M. C. Sánchez-Quevedo

In vitro and in vivo cytokeratin patterns of expression in bioengineered human periodontal mucosa.

BACKGROUND AND OBJECTIVE Development of human oral mucosa substitutes by tissue engineering may provide new therapeutic tools for the management of periodontal diseases. In this study we evaluated a fibrin-agarose human oral mucosa substitute both in vitro and in vivo. MATERIAL AND METHODS In vitro bioengineered oral mucosa substitutes were developed from irrelevant biopsy samples of human oral gingiva. In vivo evaluation of the constructed tissues was performed by implantation into athymic nude mice. The expression of several epithelial markers was assessed by microarray analysis and immunohistochemistry. RESULTS Bioengineered oral mucosa samples kept in vitro developed a multilayered epithelium that expressed several cytokeratins, including some markers of simple epithelia (cytokeratins 7, 8 and 18), along with markers of stratified epithelia (cytokeratins 5 and 13) and of cell proliferation (proliferating cell nuclear antigen). Bioengineered tissues grafted in vivo onto nude mice exhibited very good biointegration with the host, showing a cytokeratin expression pattern that was very similar to that of normal native oral mucosa controls. Histological analysis of the artificial tissues demonstrated that oral mucosa substitutes evaluated in vivo were structurally mature, showing some typical structures of human native oral mucosa such as rete ridges and chorial papillae, along with numerous blood vessels at the fibrin-agarose stromal substitute. These structures were absent in samples evaluated in vitro. CONCLUSION The results indicate that this model of human oral mucosa, constructed using fibrin-agarose scaffolds, shows similarities to native oral mucosa controls and imply that bioengineered oral mucosa substitutes could eventually be used clinically.

Epithelial and Stromal Developmental Patterns in a Novel Substitute of the Human Skin Generated with Fibrin-Agarose Biomaterials

Development of human skin substitutes by tissue engineering may offer new therapeutic alternatives to the use of autologous tissue grafts. For that reason, it is necessary to investigate and develop new biocompatible biomaterials that support the generation of a proper human skin construct. In this study, we generated a novel model of bioengineered human skin substitute using human cells obtained from skin biopsies and fibrin-agarose biomaterials and we evaluated this model both at the ex vivo and the in vivo levels. Once the dermal fibroblasts and the epithelial keratinocytes were isolated and expanded in culture, we used fibrin-agarose scaffolds for the development of a full-thickness human skin construct, which was evaluated after 1, 2, 3 and 4 weeks of development ex vivo. The skin substitutes were then grafted onto immune-deficient nude mice and analyzed at days 10, 20, 30 and 40 postimplantation using transmission electron microscopy, histochemistry and immunofluorescence. The results demonstrated that the fibrin-agarose artificial skin had adequate biocompatibility and proper biomechanical properties. A proper development of both the bioengineered dermis and epidermis was found after 30 days in vivo, although the tissues kept ex vivo and those implanted in the animal model for 10 or 20 days showed lower levels of differentiation. In summary, our model of fibrin-agarose skin equivalent was able to reproduce the structure and histological architecture of the native human skin, especially after long-term in vivo implantation, suggesting that these tissues could reproduce the native skin.

Sequential development of intercellular junctions in bioengineered human corneas

We have carried out a sequential study of intercellular junction formation and differentiation on human corneal substitutes consisting of an artificial corneal stroma and a corneal epithelium, developed by tissue engineering. To generate these artificial human corneas, we developed a corneal stroma substitute, using fibrin and agarose scaffolds with human keratocytes immersed within, then cultured the human corneal epithelium on top. Electron microscopy and immunofluorescence analyses revealed that artificial corneas with one or two epithelial cell layers did not show any formation of intercellular junctions. In contrast, several types of cell–cell junction, especially desmosomes, were found in multilayered mature corneal substitutes. Concomitantly, the expression of genes encoding for plakoglobin 3 (PKG3), desmoglein 3 (DSG3) and desmoplakin (DSP), zonula occludens 1 (ZO‐1) and 2 (ZO‐2) and connexin 37 (Cx37) was higher in multilayered artificial corneas than in immature artificial corneas, as shown by both microarray and immunofluorescence. Although expression of ZO‐1, ZO‐2 and Cx37 proteins was homogeneous, PKG3, DSG3 and DSP expression was restricted to the most apical cell layers in artificial corneas submerged in culture medium at all times, whereas expression was higher in intermediate cell layers, similar to normal human control corneas, when corneal substitutes are submitted to air–liquid culture techniques. These results suggest that cultured corneal substitutes submitted to air–liquid culture technique tend to form a well‐developed epithelium that is very similar to the epithelium of human native corneas, suggesting that these artificial corneas could eventually be used for clinical or in vitro purposes. Copyright

Evaluation of the viability of cultured corneal endothelial cells by quantitative electron probe X-ray microanalysis

Construction of artificial organs and tissues by tissue engineering is strongly dependent on the availability of viable cells. For that reason, the viability and the physiological status of cells kept in culture must be evaluated before the cells can be used for clinical purposes. In this work, we determined the viability of isolated rabbit corneal endothelial cells by trypan blue staining and quantitative electron probe X‐ray microanalysis. Our results showed that the ionic content of potassium in cultured corneal endothelial cells tended to rise initially, but significantly decreased in cells in the fifth (and final) subculture, especially in comparison to cells in the fourth subculture (P < 0.001). However, the concentration of sulfur was higher in the fifth subculture than in the fourth subculture (P < 0.001), with a nonsignificant increase in sodium in the fifth subculture (P = 0.031). These data imply a remarkable decrease in the K/Na ratio from the fourth to the fifth subculture. Our microanalytical results, along with the morphological differences between cells in the last two subcultures, are compatible with an early phase of the preapoptotic process in the fifth subculture, and suggest that cells of the first four subcultures would be better candidates for tissue engineering. J. Cell. Physiol. 211: 692–698, 2007.

X-ray microanalytical histochemistry of human circumpulpar and mantle dentine.

An X-ray microanalytical histochemistry study was carried out, on thick sections observed under scanning electron microscopy, of five freshly extracted human premolars and five molars. In particular the circumpulpar and mantle dentine were examined to determine levels of calcium, phosphorus and sulphur, the basic elements involved in the process of biomineralization. Semiquantitative analysis was subsequently performed after the appropriate ZAF (Z, atomic number; A, absorption; F, secondary fluorescence) correction. Ca/P mass ratio was found to be similar for both regions in molars and slightly higher in circumpulpar dentine in premolars implying that this parameter is independent of the processes of biomineralization in the two dentinal regions. In contrast, statistically significant differences were recorded in sulphur content upon comparing mantle and circumpulpar dentine between premolars and molars (P less than 0.02). If sulphur, which has been associated with sulphated glycosaminoglycans (GAGs) decreases as mineralization progresses it would therefore seem reasonable to posit sulphur-rich areas in both topographical regions of the matrix--more significant in premolars than molars--able to undergo subsequent mineralization if required. The possible application of these findings in remineralization therapies is suggested.

Rheological characterization of human fibrin and fibrin-agarose oral mucosa substitutes generated by tissue engineering.

In regenerative medicine, the generation of biocompatible substitutes of tissues by in vitro tissue engineering must fulfil certain requirements. In the case of human oral mucosa, the rheological properties of tissues deserve special attention because of their influence in the acoustics and biomechanics of voice production. This work is devoted to the rheological characterization of substitutes of the connective tissue of the human oral mucosa. Two substitutes, composed of fibrin and fibrin–agarose, were prepared in cell culture for periods in the range 1–21 days. The time evolution of the rheological properties of both substitutes was studied by two different experimental procedures: steady‐state and oscillatory measurements. The former allows the plastic behaviour of the substitutes to be characterized by estimating their yield stress; the latter is employed to quantify their viscoelastic responses by obtaining the elastic (G′) and viscous (G′′) moduli. The results demonstrate that both substitutes are characterized by a predominant elastic response, in which G′ (order 100 Pa) is roughly one order of magnitude larger than G′′ (order 10 Pa). But the most relevant insight is the stability, throughout the 21 days of culture time, of the rheological quantities in the case of fibrin–agarose, whereas the fibrin substitute shows a significant hardening. This result provides evidence that the addition to fibrin of a small amount of agarose allows the rheological stability of the oral mucosa substitute to be maintained. This feature, together with its viscoelastic similitude with native tissues, makes this biomaterial appropriate for potential use as a scaffold in regenerative therapies of human oral mucosa. Copyright

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. Copyright

Dentin in Severe Fluorosis: a Quantitative Histochemical Study

Dentin responds to different alterations in the enamel with hypermineralization, and is a biomarker of fluoride exposure. We hypothesized that severe fluorosis would lead to hypermineralization of the dentin when the enamel was severely affected. We used scanning electron microscopy and quantitative electron-probe microanalysis to compare dentin and enamel from healthy and fluorotic teeth. The dentin in fluorotic teeth was characterized by a highly mineralized sclerotic pattern, in comparison with control teeth (p < 0.001) and fluorotic enamel lesions (p < 0.001). Enamel near the lesions showed hypercalcification in comparison with dentin (p < 0.001). In response to the effects of severe fluorosis in the enamel, the dentin showed hypermineralization, as found in other enamel disorders. The hypermineralization response of the dentin in our samples suggests that the mechanism of the response should be taken into account in dental caries and other dental disorders associated with severe fluorosis.

Cell Viability and Prostacyclin Release in Cultured Human Umbilical Vein Endothelial Cells

Construction of efficient substitutes of human blood vessels is strongly dependent on the use of viable and fully functional cultured endothelial cells (ECs). However, very few reports have been published to date focused on the evaluation of cell viability of cultured ECs. In this work, we have determined cell viability, von Willebrand factor, and prostacyclin (PGI(2)) activity in primary cell cultures of human umbilical vein ECs, to identify the specific cell passage that is more appropriate for the development of artificial organs by tissue engineering. Cell viability was determined by quantification of the intracellular concentration of several ions by highly sensitive electron probe X-ray microanalysis, whereas von Willebrand was assayed by immunohistochemistry and PGI(2) release was quantified by radioimmunoassay. The results of our analyses demonstrate that the K/Na ratio was different for each cell passage (4.72 for the first passage, 4.55 for the second passage, and 7.82 for the third passage), suggesting that the highest cell viability corresponds to the third passage. In contrast, PGI(2) production was higher at the first two cell passages, with a significant decrease at the third passage (6.46 +/- 0.10, 5.98 +/- 0.08, and 1.62 +/- 0.05 ng/mL of supernatant for the first, second, and third passages, respectively), whereas von Willebrand expression was similar among the three cell passages analyzed in this work (64.12%, 66.66%, 65.93% of positive cells, respectively). These data suggest that cells corresponding to the second cell passage show the best ratio of viability to functionality and should therefore be used for tissue engineering protocols.

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 Whartons 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 Whartons 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.