Novel potential scaffold for periodontal tissue engineering.

OBJECTIVE: The objective of the study is characterization of novel calcium and zinc-loaded electrospun matrices to be used for periodontal regeneration. MATERIALS AND METHODS: A polymethylmetacrylate-based membrane was calcium or zinc loaded. Matrices were characterized morphologically by atomic force and scanning electron microscopy and mechanically probed by a nanoindenter. Biomimetic calcium phosphate precipitation on polymeric tissues was assessed. Cell viability tests were performed using oral mucosa fibroblasts. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests or by ANOVA and Student-Newman-Keuls multiple comparisons. RESULTS: Zinc and calcium loading on matrices did not modify their morphology but increased nanomechanical properties and decreased nanoroughness. Precipitation of calcium and phosphate on the matrix surfaces was observed in zinc-loaded specimens. Matrices were found to be non-toxic to cells in all the assays. Calcium- and zinc-loaded scaffolds presented a very low cytotoxic effect. CONCLUSIONS: Zinc-loaded membranes permit cell viability and promoted mineral precipitation in physiological conditions. Based on the tested nanomechanical properties and scaffold architecture, the proposed membranes may be suitable for cell proliferation. CLINICAL RELEVANCE: The ability of zinc-loaded matrices to promote precipitation of calcium phosphate deposits, together with their observed non-toxicity and its surface chemistry allowing covalent binding of proteins, may offer new strategies for periodontal regeneration.

Bioactive Polymeric Nanoparticles for Periodontal Therapy.

AIMS: to design calcium and zinc-loaded bioactive and cytocompatible nanoparticles for the treatment of periodontal disease. METHODS: PolymP-nActive nanoparticles were zinc or calcium loaded. Biomimetic calcium phosphate precipitation on polymeric particles was assessed after 7 days immersion in simulated body fluid, by scanning electron microscopy attached to an energy dispersive analysis system. Amorphous mineral deposition was probed by X-ray diffraction. Cell viability analysis was performed using oral mucosa fibroblasts by: 1) quantifying the liberated deoxyribonucleic acid from dead cells, 2) detecting the amount of lactate dehydrogenase enzyme released by cells with damaged membranes, and 3) by examining the cytoplasmic esterase function and cell membranes integrity with a fluorescence-based method using the Live/Dead commercial kit. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests. RESULTS: Precipitation of calcium and phosphate on the nanoparticles surfaces was observed in calcium-loaded nanoparticles. Non-loaded nanoparticles were found to be non-toxic in all the assays, calcium and zinc-loaded particles presented a dose dependent but very low cytotoxic effect. CONCLUSIONS: The ability of calcium-loaded nanoparticles to promote precipitation of calcium phosphate deposits, together with their observed non-toxicity may offer new strategies for periodontal disease treatment.

Identification of histological patterns in clinically affected and unaffected palm regions in dupuytren's disease.

Dupuytren's disease is a fibro-proliferative disease characterized by a disorder of the extracellular matrix (ECM) and high myofibroblast proliferation. However, studies failed to determine if the whole palm fascia is affected by the disease. The objective of this study was to analyze several components of the extracellular matrix of three types of tissues-Dupuytren's diseased contracture cords (DDC), palmar fascia clinically unaffected by Dupuytren's disease contracture (NPF), and normal forehand fascia (NFF). Histological analysis, quantification of cells recultured from each type of tissue, mRNA microarrays and immunohistochemistry for smooth muscle actin (SMA), fibrillar ECM components and non-fibrillar ECM components were carried out. The results showed that DDC samples had abundant fibrosis with reticular fibers and few elastic fibers, high cell proliferation and myofibroblasts, laminin and glycoproteins, whereas NFF did not show any of these findings. Interestingly, NPF tissues had more cells showing myofibroblasts differentiation and more collagen and reticular fibers, laminin and glycoproteins than NFF, although at lower level than DDC, with similar elastic fibers than DDC. Immunohistochemical expression of decorin was high in DDC, whereas versican was highly expressed NFF, with no differences for aggrecan. Cluster analysis revealed that the global expression profile of NPF was very similar to DDC, and reculturing methods showed that cells corresponding to DDC tissues proliferated more actively than NPF, and NPF more actively than NFF. All these results suggest that NPF tissues may be affected, and that a modification of the therapeutic approach used for the treatment of Dupuytren's disease should be considered.

Cell viability and proliferation capability of long-term human dental pulp stem cell cultures.

BACKGROUND AIMS: Evaluation of cell viability is one of the most important steps of the quality control process for therapeutic use of cells. The aim of this study was to evaluate the long-term cell viability profile of human dental pulp stem cell (hDPSC) subcultures (beyond 10 passages) to determine which of these passages are suitable for clinical use and to identify the cell death processes that may occur in the last passages. METHODS: Four different cell viability assays were combined to determine the average cell viability levels at each cell passage: trypan blue exclusion test, water-soluble tetrazolium 1 (WST-1), LIVE/DEAD Viability/Cytotoxicity Kit and electron probe x-ray microanalysis (EPXMA). Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and caspase 4 and BCL7C Western blotting, and cell proliferation was analyzed by WST-1 and proliferating cell nuclear antigen protein detection. RESULTS: hDPSCs showed high average cell viability levels from passages 11-14, with adequate cytoplasmic and mitochondrial functionality at these subcultures. A non-significant trend to decreased cell proliferation was found from passages 16-20. EPXMA and TUNEL analyses suggested that a pre-apoptotic process could be activated from passages 15-20 (P < 0.001), with a correlation with caspase 4 and BCL7C expression. CONCLUSIONS: hDPSCs corresponding to passages 11-14 show adequate cell function, proliferation and viability. These cells could be considered as potentially useful for clinical applications.

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.

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.