Silvio Eduardo Duailibi

Bioengineered Teeth from Cultured Rat Tooth Bud Cells

The recent bioengineering of complex tooth structures from pig tooth bud tissues suggests the potential for the regeneration of mammalian dental tissues. We have improved tooth bioengineering methods by comparing the utility of cultured rat tooth bud cells obtained from three- to seven-day post-natal (dpn) rats for tooth-tissue-engineering applications. Cell-seeded biodegradable scaffolds were grown in the omenta of adult rat hosts for 12 wks, then harvested. Analyses of 12-week implant tissues demonstrated that dissociated 4-dpn rat tooth bud cells seeded for 1 hr onto PGA or PLGA scaffolds generated bioengineered tooth tissues most reliably. We conclude that tooth-tissue-engineering methods can be used to generate both pig and rat tooth tissues. Furthermore, our ability to bioengineer tooth structures from cultured tooth bud cells suggests that dental epithelial and mesenchymal stem cells can be maintained in vitro for at least 6 days.

Tooth tissue engineering: optimal dental stem cell harvest based on tooth development.

Our long-term objective is to devise reliable methods to generate biological replacement teeth exhibiting the physical properties and functions of naturally formed human teeth. Previously, we demonstrated the successful use of tissue engineering approaches to generate small, bioengineered tooth crowns from harvested pig and rat postnatal dental stem cells (DSCs). To facilitate characterizations of human DSCs, we have developed a novel radiographic staging system to accurately correlate human third molar tooth developmental stage with anticipated harvested DSC yield. Our results demonstrated that DSC yields were higher in less developed teeth (Stages 1 and 2), and lower in more developed teeth (Stages 3, 4, and 5). The greatest cell yields and colony-forming units (CFUs) capability was obtained from Stages 1 and 2 tooth dental pulp. We conclude that radiographic developmental staging can be used to accurately assess the utility of harvested human teeth for future dental tissue engineering applications.

Cytogenetic instability of dental pulp stem cell lines

Human adult stem cells (hASCs) offer a potentially renewable source of cell types that are easily isolated and rapidly expanded for use in regenerative medicine and cell therapies without the complicating ethical problems that are associated with embryonic stem cells. However, the eventual therapeutic use of hASCs requires that these cells and their derivatives maintain their genomic stability. There is currently a lack of systematic studies that are aimed at characterising aberrant chromosomal changes in cultured ASCs over time. However, the presence of mosaicism and accumulation of karyotypic abnormalities within cultured cell subpopulations have been reported. To investigate cytogenetic integrity of cultured human dental stem cell (hDSC) lines, we analysed four expanded hDSC cultures using classical G banding and fluorescent in situ hybridisation (FISH) with X chromosome specific probe. Our preliminary results revealed that about 70% of the cells exhibited karyotypic abnormalities including polyploidy, aneuploidy and ring chromosomes. The heterogeneous spectrum of abnormalities indicates a high frequency of chromosomal mutations that continuously arise upon extended culture. These findings emphasise the need for the careful analysis of the cytogenetic stability of cultured hDSCs before they can be used in clinical therapies.

Effects of glucose and glutamine concentrations in human dental pulp stem cells viability. An approach for cell transplantation

PURPOSE To evaluate microscopic behavior and viability of dental pulp stem cells under glucose and glutamine deprivation. METHODS Human tooth tissues were minced in isolated pieces and cultured until the desired cellular proliferation for experimental phases. Cells were cultured under variations of glucose and glutamine in both serum presence and absence, and then those cells were evaluated according to number and viability by MTT assay. The confocal microscopy analyzed cytoskeleton, nucleus, and mitochondria integrity. RESULTS A low concentration of glucose favored cellular viability and microscopic behavior; the presence of glutamine in culture medium was favorable only when associated with glucose. The cellular biological potential in culture could be preserved in serum absence if nutritional requirements are adequate. CONCLUSION Cell microscopic behavior and viability have demonstrated better patterns on serum-free low glucose culture medium with glutamine deprivation.

Osteoinductive effects of preoperative dexamethasone in human dental pulp stem cells primary culture

Aim: The use of dexamethasone (DEX) in mesenchymal cell culture induces osteoblastic differentiation and, consequently, formation of mineralized tissues. Tissue engineering proposes the development of therapeutic strategies aimed at structural and functional regeneration of biological tissues. In this sense, cell characterization in vitro is critical to ensure the development of such techniques. Our objective was to evaluate the osteoinductive effect of DEX administered as a preoperative medication in primary cell culture of human dental pulp stem cell. Methodology: Cells from the third molar pulp were divided into two experimental groups, each with two preoperative medication protocols used in dental practice and differentiated by the intake of DEX in one of them. The assessment of proliferation, differentiation and viability through trypan blue, methylthiazol tetrazolium, and von Kossa and alizarin red assays, respectively, were held within fixed intervals: 7, 14, 21 and 28 days. Conclusion: This study has shown that DEX may influence in vitro human dental pulp stem cell behavior.

Tooth tissue engineering: tooth decellularization for natural scaffold

Aim: Tissue engineering is a multidisciplinary science that aims to produce replacement organs and biological substitutes. One of the techniques involves decellularizing a biological organ without altering its structure. One challenge is how to demonstrate which method would be better for this process. Methodology: Fifty premolar teeth were divided into five groups: G1 (control): solution of 10% formaldehyde; G2: phosphate buffer saline (PBS), 28 g of tetrasodium ethylenediaminetetraacetic (EDTA), sodium hypochlorite 2.5% (SH); G3: PBS, EDTA and 40v hydrogen peroxide (HP); G4: PBS, EDTA, SH, enzymatic detergent (ED); and G5: PBS, EDTA, HP, ED. Each group was analyzed by scanning electron microscopy (SEM), x-ray, measured weights and color and received statistical analysis. Conclusion: This study demonstrated that G5 was the most appropriate method to obtain a natural scaffold.

Scaling-Up of Human Dental Pulp Mesenchymal Cells on Primary Culture

Introduction: Tissue Engineering (TE) aims to bio manufacture human tissues, an endeavor that requires a large number of cells, preferably autologous ones. Typically, an autologous cell source is unable to provide the required amount of cells and it is necessary that such cells be cultured during a prolonged period of time. One of the methodologies used to obtain and expand autologous cells in culture is explants; however, explants are commonly discarded at the moment of the first cell passage. Given the difficulty in obtaining those cell sources, a good alternative would be to reuse the explants and put them back into culture after cell passage. Nevertheless, various modifications have been seen in long-term cell culture, such as modifications of gene expression, and number and amount of proteins. Objective: To evaluate changes in gene expression of extracellular matrix (ECM) and adhesion molecules (AM) in human mesenchymal dental pulp progenitor cells (hMDPC) culture of reused explants. Methods: Gene expression of cells derived from second passage reused explants culture was accessed by Real Time PCR array of Human ECM and AM. Results: Twenty-nine genes showed at least a 5-fold change increase or decrease compared to the control group. Conclusion: Long-term culture frequently induces gene expression changes in ECM or AM. Re using explants is an option to expand the cell source in order to decrease culture time. However, it is necessary to understand, map and control those changes in order to ensure clinical application safety.