María del Carmen Sánchez-Quevedo

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.

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.

Average cell viability levels of human dental pulp stem cells: an accurate combinatorial index for quality control in tissue engineering

BACKGROUND AIMS One of the most important issues in tissue engineering (TE) is the search for a suitable stem cell reservoir with optimal cell viability levels for the development of new tissues relevant for therapeutic needs. The aim of this study was to evaluate the cell viability levels of 10 sequential cell passages of human dental pulp stem cells (hDPSC) to determine their potential for TE techniques. METHODS To assess the average cell viability levels of hDPSC, four cell viability assays were used in a combinatorial approach: trypan blue exclusion test, water-soluble tetrazolium 1 assay, live/dead assay and electron probe x-ray microanalysis. RESULTS The results showed that cell viability as determined by trypan blue staining and live/dead assays was greater than 85%, with a significant decrease at the second passage (P < 0.05) and a significant increase at the ninth passage (P < 0.05). Electron probe x-ray microanalysis showed that the highest cell viability corresponded to the ninth passage, with the lowest K/Na values found at the third passage. No statistical differences were found among the different passages for the water-soluble tetrazolium 1 assay (P = 0.219). CONCLUSIONS Assessment of average cell viability levels showed that the highest viability of hDPSC was reached after nine passages, suggesting that this passage would be the most adequate for use in TE protocols.

A Combined Approach for the Assessment of Cell Viability and Cell Functionality of Human Fibrochondrocytes for Use in Tissue Engineering

Temporo-mandibular joint disc disorders are highly prevalent in adult populations. Autologous chondrocyte implantation is a well-established method for the treatment of several chondral defects. However, very few studies have been carried out using human fibrous chondrocytes from the temporo-mandibular joint (TMJ). One of the main drawbacks associated to chondrocyte cell culture is the possibility that chondrocyte cells kept in culture tend to de-differentiate and to lose cell viability under in in-vitro conditions. In this work, we have isolated human temporo-mandibular joint fibrochondrocytes (TMJF) from human disc and we have used a highly-sensitive technique to determine cell viability, cell proliferation and gene expression of nine consecutive cell passages to determine the most appropriate cell passage for use in tissue engineering and future clinical use. Our results revealed that the most potentially viable and functional cell passages were P5–P6, in which an adequate equilibrium between cell viability and the capability to synthesize all major extracellular matrix components exists. The combined action of pro-apoptotic (TRAF5, PHLDA1) and anti-apoptotic genes (SON, HTT, FAIM2) may explain the differential cell viability levels that we found in this study. These results suggest that TMJF should be used at P5–P6 for cell therapy protocols.

Ex vivo characterization of a novel tissue-like cross-linked fibrin-agarose hydrogel for tissue engineering applications

The generation of biomaterials with adequate biomechanical and structural properties remains a challenge in tissue engineering and regenerative medicine. Earlier research has shown that nanostructuration and cross-linking techniques improved the biomechanical and structural properties of different biomaterials. Currently, uncompressed and nanostructured fibrin-agarose hydrogels (FAH and NFAH, respectively) have been used successfully in tissue engineering. The aim of this study was to investigate the possibility of improving the structural and biomechanical properties of FAH and NFAH by using 0.25% and 0.5% (v/v) glutaraldehyde (GA) as a cross-linker. These non-cross-linked and cross-linked hydrogels were subjected to structural, rheological and ex vivo biocompatibility analyses. Our results showed that GA cross-linking induced structural changes and significantly improved the rheological properties of FAH and NFAH. In addition, ex vivo biocompatibility analyses demonstrated viable cells in all conditions, although viability was more compromised when 0.5% GA was used. Our study demonstrates that it is possible to control fiber density and hydrogel porosity of FAH and NFAH by using nanostructuration or GA cross-linking techniques. In conclusion, hydrogels cross-linked with 0.25% GA showed promising structural, biochemical and biological properties for use in tissue engineering.

Bone mineral density aspects in the femoral neck of hip fracture patients

Elderly people, due to neurological conditions and muscular atrophy, present a greater propensity to falls and thus are very susceptible to hip fractures. Other variables, such as osteoporosis, may also be related to the etiopathogenesis of hip fractures, although osteoporosis is in fact a concurrent disease, and merely a coadjutant cause. Nonetheless, osteoporosis can make fracture patterns more severe and interfere with osteosynthesis. Osteoporosis is the radiological image of osteopenia, a pathological concept meaning a smaller quantity of bone per unit of volume. The radiological expression of osteopenia is therefore that of bone tissue with a lower radiological density than normal. In the context of hip fractures, bone mineral density and bone architecture of the femoral neck together with protein expression profiles and cross-links of this anatomical area are of special interest which is reviewed in the current paper. Spatial variations in bone mineral density in the femoral neck were found in the literature with increased porosity from the periosteal to the endosteal region and also from the distal to the proximal part of the femoral neck. Furthermore, increased crystal size, increased cortical porosity, reduced osteocyte lacunar density and an increased Ca/P ratio associated with higher concentrations of Ca and P were described in hip fracture patients compared to control patients. Osteocalcin/collagen type 1 expression ratio and enzymatic cross-link content in high-density bone was found to be significantly lower in hip fractures compared to controls. In conclusion, further research in bone mineral density and associated parameters are of interest to deepen the understanding of osteoporotic hip fractures.

An early and late cytotoxicity evaluation of lidocaine on human oral mucosa fibroblasts

Local anesthetic drugs are extensively used in dentistry. However, the cytotoxic effects of these pharmaceutical compounds remain unclear. In this work, we have evaluated the cell viability and cell function of human oral mucosa fibroblasts exposed to different concentrations of lidocaine for increasing incubation times, using a global screening methods including structural, metabolic and microanalytical analyses. Our results demonstrate that lidocaine is able to alter cell viability and function even at low concentrations and times, although the effect of lidocaine concentration was more important than the incubation time. First, the structural analysis methods revealed that ≥5% concentrations of lidocaine are able to significantly reduce cell viability. Then, the metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and water-soluble tetrazolium salt (WST-1) assays suggest that concentrations starting from 1% were able to significantly hinder cell physiology. Finally, electron-probe X-ray microanalysis confirmed the deleterious effects of lidocaine and allowed us to demonstrate that these effects are associated to an apoptosis process of cell death. Therefore, care should be taken when lidocaine is clinically used, and the lowest efficient concentrations should always be used. Furthermore, these results suggest that the comprehensive evaluation method used in this work is accurate and efficient for screening of local anesthetics.

Qualitative and Quantitative Evaluation of a Novel Detergent-Based Method for Decellularization of Peripheral Nerves

Tissue engineering is an emerging strategy for the development of nerve substitutes for peripheral nerve repair. Especially decellularized peripheral nerve allografts are interesting alternatives to replace the gold standard autografts. In this study, a novel decellularization protocol was qualitatively and quantitatively evaluated by histological, biochemical, ultrastructural and mechanical methods and compared to the protocol described by Sondell et al. and a modified version of the protocol described by Hudson et al. Decellularization by the method described by Sondell et al. resulted in a reduction of the cell content, but was accompanied by a loss of essential extracellular matrix (ECM) molecules such as laminin and glycosaminoglycans. This decellularization also caused disruption of the endoneurial tubes and an increased stiffness of the nerves. Decellularization by the adapted method of Hudson et al. did not alter the ECM composition of the nerves, but an efficient cell removal could not be obtained. Finally, decellularization by the method developed in our lab by Roosens et al. led to a successful removal of nuclear material, while maintaining the nerve ultrastructure and ECM composition. In addition, the resulting ECM scaffold was found to be cytocompatible, allowing attachment and proliferation of adipose-derived stem cells. These results show that our decellularization combining Triton X-100, DNase, RNase and trypsin created a promising scaffold for peripheral nerve regeneration.