Ana Celeste Oliveira

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.

Generation and Characterization of Novel Magnetic Field-Responsive Biomaterials.

We report the preparation of novel magnetic field-responsive tissue substitutes based on biocompatible multi-domain magnetic particles dispersed in a fibrin–agarose biopolymer scaffold. We characterized our biomaterials with several experimental techniques. First we analyzed their microstructure and found that it was strongly affected by the presence of magnetic particles, especially when a magnetic field was applied at the start of polymer gelation. In these samples we observed parallel stripes consisting of closely packed fibers, separated by more isotropic net-like spaces. We then studied the viability of oral mucosa fibroblasts in the magnetic scaffolds and found no significant differences compared to positive control samples. Finally, we analyzed the magnetic and mechanical properties of the tissue substitutes. Differences in microstructural patterns of the tissue substitutes correlated with their macroscopic mechanical properties. We also found that the mechanical properties of our magnetic tissue substitutes could be reversibly tuned by noncontact magnetic forces. This unique advantage with respect to other biomaterials could be used to match the mechanical properties of the tissue substitutes to those of potential target tissues in tissue engineering applications.

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.

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.

Effects of four formulations of prostaglandin analogs on eye surface cells : a comparative study

We evaluated the cytotoxic effects of four prostaglandin analogs (PGAs) used to treat glaucoma. First we established primary cultures of conjunctival stromal cells from healthy donors. Then cell cultures were incubated with different concentrations (0, 0.1, 1, 5, 25, 50 and 100%) of commercial formulations of bimatoprost, tafluprost, travoprost and latanoprost for increasing periods (5 and 30 min, 1 h, 6 h and 24 h) and cell survival was assessed with three different methods: WST-1, MTT and calcein/AM-ethidium homodimer-1 assays. Our results showed that all PGAs were associated with a certain level of cell damage, which correlated significantly with the concentration of PGA used, and to a lesser extent with culture time. Tafluprost tended to be less toxic than bimatoprost, travoprost and latanoprost after all culture periods. The results for WST-1, MTT and calcein/AM-ethidium homodimer-1 correlated closely. When the average lethal dose 50 was calculated, we found that the most cytotoxic drug was latanoprost, whereas tafluprost was the most sparing of the ocular surface in vitro. These results indicate the need to design novel PGAs with high effectiveness but free from the cytotoxic effects that we found, or at least to obtain drugs that are functional at low dosages. The fact that the commercial formulation of tafluprost used in this work was preservative-free may support the current tendency to eliminate preservatives from eye drops for clinical use.