Miguel González-Andrades

Evaluation of Small Intestine Grafts Decellularization Methods for Corneal Tissue Engineering

Advances in the development of cornea substitutes by tissue engineering techniques have focused on the use of decellularized tissue scaffolds. In this work, we evaluated different chemical and physical decellularization methods on small intestine tissues to determine the most appropriate decellularization protocols for corneal applications. Our results revealed that the most efficient decellularization agents were the SDS and triton X-100 detergents, which were able to efficiently remove most cell nuclei and residual DNA. Histological and histochemical analyses revealed that collagen fibers were preserved upon decellularization with triton X-100, NaCl and sonication, whereas reticular fibers were properly preserved by decellularization with UV exposure. Extracellular matrix glycoproteins were preserved after decellularization with SDS, triton X-100 and sonication, whereas proteoglycans were not affected by any of the decellularization protocols. Tissue transparency was significantly higher than control non-decellularized tissues for all protocols, although the best light transmittance results were found in tissues decellularized with SDS and triton X-100. In conclusion, our results suggest that decellularized intestinal grafts could be used as biological scaffolds for cornea tissue engineering. Decellularization with triton X-100 was able to efficiently remove all cells from the tissues while preserving tissue structure and most fibrillar and non-fibrillar extracellular matrix components, suggesting that this specific decellularization agent could be safely used for efficient decellularization of SI tissues for cornea TE applications.

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. Copyright

Wharton's Jelly Stem Cells: A Novel Cell Source for Oral Mucosa and Skin Epithelia Regeneration

Perinatal stem cells such as human umbilical cord Whartons jelly stem cells (HWJSCs) are excellent candidates for tissue engineering because of their proliferation and differentiation capabilities. However, their differentiation potential into epithelial cells at in vitro and in vivo levels has not yet been reported. In this work we have studied the capability of HWJSCs to differentiate in vitro and in vivo to oral mucosa and skin epithelial cells using a bioactive three‐dimensional model that mimics the native epithelial‐mesenchymal interaction. To achieve this, primary cell cultures of HWJSCs, oral mucosa, and skin fibroblasts were obtained in order to generate a three‐dimensional heterotypical model of artificial oral mucosa and skin based on fibrin‐agarose biomaterials. Our results showed that the cells were unable to fully differentiate to epithelial cells in vitro. Nevertheless, in vivo grafting of the bioactive three‐dimensional models demonstrated that HWJSCs were able to stratify and to express typical markers of epithelial differentiation, such as cytokeratins 1, 4, 8, and 13, plakoglobin, filaggrin, and involucrin, showing specific surface patterns. Electron microscopy analysis confirmed the presence of epithelial cell‐like layers and well‐formed cell‐cell junctions. These results suggest that HWJSCs have the potential to differentiate to oral mucosa and skin epithelial cells in vivo and could be an appropriate novel cell source for the development of human oral mucosa and skin in tissue engineering protocols.

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.

Transparency in a fibrin and fibrin-agarose corneal stroma substitute generated by tissue engineering.

Purpose: To examine the transparency characteristics at different times of development in the culture of 2 different types of human corneal stroma substitutes generated by tissue engineering using human fibrin or human fibrin and 0.1% agarose, with human keratocytes entrapped within. Methods: The transparency of these artificial corneal stromas was analyzed after 1, 7, 14, 21, and 28 days of development in culture by determining the scattering and absorption coefficients from the spectral reflectance data of each sample using the Kubelka–Munk equations. Results: The scattering coefficient of both types of bioengineered tissues tended to increase with culture time and wavelength until 550 nm, whereby a slight decrease was observed for longer wavelengths. In general, the spectral distribution of the Kubelka–Munk scattering coefficient of the fibrin–agarose corneal constructs was more stable than that of the fibrin constructs. The absorption coefficient of the human fibrin and fibrin–agarose corneal substitutes tended to decrease with increasing wavelength, and their absolute values were higher for fibrin–agarose than for fibrin scaffolds, especially for short wavelengths. In addition, the spectral transmittance behavior of both types of tissue analyzed was similar to the ones of the theoretical and control corneas, with absolute values above 90% for all wavelengths at 28 days of development. Conclusions: The transparency, scattering, and absorption of both fibrin and fibrin–agarose corneal stroma substitutes indicate that these new biomaterials could be adequate for clinical use.

UV absorbance of a bioengineered corneal stroma substitute in the 240-400 nm range.

Purpose: To determine the UV absorbance of a bioengineered human corneal stroma construct based on fibrin and fibrin-agarose scaffolds in the 240-400 nm range. Methods: Three types of artificial substitutes of the human corneal stroma were developed by tissue engineering using fibrin and fibrin with 0.1% and 0.2% agarose scaffolds with human keratocytes immersed within. After 28 days of culture, the UV absorbance of each sample was determined using a spectrophotometer. The thickness of corneal stroma samples was determined by light microscope. Results: For all the 3 types of corneal stroma substitutes studied, the range of the UV absorbance values was similar to that of the native human corneal stroma, although the fibrin with 0.1% agarose stroma substitute had the best UV filtering properties. The higher UV absorbance of the artificial substitute of the human corneal stroma was in the UV-B and -A ranges, suggesting that these artificial tissues could have potential clinical usefulness and proper UV light-absorption capabilities. Conclusion: Our data suggest that the bioengineered human corneal substitute of fibrin with 0.1% agarose is an effective absorber of harmful UV radiation and could therefore be potentially useful.

Controlling the 3D architecture of Self-Lifting Auto-generated Tissue Equivalents (SLATEs) for optimized corneal graft composition and stability

Ideally, biomaterials designed to play specific physical and physiological roles in vivo should comprise components and microarchitectures analogous to those of the native tissues they intend to replace. For that, implantable biomaterials need to be carefully designed to have the correct structural and compositional properties, which consequently impart their bio-function. In this study, we showed that the control of such properties can be defined from the bottom-up, using smart surface templates to modulate the structure, composition, and bio-mechanics of human transplantable tissues. Using multi-functional peptide amphiphile-coated surfaces with different anisotropies, we were able to control the phenotype of corneal stromal cells and instruct them to fabricate self-lifting tissues that closely emulated the native stromal lamellae of the human cornea. The type and arrangement of the extracellular matrix comprising these corneal stromal Self-Lifting Analogous Tissue Equivalents (SLATEs) were then evaluated in detail, and was shown to correlate with tissue function. Specifically, SLATEs comprising aligned collagen fibrils were shown to be significantly thicker, denser, and more resistant to proteolytic degradation compared to SLATEs formed with randomly-oriented constituents. In addition, SLATEs were highly transparent while providing increased absorption to near-UV radiation. Importantly, corneal stromal SLATEs were capable of constituting tissues with a higher-order complexity, either by creating thicker tissues through stacking or by serving as substrate to support a fully-differentiated, stratified corneal epithelium. SLATEs were also deemed safe as implants in a rabbit corneal model, being capable of integrating with the surrounding host tissue without provoking inflammation, neo-vascularization, or any other signs of rejection after a 9-months follow-up. This work thus paves the way for the de novo bio-fabrication of easy-retrievable, scaffold-free human tissues with controlled structural, compositional, and functional properties to replace corneal, as well as other, tissues.

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.

The Italian Catquest-9SF cataract questionnaire: translation, validation and application

BackgroundTo validate the Catquest-9SF questionnaire in Italian, assess the change in visual disability with cataract surgery and determine the correlation between pre-operative Catquest-9SF scores and Lens Opacities Classification System (LOCS) III cataract grading.MethodsProspective, questionnaire validation study. The Catquest-9SF questionnaire was forward and back translated and completed by 209 Italian patients before and three months following cataract surgery. Rasch analysis was used to assess its psychometric properties.ResultsThe Italian Catquest-9SF demonstrated ordered response categories, unidimensionality (item fit statistics range: 0.73–1.34), adequate person separation (2.04), and no differential item functioning. Mistargeting was evident with a mean difference in item difficulty and person ability of 2.04 logits but improved with inclusion of pre-operative data only. There was a statistically significant (Friedman tests, p < 0.001) median improvement in visual disability of 1.92, 3.57, 1.44 and 2.94 logits in patients undergoing first eye surgery with and without ocular comorbidity, and second eye surgery with and without ocular comorbidity respectively. There was no statistically significant difference in the improvements among the four groups (Kruskal-Wallis H test, X2(3) = 5.445, p = 0.142). There was no correlation between Catquest-9SF scores and nuclear opalescence (rs = 0.049, p = 0.478), nuclear colour (rs = 0.008, p = 0.909), cortical (rs = 0.066, p = 0.341), and posterior subcapsular components (rs = 0.048, p = 0.494).ConclusionsThe Italian Catquest-9SF demonstrated good psychometric properties and is suitable for use in Italian speaking patients. There were similar improvements in visual disability in patients undergoing first or second eye surgery, with or without ocular comorbidity. There was no correlation between pre-operative Catquest-9SF scores and LOCS III cataract grading.