Ascorbic acid enhances bone parameter expression in human gingival mesenchymal stem cells.

Ascorbic acid (AS), also known as vitamin C or ascorbate, is an essential dietary nutrient which plays a vital role in biological processes through various different mechanisms, in particular for the biosynthesis of collagen. The aim of the study was to establish the possibility of enhancing the osteogenic differentiation potential by manipulating the cellular micro-environment, through AS supplementation in human gingival mesenchymal stem cells (hGMSCs) at different concentrations, such as 60 and 90 µg/mL, for three weeks. Human GMSCs are considered a stem cell population, easily obtainable and displaying a remarkable immunotherapeutic potential and regenerative repair expression. Osteogenic differentiation level induced from AS was assayed by histochemical characterization, using light microscopy through Alizarin red S staining. The transcript levels of Collagen 1A1 (COL1A1), runtrelated transcription factor 2 (RUNX2), bone morphogenetic protein 2/4 (BMP2/4), osteopontin (OPN) and osteonectin (SPARC) were determined by quantitative RT-PCR. Protein expression of COL1A1, RUNX2, BMP2/4, OPN, SPARC were studied through Western blotting and confocal laser scanning microscopy (CLSM). Our results demonstrate that AS supports osteogenic differentiation in stem cells from gingiva niche as shown by osteogenic marker upregulation and by de novo production of calcium phosphate deposits as revealed by Alizarin red S staining. In summary, the results of the current study provide evidence that hGMSCs undergo osteogenic differentiation with AS treatment, for that reason AS could be a promising candidate for the prevention and healing of bone-related diseases.

Human periodontal ligament stem cells cultured onto cortico-cancellous scaffold drive bone regenerative process.

The purpose of this work was to test, in vitro and in vivo, a new tissue-engineered construct constituted by porcine cortico-cancellous scaffold (Osteobiol Dual Block) (DB) and xeno-free ex vivo culture of human Periodontal Ligament Stem Cells (hPDLSCs). hPDLSCs cultured in xeno-free media formulation preserved the stem cells' morphological features, the expression of stemness and pluripotency markers, and their ability to differentiate into mesenchymal lineage. Transmission electron microscopy analysis suggested that after one week of culture, both noninduced and osteogenic differentiation induced cells joined and grew on DB secreting extracellular matrix (ECM) that in osteogenic induced samples was hierarchically assembled in fibrils. Quantitative RT-PCR (qRT-PCR) showed the upregulation of key genes involved in the bone differentiation pathway in both differentiated and undifferentiated hPDLSCs cultured with DB (hPDLSCs/DB). Functional studies revealed a significant increased response of calcium transients in the presence of DB, both in undifferentiated and differentiated cells stimulated with calcitonin and parathormone, suggesting that the biomaterial could drive the osteogenic differentiation process of hPDLSCs. These data were confirmed by the increase of gene expression of L-type voltage-dependent Ca2+ (VDCCL), subunits α1C and α2D1 in undifferentiated cells in the presence of DB. In vivo implantation of the hPDLSCs/DB living construct in the mouse calvaria evidenced a precocious osteointegration and vascularisation process. Our results suggest consideration of DB as a biocompatible, osteoinductive and osteoconductive biomaterial, making it a promising tool to regulate cell activities in biological environments and for a potential use in the development of new custom-made tissue engineering.

miR-2861 is involved in osteogenic commitment of human periodontal ligament stem cells grown onto 3D scaffold.

miR-2861 endorsing osteoblast differentiation through the overexpression of Runt-related transcription factor 2 (RUNX2) protein has been recently described. In this study we evaluated: the performance of living construct, composed by human Periodontal Ligament Stem Cells (hPDLSCs) and 3D scaffold (EXg), and the behaviour of miR-2861/RUNX2 expression pathway on the osteogenic commitment. Human PDLSCs were seeded with and without EXg scaffold and cultured under basal and osteogenic conditions. Morphological features, adhesiveness and differentiation abilities were analysed using scanning electron and confocal laser scanning microscopy. Time-course of RUNX2, ALP, OPN and miR-2861 were evaluated through RT-PCR analysis. Our results highlighted that the osteogenic differentiation was mostly obvious in the hPDLSCs, grown onto 3D scaffold in presence of osteoinductive medium. Moreover, the overexpression of miR-2861 and RUNX2 in hPDLSCs cultured in presence of EXg under osteogenic and standard conditions was demonstrated. In synthesis, the increased expression of miR-2861/RUNX2 provides new insights regarding miRNA signaling network in the presence of scaffold providing an additional method to evaluate the performance of biomaterial in bone regeneration.

Osteogenic potential of dualblocks cultured with human periodontal ligament stem cells: in vitro and synchrotron microtomography study.

BACKGROUND AND OBJECTIVE: In the present study, the early stages of in vitro bone formation in collagenated porcine scaffolds cultured with human periodontal ligament cells were investigated. The comparison between the osteogenic potential of this structure in basal and differentiating culture media was explored to predict the mechanism of its biological behavior as graft in human defect. Results were validated by synchrotron radiation X-Ray phase contrast computed microtomography (micro-CT). As the periodontal disease plays a key role in systemic and oral diseases, it is crucial to find advanced therapeutic clinical interventions to repair periodontal defects. This has been recently explored using cells and tissues developed in vitro that should ideally be immunologically, functionally, structurally and mechanically identical to the native tissue. MATERIAL AND METHODS: In vitro cultures of human periodontal ligament cells, easily obtained by scraping of alveolar crestal and horizontal fibers of the periodontal ligament, were seeded on to collagenated porcine blocks constituted by natural cancellous and cortical bone. 3D images were obtained by synchrotron radiation micro-CT and processed with a phase-retrieval algorithm based on the transport of intensity equation. RESULTS: Starting from the second week of culture, newly formed mineralized bone was detected in all the scaffolds, both in basal and differentiating media. Bone mineralization was proved to occur preferentially in the trabecular portion and in differentiating media. CONCLUSION: The chosen method, supported by phase contrast micro-CT analysis, successfully and quantitatively monitored the early stages of bone formation and the rate of the bioscaffold resorption in basal and differentiating culture media.

A cytotoxic analysis of a sardinian plant extract cream on human oral primary cell cultures: an in vitro study.

Wound healing agents support the natural healing process, reduce trauma and likelihood of secondary infections and hasten wound closure. The aim of this work was to evaluate the effect of different concentration of a new Sardinian plant cream (RD7) on two human primary cultures: Periodontal Ligament Stem Cells (hPDLSCs) and Gingival Fibroblasts (hGFs) derived from oral tissues in terms of morphological changes, cell proliferation and wound healing properties. RD7, is an interactive dressing containing phytocomplex derived from Sardinian endemic or not, medicinal plant extracts, with an important anti-radical, anti-inflammatory and antiseptic activity finalized to rapidly promote tissue regeneration and the formation of granulation tissue. hPDLSCs and hGFs were seeded at different concentrations (0.5, 1, 2.5 and 5 mg/ml) of RD7. The cell proliferation and viability was evaluated using colorimetric assays (MTT assay) and trypan blue exclusion test. Meanwhile, the morphological cell changes were evaluated by means of optic (OM) and scanning electronic microscopes (SEM). The induction of the migratory properties was evaluated by means of wound healing assay. In vitro results, using hPDLSCs and hGFs, showed a decrease of cell growth starting at 24 h of incubation, at high concentrations (2.5 mg/ml and 5 mg/ml). This cell growth reduction was associated to evident morphological changes, whilst, at low concentrations (0.5 and 1 mg/ml) a typical unchanged morphology of both hPDLSCs and hGFs was shown. Wound healing assay showed a complete wound full closure occurring after 24 h of treatment in samples treated with low concentration of RD7. The results of the present work indicate that low concentrations of RD7 have no cytotoxicity effect, stimulate cell proliferation and contribute to induce the migratory properties in hPDLSCs and hGFs, therefore it could be considered a new product for use in clinical practice.

Pro-inflammatory cytokine release and cell growth inhibition in primary human oral cells after exposure to endodontic sealer.

AIM: To assay the toxicity of the single-methacrylate-based sealer urethane dimethacrylate (UDMA) (EndoRez) in terms of cell growth and pro-inflammatory cytokines release, in expanded ex vivo human dental pulp stem cells (hDPSCs), human periodontal ligament stem cells (hPDLSCs), human gingival fibroblasts (hGFs) and human osteoblasts (hOSTs). METHODOLOGY: Dental pulp and periodontal ligament stem cells, osteoblasts and fibroblasts were derived from five young donors. After in vitro isolation, hDPSCs, hPDLSCs, hGFs and hOSTs were seeded to resin-based sealers for 24, 48, 72 h up to 1 week. The morphological features and the cell growth and the release of pro-inflammatory interleukin (IL)6, IL8, IL12 and tumour necrosis factor (TNF) α were analysed. Differences in cell growth and in interleukin secretion were analysed for statistical significance with two-way anova tests for multiple comparisons. RESULTS: Exposure to endodontic sealer based on UDMA resulted in a 50% decrease in survival oral cells at 24 h of incubation. No evident morphological changes were present in cell cultures examined. After 48 h, 72 h and 1-week culture time, a progressive cell growth was evident. A significant up-regulation of IL6, IL8, IL12 and TNFα cytokines in cells in contact with the dental sealer compared to the control was observed. CONCLUSION: In vitro, EndoRez interacted with primary human hDPSCs, hPDLSCs, hGFs and hOSTs causing damage to biological system evidenced through cell growth inhibition and up-regulation of IL6, IL8, IL12 and TNFα proinflammatory mediators.

Influence of titanium laser surface geometry on proliferation and on morphological features of human mandibular primary osteoblasts.

The aim of this study is to assess in vitro the proliferation and the morphological changes of primary osteoblast-like cells (HOst) seeded on titanium dish grade 4 and 5 with different roughness and different titanium grade: machined (M), sandblasted (SBT), laser-treated with pitches of 20-microm diameter and 30-microm interpore distance. The titanium disks were divided into two groups: group A (titanium grade 4) and Group B (titanium Grade 5), respectively. Proliferation rate of attached cells was evaluated at different time (24, 48, 72 h and 1 week) by the quantitative colorimetric MTT assay. Our results showed a cell growth decrease evident in M titanium surfaces in both Groups A and B, while the cells seeded on the STB and laser disks displayed an increase of cells growth, more evident in laser titanium surfaces in groups A and B. Morphological changes of the biocomplex cells/titanium was assessed by light, scanning and confocal microscopy. In fact, the microscopic analysis helped to clarify the behavior of the cells in contact with the titanium surfaces, in particular the M surface induced significant morphological changes, which were less evident in the SBT surfaces. Laser-engineered porous titanium surfaces promoted viability and proliferation of the osteoblasts. In particular, hemispherical porosity of 20 microm could be responsible for the higher HOst activation, in terms of cells proliferation, adhesion and morphological features.

Morphological analysis and interleukin release in human gingival fibroblasts seeded on different denture base acrylic resins.

The development of different types of materials with application in practice dentistry is an area of intense growth and research due to its importance in oral health. Among the diverse materials currently used in restoration or in dentures, the acrylic based resins have been widely employed. The release of toxic components and the changes on their physical and mechanical properties actually represent a goal of intensive research. In vivo analysis showed that the surface roughness of the acrylic resin represents a factor that could stimulate bacteria colonization and soft tissue inflammation. For this purpose, in this work, we have analyzed the cell response to acrylic based resins Ivoclar, Tokuso and Coldpack in basal conditions, unpolished, and after the polished procedure performed to reduce the surface roughness. Our in vitro results using human gingival fibroblasts (HGFs) showed a decrease of cell growth, evaluated by MTT assay starting at 24 h of incubation, in samples seeded on resins in basal conditions and after the polished procedure. This cell growth reduction was associated to evident morphological changes in unpolished materials. After 24 h of culture in presence of polished and unpolished resins a spontaneous release was present of pro-inflammatory cytokines such as interleukin-6 (IL-6) and -8 (IL-8), which was higher in unpolished resins, indicating that the polished procedure, minimizing the cytotoxicity process, could contribute to reduce the gingival inflammation processes.