Ingrid Garzón

Combination of fibrin-agarose hydrogels and adipose-derived mesenchymal stem cells for peripheral nerve regeneration

OBJECTIVE The objective was to study the effectiveness of a commercially available collagen conduit filled with fibrin-agarose hydrogels alone or with fibrin-agarose hydrogels containing autologous adipose-derived mesenchymal stem cells (ADMSCs) in a rat sciatic nerve injury model. APPROACH A 10 mm gap was created in the sciatic nerve of 48 rats and repaired using saline-filled collagen conduits or collagen conduits filled with fibrin-agarose hydrogels alone (acellular conduits) or with hydrogels containing ADMSCs (ADMSC conduits). Nerve regeneration was assessed in clinical, electrophysiological and histological studies. MAIN RESULTS Clinical and electrophysiological outcomes were more favorable with ADMSC conduits than with the acellular or saline conduits, evidencing a significant recovery of sensory and motor functions. Histological analysis showed that ADMSC conduits produce more effective nerve regeneration by Schwann cells, with higher remyelination and properly oriented axonal growth that reached the distal areas of the grafted conduits, and with intensely positive expressions of S100, neurofilament and laminin. Extracellular matrix was also more abundant and better organized around regenerated nerve tissues with ADMSC conduits than those with acellular or saline conduits. SIGNIFICANCE Clinical, electrophysiological and histological improvements obtained with tissue-engineered ADMSC conduits may contribute to enhancing axonal regeneration by Schwann cells.

Transdifferentiation potentiality of human Wharton's jelly stem cells towards vascular endothelial cells†

Human Whartons jelly stem cells (HWJSC) emerged as a potential source of viable cells for use in tissue engineering. In this work, we have analyzed the transdifferentiation capabilities of HWJSC towards transdifferentiated endothelial‐like cells (Tr‐ELC) in order to establish the potential usefulness of these cells in vascular tissue engineering. Our results show that Tr‐ELC became more polygonal and less proliferative than HWJSC, resembling the structure and proliferation rate of the endothelial cells. In addition, the markers of mesenchymal undifferentiation CD9, E‐cad, PODXL, and SSEA‐4 are downregulated in Tr‐ELC, suggesting that these cells can be in the process of adult differentiation. Besides, RT‐PCR and microarray analyses revealed that some genes with a role in defining the endothelial phenotype and structure are upregulated (VEGF‐R1, EDF1, AAMP, CD31, CD34, CDH5, and ICAM2) or downregulated (VEGF) in Tr‐ELC, although a number of genes related to relevant endothelial cell functions (CD36, ECE2, VWF, THBD, PGI2, ECE1, and ACE) did not change or were only partially induced. All this implies that HWJSC are able to efficiently transdifferentiate towards Tr‐ELC at the phenotypical level following a hierarchical pattern of gene activation, with an earlier induction of morphological and phenotypical genes. J. Cell. Physiol. 223:640–647, 2010.

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.

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.

A Novel Histochemical Method for a Simultaneous Staining of Melanin and Collagen Fibers

For the histopathologic diagnosis of melanocytic lesions, it could be necessary to identify the melanin pigment because its visualization is unspecific with hematoxylin-eosin (HE). The Fontana-Masson (FM) technique is used in histopathology in this type of lesion, which allows the identification of the pigment, but it loses all the morphologic parameters. The authors describe a modification to the FM method, for the evaluation of the morphology, the argentaffin reaction of the melanin, and collagens fibers of the extracellular matrix simultaneously, for which they have developed the Fontana-Masson picrosirius (FMPS) method. Biopsies of different melanocytic lesions were used for the performance of the HE, FM, and FMPS methods. The pixel intensity of the reaction for melanin, collagen, and epithelium was determined with ImageJ software. The FMPS method allows the evaluation of morphological characteristics, identifying the melanin pigment and collagen fibers with high intensity simultaneously. This method does not differ significantly from FM in the identification of melanin, maintaining its sensitivity and specificity. In addition, it does not differ in the demonstration of the morphology with HE. However, FMPS is significantly superior in the identification of collagen fibers. The FMPS method combines morphological and histochemical parameters that could be useful in the study of pigmented lesions of melanocytic origin.

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

Differential expression of GAP‐43 and neurofilament during peripheral nerve regeneration through bio‐artificial conduits

Nerve conduits are promising alternatives for repairing nerve gaps; they provide a close microenvironment that supports nerve regeneration. In this sense, histological analysis of axonal growth is a determinant to achieve successful nerve regeneration. To evaluate this process, the most‐used immunohistochemical markers are neurofilament (NF), β‐III tubulin and, infrequently, GAP‐43. However, GAP‐43 expression in long‐term nerve regeneration models is still poorly understood. In this study we analysed GAP‐43 expression and its correlation with NF and S‐100, using three tissue‐engineering approaches with different regeneration profiles. A 10 mm gap was created in the sciatic nerve of 12 rats and repaired using collagen conduits or collagen conduits filled with fibrin–agarose hydrogels or with hydrogels containing autologous adipose‐derived mesenchymal stem cells (ADMSCs). After 12 weeks the conduits were harvested for histological analysis. Our results confirm the long‐term expression of GAP‐43 in all groups. The expression of GAP‐43 and NF was significantly higher in the group with ADMSCs. Interestingly, GAP‐43 was observed in immature, newly formed axons and NF in thicker and mature axons. These proteins were not co‐expressed, demonstrating their differential expression in newly formed nerve fascicles. Our descriptive and quantitative histological analysis of GAP‐43 and NFL allowed us to determine, with high accuracy, the heterogenic population of axons at different stages of maturation in three tissue‐engineering approaches. Finally, to perform a complete assessment of axonal regeneration, the quantitative immunohistochemical evaluation of both GAP‐43 and NF could be a useful quality control in tissue engineering. Copyright

Evaluation of myelin sheath and collagen reorganization pattern in a model of peripheral nerve regeneration using an integrated histochemical approach

Peripheral nerves are complex histological structures that can be affected by a variety of conditions with different degree of axonal degeneration and demyelination. For the study of peripheral nerve regeneration in pathology and tissue engineering, it is necessary to evaluate the regeneration, remyelination and extracellular matrix reorganization of the neural tissue. Currently, different histochemical techniques must be used in parallel, and a correlation among their findings should be further performed. In this work, we describe a new histochemical method for myelin and collagen fibers based on luxol fast blue and picrosirius methods, for the evaluation of the morphology, the myelin sheath and the collagen fiber reorganization using a model of peripheral nerve regeneration. Whole brain, normal sciatic nerve and regenerating peripheral nerve samples were fixed in 10% neutral buffered formalin and paraffin-embedded, for the performance of the hematoxylin-eosin stain, the Luxol fast blue method and the new histochemical method for myelin and collagen. The results of this technique revealed that this new histochemical method allowed us to properly evaluate histological patterns, and simultaneously observe the histochemical reaction for myelin sheath and collagen fibers in normal tissue, and during the regeneration process. In conclusion, this new method combines morphological and histochemical properties that allowed us to determine with high accuracy the degree of remyelination and collagen fibers reorganization. For all these reasons, we hypothesize that this new histochemical method could be useful in pathology and tissue engineering.

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.