos disponible para trasplantar a estos pacientes. En este trabajo hemos desarrollado un nuevo modelo de mucosa oral artificial generada mediante ingeniería tisular usando un soporte de fibrina-agarosa. Para ello, se han generado cultivos primarios de fibroblastos de la mucosa oral humana y queratinocitos a partir de pequeñas biopsias de mucosa oral normal y aplicándoles tratamiento senzimáticos. Después, se ha determinado la viabilidad de las células cultivadas mediante microanálisis por rayos-X, demostrando que la mayoría de las células de los cultivos primarios estaban vivas y tenían elevados K/Na ratios. Una vez que la viabilidad celular fue determinada, se usaron los fibroblastos y queratinocitos cultivados para desarrollar un constructo de mucosa oral sobre una matriz extracelular de (..) (AU)

Reconstruction of large oral mucosa defects is often challenging, since the shortage of healthy oral mucosa to replace the excised tissues. This way, tissue ingineering techniques may provide a source of autologoustissues available for transplant in these patients. In this work, wehave developed a new model for artificial oral mucosa generated by tissue engineering using a fibrin-agarosa scaffold. For that purpose, we have generated primary cultures of human oral mucosa fibroblasts and keratinocytes from small biopsies of normal mucosa oral using enzymatic treatments. Then, we have determined the viability of cultured cells by electron probe quantitative X-ray microanalysis, and we have demonstrated that most of the cells in the primary cultures were alive and hd high K/Na ratios. Once cell viability was determined, we used cultured fibroblasts and keratinocytes to develop an artificial oral mucosaconstruct by using a fibrin-agarosa extracellular matrix and a sequential culture technique using porous culture inserts. Histological analysis of the artificial tissues showed high similarities with normal oral (..) (AU)

[Generation of a substitute for human oral mucosa and verification of its viability by tissue-engineering]. / Generación de un sustituto de mucosa oral humana y comprobación de su viabilidad mediante ingeniería tisular.

Reconstruction of large oral mucosa defects is often challenging, since the shortage of healthy oral mucosa to replace the excised tissues. This way, tissue ingineering techniques may provide a source of autologous tissues available for transplant in these patients. In this work, we have developed a new model for artificial oral mucosa generated by tissue engineering using a fibrin-agarosa scaffold. For that purpose, we have generated primary cultures of human oral mucosa fibroblasts and keratinocytes from small biopsies of normal mucosa oral using enzymatic treatments. Then, we have determined the viability of cultured cells by electron probe quantitative X-ray microanalysis, and we have demonstrated that most of the cells in the primary cultures were alive and hd high K/Na ratios. Once cell viability was determined, we used cultured fibroblasts and keratinocytes to develop an artificial oral mucosa construct by using a fibrin-agarosa 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 de 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.

Pluripotential differentiation capability of human adipose-derived stem cells in a novel fibrin-agarose scaffold.

The potentiality of adipose-derived stem cells (ASCs) cultured on 2D systems has been previously established. Nevertheless, very little is known so far about the differentiation potentiality of ASCs in 3D culture systems using biomaterials. In this work, we have evaluated the transdifferentiation capabilities of ASCs cultured within a novel fibrin-agarose biomaterial by histological analysis, histochemistry and immunofluorescence. Our results showed that 3D fibrin-agarose biomaterial is highly biocompatible and supports the transdifferentiation capabilities of ASCs to the osteogenic, chondrogenic, adipogenic, and neurogenic lineages.