Is the bone tissue of the femoral neck demineralised in patients with hip fracture?

The aim of this study is to establish the falsifiability of the "osteoporotic hypothesis" for hip fracture, according to which the bone density and mineral composition of bone tissue in patients with hip fracture is poorer than when no such fracture is present, and that this circumstance is relevant to the occurrence of a fracture. The study population consisted of forty patients treated with arthroplasty. Twenty patients with femoral neck fracture and another twenty with hip osteoarthritis received the same diagnostic protocol and the same antibiotic, anaesthetic, surgical and antithrombotic prophylaxis. Levels of calcium (Ca), phosphorus (P) and vitamin D in blood, amongst other values, were determined, and five samples of bone tissue from the proximal femoral metaphysis were obtained and characterised by optical microscopy and microanalytical analysis. No statistically significant differences were observed between the two groups with respect to the trabecular number, area or thickness, or inter-trabecular distance. However, there were differences in the length of the trabeculae, which was greater in the patients with hip osteoarthritis (p = 0.002), but not when the groups were compared by gender. When compared by age, a greater inter-trabecular distance was observed in the patients aged over 75 years (p = 0.036) but there were no differences in the remaining parameters. Serum levels of Ca (p = 0.03), P (p < 0.01) and vitamin D (p < 0.01) were lower in the fracture group. In the quantitative microanalytical analysis, no significant differences were observed in bone levels of Ca or P or in the Ca/P index, nor was there any correlation between serum and levels of bone Ca or P (Ca-0.197:p = 0.314;P-0.274:p = 0.158).Multiple linear regression revealed no correlation between the diagnoses, vitamin D and bone levels of Ca or P. Despite the reduced serum levels of Ca and P in the patients with hip fracture, no correlation was observed with bone levels of Ca and P,which were similar in both groups. There were differences in the organic bone structure, in terms of length and inter-trabecular distance. For patients with osteoporosis, treatment should be aimed at increasing the synthesis of bone trabeculae to reinforce their structure. Nevertheless, no such treatment can prevent falls, and therefore no reduction in hip fractures amongst this population can be assured.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Volumetric and ionic regulation during the in vitro development of a corneal endothelial barrier.

Corneal endothelium is responsible for generating an ion flux between the corneal stroma and the anterior chamber of the eye that is necessary for the cornea to remain transparent. However, the ion transport regulatory mechanisms that develop during the formation of the endothelial barrier are not known. In this study, we determined the influence of cell confluence on cell volume and intracellular ionic content on the corneal endothelial cells of rabbits. Our results demonstrate that non-confluent endothelial cells display a hypertrophic volume increase, with higher intracellular contents of potassium and chlorine than those of confluent cells. In contrast, when cells reach confluence and the endothelial barrier forms, cell volume decreases and the intracellular contents of potassium and chlorine decrease. Our genetic analysis showed a higher expression of CFTR and CA2 genes in non-confluent cells, and of the gene KCNC3 in confluent cells. These results suggest that the normal ionic current that keeps the corneal stroma dehydrated and transparent is regulated by cell-cell contacts and endothelial cell confluence, and could explain why the loss of corneal endothelial cells is often associated with corneal edema and even blindness.

Time-course study of histological and genetic patterns of differentiation in human engineered oral mucosa.

The lack of sufficient oral mucosa available for intra-oral grafting is a major surgical problem, and new sources of oral tissues for clinical use are needed. In this regard, some models of engineered oral mucosa have been reported to date, but little is known about the structural and genetic mechanisms that occur during the process of development and maturation of these tissue substitutes. We have carried out a time-course study of the genes and morphological patterns of cell and tissue differentiation that develop in oral mucosa constructs after 3, 7, 11 and 21 days of development. Our electron microscopy and microarray analyses demonstrated that the oral mucosa constructs generated by tissue engineering undergo a progressive process of cell differentiation with the sequential formation and maturation of several layers of epithelium (with expression of stratifin, sciellin, involucrin, trichohyalin and kallikrein 7), intercellular junctions (with expression of plakophilin, desmocollin, desmoglein and cadherins), cytokeratins, a basement membrane (laminins, collagen IV) and the extracellular matrix (biglycan, matrix metalloproteinases). In conclusion, although the level and type of keratinization developed in vitro could be different, the oral mucosa substitutes were very similar to the native tissues.

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.

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

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

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.

Incontinentia pigmenti: a case report.

Incontinentia pigmenti (IP) is a genodermatosis with an X-linked dominant mode of inheritance, characterized by ectodermal, mesodermal, neurological, ocular, and dental manifestations. The purpose of this case study was to report the oral and dental manifestations of an IP case in a Venezuelan pediatric patient. A 9 year-old Venezuelan girl was evaluated. She showed macular pigmented lesions in her face, trunk, back, legs, and fingers as well as abnormal hair distribution, alopecia on the vertex, and hypoplasia of eyebrows. During the dental examination, conical shaped-teeth and delayed dental eruption was evidenced. The microanalytical examination showed dentin without significant alterations in the mineralization except for hypermineralization in focal areas. In addition, a decrease in the enamel mineralization was observed.