Assessment of the regenerative potential of macro-porous chitosan-calcium simvastatin scaffolds on bone cells

Abstract This study evaluated the bioactive potential of a macro-porous chitosan scaffold incorporated with calcium hydroxide (CH-Ca) and functionalized with bioactive doses of simvastatin (SV) for bone tissue regeneration. Initially, the bioactive dose of SV in osteoblastic cells (SAOS-2) was determined. For the direct contact experiment, SAOS-2 cells were plated on scaffolds to assess cell viability and osteogenic differentiation. The second assay was performed at a distance using extracts from scaffolds incubated in culture medium to assess the effect of conditioned medium on viability and osteogenic differentiation. The initial screening showed that 1 μM SV presented the best biostimulating effects, and this dose was selected for incorporation into the CH-Ca and pure chitosan (CH) scaffolds. The cells remained viable throughout the direct contact experiment, with the greatest cell density in the CH-Ca and CH-Ca-SV scaffolds because of their higher porosity. The CH-Ca-SV scaffold showed the most intense bio-stimulating effect in assays in the presence and absence of osteogenic medium, leading to an increased deposition of mineralized matrix. There was an increase in the viability of cells exposed to the extracts for CH-Ca, CH-SV, and CH-Ca-SV during the one-day period. There was an increase in ALP activity in the CH-Ca and CH-Ca-SV; however, the CH-Ca-SV scaffold resulted in an intense increase in the deposition of mineralized nodules, approximately 56.4% at 7 days and 117% at 14 days, compared with CH (control). In conclusion, functionalization of the CH-Ca scaffold with SV promoted an increase in bioactivity, presenting a promising option for bone tissue regeneration.

Chronic exposure to lipopolysaccharides as an in vitro model to simulate the impaired odontogenic potential of dental pulp cells under pulpitis conditions

Abstract Simulating a bacterial-induced pulpitis environment in vitro may contribute to exploring mechanisms and bioactive molecules to counteract these adverse effects. Objective To investigate the chronic exposure of human dental pulp cells (HDPCs) to lipopolysaccharides (LPS) aiming to establish a cell culture protocol to simulate the impaired odontogenic potential under pulpitis conditions. Methodology HDPCs were isolated from four healthy molars of different donors and seeded in culture plates in a growth medium. After 24 h, the medium was changed to an odontogenic differentiation medium (DM) supplemented or not with E. coli LPS (0 - control, 0.1, 1, or 10 µg/mL) (n=8). The medium was renewed every two days for up to seven days, then replaced with LPS-free DM for up to 21 days. The activation of NF-κB and F-actin expression were assessed (immunofluorescence) after one and seven days. On day 7, cells were evaluated for both the gene expression (RT-qPCR) of odontogenic markers (COL1A1, ALPL, DSPP, and DMP1) and cytokines (TNF, IL1B, IL8, and IL6) and the production of reactive nitrogen (Griess) and oxygen species (Carboxy-H2DCFDA). Cell viability (alamarBlue) was evaluated weekly, and mineralization was assessed (Alizarin Red) at 14 and 21 days. Data were analyzed with ANOVA and post-hoc tests (α=5%). Results After one and seven days of exposure to LPS, NF-κB was activated in a dose-dependent fashion. LPS at 1 and 10 µg/mL concentrations down-regulated the gene expression of odontogenic markers and up-regulated cytokines. LPS at 10 µg/mL increased both the production of reactive nitrogen and oxygen species. LPS decreased cell viability seven days after the end of exposure. LPS at 1 and 10 µg/mL decreased hDPCs mineralization in a dose-dependent fashion. Conclusion The exposure to 10 µg/mL LPS for seven days creates an inflammatory environment that is able to impair by more than half the odontogenic potential of HDPCs in vitro, simulating a pulpitis-like condition.

Proteolytic activity and degradation of bovine versus human dentin matrices

Abstract Non-human teeth have been commonly used in research as replacements for human teeth, and potential dissimilarities between the dental tissues should be considered when interpreting the outcomes. Objective: To compare the proteolytic activity and degradation rate of bovine and human dentin matrices. Methodology: Dentin beam specimens were obtained from human molars (n=30) and bovine incisors (n=30). The beams were weighed hydrated and after complete dehydration to obtain the mineralized wet and dry masses. Then, the beams were demineralized in 10 wt% phosphoric acid. Next, 15 beams from each substrate were randomly selected and again dehydrated and weighed to obtain the initial demineralized dry mass (DM). Then, the beams were stored in saliva-like buffer solution (SLBS) for 7, 14 and 21 days. SLBS was used to evaluate hydroxyproline (HYP) release after each storage period. The remaining beams of each substrate (n=15) were tested for initial MMP activity using a colorimetric assay and then also stored in SLBS. DM and MMP activity were reassessed after 7, 14 and 21 days of incubation. The data were subjected to two-way ANOVA tests with repeated measures complemented by Bonferroni's tests. Unpaired two-tailed t-tests were also used (p<0.05). Results: Similar water and inorganic fractions were found in human and bovine dentin, while human dentin had a higher protein content. The most intense proteolytic activity and matrix deterioration occurred short after dentin was demineralized. Both substrates exhibited a sharp reduction in MMP activity after seven days of incubation. Although human dentin had higher MMP activity levels, greater HYP release and DM loss after seven days than bovine dentin, after 14 and 21 days, the outcomes were not statistically different. Conclusion: Bovine dentin is a suitable substrate for long-term studies involving the degradation of dentin matrices.

Bioactivity effects of extracellular matrix proteins on apical papilla cells

Abstract Potent signaling agents stimulate and guide pulp tissue regeneration, especially in endodontic treatment of teeth with incomplete root formation. Objective This study evaluated the bioactive properties of low concentrations of extracellular matrix proteins on human apical papilla cells (hAPCs). Methodology Different concentrations (1, 5, and 10 µg/mL) of fibronectin (FN), laminin (LM), and type I collagen (COL) were applied to the bottom of non-treated wells of sterilized 96-well plates. Non-treated and pre-treated wells were used as negative (NC) and positive (PC) controls. After seeding the hAPCs (5×103 cells/well) on the different substrates, we assessed the following parameters: adhesion, proliferation, spreading, total collagen/type I collagen synthesis and gene expression (ITGA5, ITGAV, COL1A1, COL3A1) (ANOVA/Tukey; α=0.05). Results We observed greater attachment potential for cells on the FN substrate, with the effect depending on concentration. Concentrations of 5 and 10 µg/mL of FN yielded the highest cell proliferation, spreading and collagen synthesis values with 10 µg/mL concentration increasing the ITGA5, ITGAV, and COL1A1 expression compared with PC. LM (5 and 10 µg/mL) showed higher bioactivity values than NC, but those were lower than PC, and COL showed no bioactivity at all. Conclusion We conclude that FN at 10 µg/mL concentration exerted the most intense bioactive effects on hAPCs.