Several types of promising cell-based therapies for tissue regeneration have been developing worldwide. However, for successful therapeutical application of cells in this field, appropriate scaffolds are also required. Recently, the research for suitable scaffolds has been focusing on polymer hydrogels due to their similarity to the extracellular matrix. The main limitation regarding amino acid-based hydrogels is their difficult and expensive preparation, which can be avoided by using poly(aspartamide) (PASP)-based hydrogels. PASP-based materials can be chemically modified with various bioactive molecules for the final application purpose. In this study, dopamine containing PASP-based scaffolds is investigated, since dopamine influences several cell biological processes, such as adhesion, migration, proliferation, and differentiation, according to the literature. Periodontal ligament cells (PDLCs) of neuroectodermal origin and SH-SY5Y neuroblastoma cell line were used for the in vitro experiments. The chemical structure of the polymers and hydrogels was proved by ¹H-NMR and FTIR spectroscopy. Scanning electron microscopical (SEM) images confirmed the suitable pore size range of the hydrogels for cell migration. Cell viability assay was carried out according to a standardized protocol using the WST-1 reagent. To visualize three-dimensional cell distribution in the hydrogel matrix, two-photon microscopy was used. According to our results, dopamine containing PASP gels can facilitate vertical cell penetration from the top of the hydrogel in the depth of around 4 cell layers (~150 μm). To quantify these observations, a detailed image analysis process was developed and firstly introduced in this paper.

Background: Triethylene glycol dimethacrylate (TEGDMA) monomers released from resin matrix are toxic to dental pulp cells, induce apoptosis, oxidative stress and decrease viability. Recently, mitochondrial complex I (CI) was identified as a potential target of TEGDMA. In isolated mitochondria supported by CI, substrates oxidation and ATP synthesis were inhibited, reactive oxygen species production was stimulated. Contrary to that, respiratory Complex II was not impaired by TEGDMA. The beneficial effects of electron carrier compound methylene blue (MB) are proven in many disease models where mitochondrial involvement has been detected. In the present study, the bioenergetic effects of MB on TEGDMA-treated isolated mitochondria and on human dental pulp stem cells (DPSC) were analyzed. Methods: Isolated mitochondria and DPSC were acutely exposed to low millimolar concentrations of TEGDMA and 2 μM concentration of MB. Mitochondrial and cellular respiration and glycolytic flux were measured by high resolution respirometry and by Seahorse XF extracellular analyzer. Mitochondrial membrane potential was measured fluorimetrically. Results: MB partially restored the mitochondrial oxidation, rescued membrane potential in isolated mitochondria and significantly increased the impaired cellular O₂ consumption in the presence of TEGDMA. Conclusion: MB is able to protect against TEGDMA-induced CI damage, and might provide protective effects in resin monomer exposed cells.

Ectomesenchymal stem cells derived from the dental pulp are of neural crest origin, and as such are promising sources for cell therapy and tissue engineering. For safe upscaling of these cells, microcarrier-based culturing under dynamic conditions is a promising technology. We tested the suitability of two microcarriers, non-porous Cytodex 1 and porous Cytopore 2, for culturing well characterized dental pulp stem cells (DPSCs) using a shake flask system. Human DPSCs were cultured on these microcarriers in 96-well plates, and further expanded in shake flasks for upscaling experiments. Cell viability was measured using the alamarBlue assay, while cell morphology was observed by conventional and two-photon microscopies. Glucose consumption of cells was detected by the glucose oxidase/Clark-electrode method. DPSCs adhered to and grew well on both microcarrier surfaces and were also found in the pores of the Cytopore 2. Cells grown in tissue culture plates (static, non-shaking conditions) yielded 7 × 105 cells/well. In shake flasks, static preincubation promoted cell adhesion to the microcarriers. Under dynamic culture conditions (shaking) 3 × 107 cells were obtained in shake flasks. The DPSCs exhausted their glucose supply from the medium by day seven even with partial batch-feeding. In conclusion, both non-porous and porous microcarriers are suitable for upscaling ectomesenchymal DPSCs under dynamic culture conditions.

Chronic periodontitis is a bacterial infection associated with dentally adherent biofilm (plaque) accumulation and age-related comorbidities. The disease begins as an inflammatory exudate from gingival margins, gingival crevicular fluid (GCF) in response to biofilm lysine. After a week of experimental gingivitis (no oral hygiene), biofilm lysine concentration was linearly related to biofilm accumulation (plaque index) but to GCF as an arch-shaped double curve which separated 9 strong from 6 weak GCF responders (hosts). Host DNA was examined for single nucleotide polymorphisms (SNPs) of alleles reported in 7 periodontitis-associated genes. Across all 15 hosts, an adenine SNP (A) at IL1B-511 (rs16944), was significant for strong GCF (Fisher’s exact test, p < 0.05), and a thymidine SNP (T) at IL1B+3954 (rs1143634) for weak GCF provided 2 hosts possessing IL6-1363(T), rs2069827, were included. The phenotype of IL1B+3954(T) was converted from weak to strong in one host, and of the non-T allele from strong to weak in the other (specific epistasis, Fisher’s exact test, p < 0.01). Together with homozygous alternate or reference SNPs at IL10-1082 or CD14-260 in 4 hosts, all hosts were identified as strong or weak GCF responders. The GCF response is therefore a strong or weak genetic trait that indicates strong or weak innate immunity in EG and controllable or uncontrollable periodontal disease, dental implant survival and late-life comorbidities.

Direct neuronal reprogramming offers an alternative to induced pluripotent stem cell-based differentiation by converting somatic cells directly into neurons without passage through pluripotency. However, commonly used fibroblast-based protocols are often slow and inefficient. Here, we evaluated human dental pulp stem cells (DPSCs), which originate from the cranial neural crest and possess intrinsic neurogenic potential, as a developmentally relevant source of induced neurons (iNs). Using an all-in-one lentiviral vector, we converted DPSCs into iNs within 17 days, compared with 28 days for fibroblasts reprogrammed with the same vector, and achieved significantly higher neuronal purity under the respective established protocols. Multi-omic profiling revealed coordinated suppression of mesenchymal and cell-cycle programs and induction of neuronal, synaptic, and metabolic pathways. Single-nucleus RNA sequencing resolved fibroblast-like, transitional, maturing neuronal, GABAergic-like, and alternative fates, while trajectory inference suggested divergent neuronal and non-neuronal conversion paths. Whole-cell recordings showed that a subset of DPSC-iNs developed early neuronal excitability and voltage-gated inward and outward currents. Together, our findings establish DPSCs as an accessible and developmentally relevant source for rapid direct neuronal conversion. This integrated molecular, single-nucleus, and electrophysiological characterization defines the cellular heterogeneity of DPSC-to-neuron reprogramming and provides a framework for protocol refinement and future patient-specific disease modeling.