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.

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.

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.

[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.

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.

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.

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.

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.

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.

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.

Effect of dietary (n-9), (n-6) and (n-3) fatty acids on membrane lipid composition and morphology of rat erythrocytes.

Studies focused on the intake of different dietary fats have shown changes in membrane lipid composition and, as a result, alterations in membrane physical properties. These changes affect erythrocyte morphology, receptor activity and oxygen transport, among others. Here, we compare the effects of diets exclusively differing in the type of fat (olive oil rich in monounsaturates, sunflower oil rich in n-6 polyunsaturates and fish oil rich in n-3 polyunsaturates) on fatty acid composition of plasma and erythrocyte membranes and erythrocyte morphology under scanning electron microscopy in rats. Monounsaturates are highest in animals fed olive oil diets; as are linoleic and arachidonic acids in sunflower oil-fed animals and n-3 PUFAs in fish oil-fed animals. The lowest levels of arachidonic acid are found in fish oil-fed animals and so are n-3 PUFAs in sunflower oil-fed animals. Our results show that sunflower oil-fed animals present lower discocyte, the major cell shape related to tissue oxygen supply, and higher codocyte percentages than olive oil- and fish oil-fed groups. Echinocyte percentage is higher in fish oil-fed animals with respect to the other two groups. The collective data indicate that olive oil elevates monounsaturates and the number of discocytes, pointing out a possible beneficial aspect of this dietary fat.

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.

Cell surface patterns in normal human oral gingival epithelium. A quantitative scanning electron microscopy approach.

We used scanning electron microscopy to study the morphological surface patterns of cells that cover the attached gingiva and intervestibular papilla of the human oral gingival epithelium. Five patterns are described on the basis of the overall appearance of morphological surface markers: microvilli, parallel, fingerprint, reticular and pitted. Statistical analyses detected significant differences in the frequency of each pattern in both regions of the oral gingival epithelium, and showed the reticular and fingerprint types to predominate. We propose that our description of the different morphological surface types may be of use as a standard for subsequent cytological studies and characterizations of morphological alterations in diseased gingiva.

Regional differences in cell surface patterns in normal human sulcular epithelium.

Studies with scanning electron microscopy in the normal human sulcular epithelium are scarce, and no precise information exists about cell surface patterns along the epithelium, the frequencies of these patterns, or possible regional differences within the mouth. In five periodontal biopsy specimens each from the anterior and posterior region of the mouth, we observed three cell patterns on the basis of the overall appearance of morphological surface markers in the coronal and apical zones of sulcular epithelium: microvilli; microplicae; and pits. The percentage of keratinocytes showing the microvillous pattern in the surface of apical sulcular epithelium of the posterior region of the mouth was significantly higher than in the anterior region. We posit that the presence, in the bottom of the normal sulcular epithelium in the posterior region of the mouth, of mainly microvillous keratinocytes (the most undifferentiated and least desquamative type of keratinocyte, and thus the most vulnerable to bacterial colonization) can be associated with observations of longitudinal clinical studies of periodontal disease, which suggest that more severe clinical findings are found in the region of the molars.

Mineralization of human premolar occlusal fissures. A quantitative histochemical microanalysis.

The mechanisms of cariogenesis in occlusal fissures remain elusive because of limited information about fissure structure and wall mineralization. The purpose of the present study was to determine the correlation between morphological patterns in occlusal fissures in human premolars and quantitative histochemical patterns of mineralization in the walls of these formations. We used scanning electron microscopy and quantitative X-ray microanalysis with the peak-to-local background ratio method and microcrystalline calcium salts as standards. We distinguished three morphological patterns of fissures in scanning electron microscopic images. The wall of the fissures was less mineralized than the control enamel in all three types of fissures. Because the fissure walls are hypomineralized, we suggest that practicing dentists should take into account the degree of mineralization when they are preparing the fissures for the application of sealant.

Electron probe microanalysis of permanent human enamel and dentine. A methodological and quantitative study.

Sample preparation of dental tissues for quantitative electron microprobe analysis has not been critically examined because of the highly mineralized nature of these structures. The present study was designed to establish the most suitable method for the electron probe quantitative determination of calcium in human permanent enamel and dentine while preserving the morphological features. Comparisons of quantitative data obtained with air-drying and freeze-drying methods showed that calcium in enamel was more accurately measured in specimens prepared with cryopreservation and freeze-drying. No significant differences between the methods tested were found in dentine although cryopreservation and freeze-drying yielded less statistical variability. Moreover this approach did not modify morphological features of interest. We recommend this combination of processing techniques for human permanent teeth not only because it was found the most accurate and least variable in determining calcium concentration, but also because of its potential usefulness in studies of alterations in tooth mineralization.

Scanning electron microscopy and calcification in amelogenesis imperfecta in anterior and posterior human teeth.

Teeth fragments from members of a family clinically and genetically diagnosed as having amelogenesis imperfecta were studied by scanning electron microscopy and X-ray microprobe analysis to establish the morphological patterns and the quantitative concentration of calcium in the enamel of anterior (canine, incisor) and posterior (premolar and molar) teeth. The prism patterns in the enamel of teeth from both regions were parallel or irregularly decussate, with occasional filamentous prisms accompanied by small, irregularly rounded formations. Prismless enamel showed the R- and P-type patterns. Calcium levels in enamel of amelogenesis imperfecta and control teeth differed significantly between anterior and posterior teeth, indicating that the factors that influence normal mineralization in different regions of the dental arch are not altered in the process of amelogenesis imperfecta.

Cutaneous sarcoidosis and foreign bodies. An electron probe roentgenographic microanalytic study.

A 60-year-old woman had extensive and recurrent foreign-body granulomatous inflammation of her forehead. The granulomata contained double refractile crystalline material. Electron probe roentgenographic microanalysis identified silicon, calcium, aluminum, potassium, and sulphur in the crystalline material. Silicon-rich particles are generally considered exogenous. A diagnosis of sarcoidosis was confirmed by the presence of bilateral hilar and mediastinal lymphadenopathy due to biopsy-proven nodal replacement by noninfectious noncaseating granulomata. We conclude that foreign matter may operate as a nidus for the formation of granuloma in sarcoidosis and that the presence of polarizable matter in a sarcoid granuloma does not rule out sarcoidosis.

The crystalline pattern of calcium in different topographical regions of the otoconial membrane. An electron probe and spectroscopic diffraction study.

Quantitative electron probe microanalysis and electron spectroscopic diffraction analysis was used to determine the gradient of distribution of calcium and its crystalline pattern at different levels (lower gelatinous membrane, upper gelatinous membrane and otoliths) in the otoconial membrane of adult OF1 mice. Our quantitative electron probe microanalytical data, obtained with scanning-transmission electron microscopy, indicated that there was a gradient in calcium concentration which increased from the vestibular surface towards the otoliths. Differences between the three regions of the otoconial membrane were statistically significant in both the utricle and saccule. Our results with electron spectroscopic diffraction revealed an increasing crystalline development from the lower gelatinous membrane towards the otoliths. Our findings with both techniques suggest that the gelatinous membrane is involved in the maturation and crystallization of the otoliths.

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.