OBJECTIVES:Vitamin D3 status profoundly affects oral health and disease. Over the last decade, evidence has emerged that vitamin D3 activation can also occur in peripheral tissues. This narrative review was conducted to critically analyze the state of the art about the presence and activity of enzymes involved in the bioactivation of vitamin D3 in the oral tissues. DESIGN:A comprehensive literature search was performed in PubMed and Google Scholar. The search included articles published in English without any time limit. Keywords included but not limited to the combinations of: "vitamin D3", "25(OH)D3", "1,25(OH)2D3", "oral tissues", "megalin", "CYP27B1", "extrarenal", and "antimicrobial peptides". Further studies were identified by screening the reference lists of the relevant publications. RESULTS:Various vitamin D3 metabolites influence the inflammatory response and the production of antimicrobial peptides in various oral cells. The enzyme CYP27B1, which is responsible for the conversion of 25(OH)D3 into 1,25(OH)2D3 and its bioactivation, is present in various oral tissues and cells. The existence and physiological significance of local vitamin D3 activation in oral tissues remain unclear. Most of 25(OH)D3 is bound to the vitamin D-binding protein (DBP) and must be dissociated for activation. It is unclear whether and how this uncoupling occurs in oral tissue. CONCLUSION:Currently, there is rather indirect evidence that vitamin D3 could be bioactivated in oral tissues. Further studies on the local conversion of vitamin D3 to 25(OH)D3 and, subsequently, to 1,25(OH)2D3 in oral tissue, their regulation, and the role of free and bioavailable vitamin D3 metabolites are required.

Abstract Background Human gingiva-derived mesenchymal stromal cells (hG-MSCs) are promising candidates for cell-based therapy due to their immunomodulatory ability. Culturing in 3D hydrogels has emerged as a promising strategy to improve cell survival in the host and enhance clinical efficacy. This study assesses how embedding hG-MSCs in “VitroGel ® MSC” hydrogel affects their immunomodulatory profile in various inflammatory environments, compared with conventional 2D cultures. Methods hG-MSCs from six donors were either 3D-embedded in commercially available “VitroGel ® MSC”, cultured on “VitroGel ® MSC”, or in 2D on tissue culture plastic under resting conditions or in the presence of interferon (IFN)-γ, interleukin (IL)-1β, or tumor necrosis factor (TNF)-α. Metabolic activity and cell viability were assessed by MTT assay and live/dead staining, respectively. Gene expression of IL-8, cyclooxygenase (COX)-2, indoleamine-2,3-dioxygenase (IDO)-1, programmed cell death ligand 1 (PD-L1), PD-L2, and TNF-stimulated gene (TSG)-6 were analyzed by RT-qPCR, and IL-8 and prostaglandin E 2 (PGE 2 ) protein levels were determined by ELISA. Results Metabolic activity was reduced in 3D-embedded hG-MSCs independently of the environment, but only in the presence of IFN-γ and TNF-α, a slight decrease in cell viability was observed. Hydrogel-embedded and -exposed hG-MSCs exhibited increased IL-8 expression under resting and IFN-γ conditions, and consistently upregulated COX-2 and PGE 2 . The immunomodulatory mediator TSG-6 was elevated in 3D-embedded hG-MSCs, while the expression of IDO-1, PD-L1, and PD-L2 upon transition to 3D hydrogel was differently altered depending on the inflammatory environments. Conclusions These results demonstrate that 3D microenvironments reshape hG-MSC immunomodulatory potential, underscoring the relevance of 3D models for optimizing MSC-based therapies.

Background/Objectives: Formation of tight contacts between oral soft tissue and dental implants is a significant challenge in contemporary implantology. An essential role in this process is played by oral epithelial cells. In the present study, we investigated how titanium and zirconia surfaces with different roughness influence various parameters of oral epithelial cells in vitro. Methods: We used the human oral squamous carcinoma Ca9-22 cell line and cultured them on the following surfaces: machined smooth titanium (TiM) and zirconia (ZrM) surfaces, as well as sandblasted and acid-etched titanium moderately rough (SLA) and zirconia (ZLA) surfaces. Cell proliferation/viability was measured by CCK-8 assay, and cell morphology was analyzed by fluorescent microscopy. The gene expression of interleukin (IL)-8, intercellular adhesion molecule (ICAM)-1, E-cadherin, integrin (ITG)-α6, and ITG-β4 was measured by qPCR, and the content of IL-8 in conditioned media by ELISA. Results: At the initial culture phase, cell proliferation was promoted by rougher surfaces. Differences in cell attachment were observed between machined and moderately rough surfaces. Machined surfaces were associated with slightly higher IL-8 levels (p < 0.05). Furthermore, both ZLA and SLA surfaces promoted the expression of (ITG)-α, ITG-β4, and ICAM-1 in Ca9-22 cells (p < 0.05). Surface material had no impact on the investigated parameters. Conclusions: Under the limitations of this in vitro study, some properties of oral epithelial cells, particularly the immunological and barrier function, are moderately modified by roughness but not by material. Hence, the roughness of the implant surface might play a role in the quality of the peri-implant epithelium.

Isolating mesenchymal stromal cell (MSC) subpopulations with improved quality based on specific surface markers, such as CD146, is one approach to enhance their therapeutic potential. However, there is limited information on the cytokine-boosted immunomodulatory mechanisms between CD146-expressing and non-expressing MSCs across various inflammatory conditions. This study seeks to address this gap. MSCs from the human periodontal ligament (hPDL-MSCs) were treated with interleukin-(IL)-1β, interferon-(IFN)-γ, or tumor necrosis factor-(TNF)-α, investigating the expression of IDO-1, PD-L1, PTGS-2, and TSG-6 between CD146+ and CD146− hPDL-MSCs. Additionally, the expression of the immunomediators was compared between CD146-depleted and -enriched populations, generated by magnetic-bead-sorting. CD146+ hPDL-MSCs exhibited a significantly higher proportion of IDO-1+ cells, enhanced expression levels in the presence of IL-1β or TNF-α, and higher IL-1β, IFN-γ, or TNF-α-induced PD-L1 protein expression. Conversely, the proportion of TSG-6+ hPDL-MSCs was significantly lower in CD146+ cells under basal conditions. In CD146-enriched hPDL-MSCs, the immunomediator expression was minimally elevated, with significant differences under specific conditions: a higher PD-L1 protein expression under basal conditions and in the presence of IFN-γ or TNF-α, as well as higher or lower PGE2 levels with TNF-α or IL-1β, respectively. These results suggest that the immunomediator expression in hPDL-MSCs alters with different CD146 surface expression. CD146+ hPDL-MSCs do not always show higher levels of immunomediator expression. Further research is necessary to isolate hPDL-MSC subpopulations with optimal potential for therapeutic applications.

Regeneration of periodontal epithelium remains a major focus in current dental research, with various exogenous substitute materials being applied in clinical practice. Yet, the highly organized structure of native tissue still poses considerable challenges for biomaterials attempting to mimic the original environment. In this study, we investigated the effects of a newly developed gelatin/polycaprolactone nanofiber (GPF) and a micro-scaled collagen matrix (CM) on the biological behavior of oral epithelial Ca9-22 cells, aiming to assess the clinical applicability of the materials and conducted a preliminary exploration of the interplay between the Ca9-22 cells and the material properties. The oral epithelial Ca9-22 cell line was cultured onto the GPF, CM, and tissue culture plate (TCP) for 3, 7, and 14 days. Cell morphology, attachment proliferation/viability, the gene expression of keratin 14 (KRT14), keratin 10 (KRT10), integrin β-1 (ITGB-1), intercellular adhesion molecule 1 (ICAM-1), interleukin 8 (IL-8) and interleukin 1β (IL-1β), the levels of IL-8 proteins were evaluated. Ca9-22 cells exhibited distinct adhesion morphology and distribution patterns on two biomaterials. After 3 days of culturing on GPF, Ca9-22 cells demonstrated higher levels of proliferation/viability compared to those on CM. In most situations, except KRT10, both materials effectively stimulated gene and protein expression related to epithelial regeneration and wound healing, especially in the early stage of culture. Compared to CM, GPF demonstrated a stronger stimulation of KRT14 expression at day 3 and a more significant enhancement of KRT10 expression after 7 and 14 days. However, it was less effective at promoting IL-8 expression after 3 days than the former. The gene expression of KRT10 was suppressed by CM at day 7. The IL-8 protein production was the highest in cells grown on CM. The morphology and cellular functions of oral epithelial cells differed between GPF and CM. Both materials are capable of promoting epithelial regeneration; however, GPF is more conducive to functional stratification of newly formed epithelium, while CM holds a more sustained effect on epithelial proliferation.

Low-level laser therapy (LLLT) is gaining attention as an effective adjunct to non-surgical periodontal treatment. This study evaluates the potential of LLLT to reduce bacterial load in a clinically relevant in vitro subgingival biofilm model and its impact on the inflammatory response. A subgingival biofilm model consisting of seven bacterial species was established. Primary human gingival fibroblasts (GFs) and periodontal ligament cells (PDLs) were cultured. Both biofilms and host cells were treated with the DenLase Diode Laser (980 nm) under various clinically relevant settings. The composition and structure of the seven-species biofilms were evaluated using quantitative PCR and fluorescence microscopy, respectively. The inflammatory response in host cells was analyzed by measuring the gene and protein expression levels of various inflammatory mediators. Laser treatment at power outputs ranging from 0.3 to 2 W had no significant effect on biofilm composition or architecture. LLLT, particularly at higher power settings, reduced the viability in both GFs and PDLs up to 70%. Gene expression levels of inflammatory mediators were only minimally influenced by laser treatment. However, LLLT significantly decreased the secretion of all examined cytokines. These findings suggest that LLLT with a 980 nm diode laser, under clinically relevant conditions, exerts anti-inflammatory rather than antimicrobial effects.

The oral epithelium is essential for maintaining oral health and plays a key role in the onset and progression of periodontitis. It serves as both a mechanical and immunological barrier and possesses antimicrobial activity. Vitamin D3, a hormone with known immunomodulatory functions, may influence oral epithelial responses. This study investigated the effects of two vitamin D3 metabolites on key immunological and antimicrobial functions of oral epithelial cells, both under basal conditions and during bacterial challenge. Ca9-22 oral epithelial cells were treated with 1,25(OH)2D3 or 25(OH)D3 in the presence or absence of Tannerella forsythia, Fusobacterium nucleatum, or Porphyromonas gingivalis. Inflammatory responses were assessed by measuring gene and protein expression of IL-1β and IL-8. Antimicrobial activity was evaluated via expression of LL-37, hBD-2, and hBD-3, as well as direct bacterial killing assays. Expression of epithelial integrity markers E-cadherin and ICAM-1 was also analyzed. Vitamin D3 metabolites reduced IL-8 expression and significantly increased LL-37 expression and production in Ca9-22 cells. Both forms enhanced antimicrobial activity against all tested pathogens and modulated epithelial integrity markers. Vitamin D3 positively regulates antimicrobial and barrier functions in oral epithelial cells, suggesting a potential role in supporting oral health and preventing periodontitis progression.

IntroductionMesenchymal stromal cells (MSCs) are promising therapeutic candidates in regenerative medicine and the treatment of inflammatory diseases, yet their therapeutic effectiveness is limited by their heterogeneity. Clinical outcomes may be enhanced by isolating MSC subpopulations based on surface markers, including CD146. Many in vitro studies have investigated various cellular properties of MSC subpopulations that are enriched in CD146-expressing cells (CD146Enr.) compared to those that are depleted in CD146-expressing cells (CD146Depl.) and/or heterogeneous populations. Hence, this review aimed to systematically explore the basic cellular characteristics of MSC populations with different levels of CD146-expressing cells.MethodsTwo electronic databases were searched until 9 September 2024. Studies were screened using PICO-based eligibility criteria whilst following PRISMA guidelines. Risk of bias was assessed by evaluating reporting and methodological criteria, modified from Samuel et al. A Meta-analysis was performed on four studies on population doubling time (PDT) and five studies on colony-forming (CF) potential comparing CD146Enr. with CD146Depl. populations.ResultsA total of 29 in vitro studies were covered by this systematic review. PDT was slightly higher in CD146Enr. MSCs compared to CD146Depl. MSCs, but without statistical significance (2.52 hours, 95% CI -7.69, 12.74, p = 0.63, n = 19 donors). Contrary, CD146Enr. populations displayed significantly higher CF potential (1.29, 95% CI 0.41, 2.16, p = 0.004, n = 25 donors). All four studies assessing migration reported enhanced migratory potential in CD146Enr. populations. Results from tri-lineage differentiation, proliferation, and immunomodulation were highly variable across studies.ConclusionOverall, this systematic review indicates that CD146Enr. MSCs demonstrate only partially enhanced cellular characteristics, depending on the investigated study. The substantial heterogeneity across included studies limits firm conclusions. To enable robust comparisons and to fully evaluate the clinical potential of CD146Enr.MSCs, standardized experimental protocols and outcome measures are needed.

Over the past few years, biomaterial-based periodontal tissue engineering has gained popularity. An ideal biomaterial for treating periodontal defects is expected to stimulate periodontal-derived cells, allowing them to contribute most efficiently to tissue reconstruction. The present study focuses on evaluating the in vitro behavior of human periodontal ligament-derived stromal cells (hPDL-MSCs) when cultured on gelatin/Polycaprolactone prototype (GPP) and volume-stable collagen matrix (VSCM). Cells were cultured onto the GPP, VSCM, or tissue culture plate (TCP) for 3, 7, and 14 days. Cell morphology, adhesion, proliferation/viability, the gene expression of Collagen type I, alpha1 (COL1A1), Vascular endothelial growth factor A (VEGF-A), Periostin (POSTN), Cementum protein 1 (CEMP1), Cementum attachment protein (CAP), Interleukin 8 (IL-8) and Osteocalcin (OCN), and the levels of VEGF-A and IL-8 proteins were investigated. hPDL-MSCs attached to both biomaterials exhibited a different morphology compared to TCP. GPP exhibited stronger capabilities in enhancing cell viability and metabolic activity compared to VSCM. In most cases, the expression of all investigated genes, except POSTN, was stimulated by both materials, with GPP having a superior effect on COL1A1 and VEGF-A, and VSCM on OCN. The IL-8 protein production was slightly higher in cells grown on VSCM. GPP also exhibited the ability to absorb VEGF-A protein. The gene expression of POSTN was promoted by GPP and slightly suppressed by VSCM. In summary, our findings indicate that GPP electrospun nanofibers effectively promote the functional performance of PDLSCs in periodontal regeneration, particularly in the periodontal ligament and cementum compartment.

IntroductionMesenchymal stromal cells (MSCs) with high expression of CD146 have superior properties for tissue regeneration. However, high variability in the rate of CD146+ cells among donors is observed. In this study, the possible reasons behind this variability in human periodontal ligament MSCs (hPDL-MSCs) were explored.MethodshPDL-MSCs were isolated from 22 different donors, and rates of CD146+ cells were analyzed by flow cytometry. Furthermore, populations with various rates of CD146+ cells were isolated with magnetic separation. The dependency of cell proliferation, viability, cell cycle, and osteogenic differentiation on the rates of CD146+ cells was investigated. Besides, the effects of various factors, like cell density, confluence, and inflammatory environment on the CD146+ rate and expression were analyzed.ResultsThe rate of CD146+ cells exhibited high variability between donors, with the percentage of CD146+ cells ranging from 3% to 67%. Higher percentage of CD146+ cells was associated with higher proliferation, presumably due to the higher percentage of cells in the S-phase, and higher osteogenic differentiation potential. Prolonged cell confluence and higher cell seeding density led to the decline in the rate of CD146+ cells. The surface rate of CD146 in hPDL-MSCs was stimulated by the treatment with interleukin-1β and tumor necrosis factor-α, and inhibited by the treatment with interferon-γ.ConclusionThese results suggest that hPDL-MSCs with high rate of CD146+ cells are a promising subpopulation for enhancing the effectiveness of MSC-based regenerative therapies, however the rate of CD146 is affected by various factors, which must be considered for cell propagation and their potential application in vivo.

IntroductionHuman periodontal ligament-derived mesenchymal stromal cells (hPDL-MSCs) possess a strong ability to modulate the immune response, executed via cytokine-boosted paracrine and direct cell-to-cell contact mechanisms. This reciprocal interaction between immune cells and hPDL-MSCs is influenced by 1,25-dihydroxyvitamin-D3 (1,25(OH)2D3). In this study, the participation of different immunomodulatory mechanisms on the hPDL-MSCs-based effects of 1,25(OH)2D3 on CD4+ T lymphocytes will be elucidated using different co-culture models with various cytokine milieus.Material and methodshPDL-MSCs and CD4+ T lymphocytes were co-cultured indirectly and directly with inserts (paracrine interaction only) or directly without inserts (paracrine and direct cell-to-cell contact interaction). They were stimulated with TNF-α or IL-1β in the absence/presence of 1,25(OH)2D3. After five days of co-cultivation, the CD4+ T lymphocyte proliferation, viability, and cytokine secretion were analyzed. Additionally, the gene expression of soluble and membrane-bound immunomediators was determined in hPDL-MSCs.ResultsIn the indirect and direct co-culture model with inserts, 1,25(OH)2D3 decreased CD4+ T lymphocyte proliferation and viability. The direct co-culture model without inserts caused the opposite effect. 1,25(OH)2D3 mainly decreased the CD4+ T lymphocyte-associated secretion of cytokines via hPDL-MSCs. The degree of these inhibitions varied between the different co-culture setups. 1,25(OH)2D3 predominantly decreased the expression of the soluble and membrane-bound immunomediators in hPDL-MSCs to a different extent, depending on the co-culture models. The degree of all these effects depended on the absence and presence of exogenous TNF-α and IL-1β.ConclusionThese data assume that 1,25(OH)2D3 differently affects CD4+ T lymphocytes via the paracrine and direct cell-to-cell contact mechanisms of hPDL-MSCs, showing anti- or pro-inflammatory effects depending on the co-culture model type. The local cytokine microenvironment seems to be involved in fine-tuning these effects. Future studies should consider this double-edged observation by executing different co-culture models in parallel.

Abstract Background Mesenchymal stromal cells (MSCs) isolated from the periodontal ligament (hPDL-MSCs) have a high therapeutic potential, presumably due to their immunomodulatory properties. The interaction between hPDL-MSCs and immune cells is reciprocal and executed by diverse cytokine-triggered paracrine and direct cell-to-cell contact mechanisms. For the first time, this study aimed to directly compare the contribution of various mechanisms on this reciprocal interaction using different in vitro co-culture models at different inflammatory milieus. Methods Three co-culture models were used: indirect with 0.4 μm-pored insert, and direct with or without insert. After five days of co-culturing mitogen-activated CD4+ T lymphocytes with untreated, interleukin (IL)-1β, or tumor necrosis factor (TNF)-α- treated hPDL-MSCs, the CD4+ T lymphocyte proliferation, viability, and cytokine secretion were investigated. The gene expression of soluble and membrane-bound immunomediators was investigated in the co-cultured hPDL-MSCs. Results Untreated hPDL-MSCs decreased the CD4+ T lymphocyte proliferation and viability more effectively in the direct co-culture models. The direct co-culture model without inserts showed a strikingly higher CD4+ T lymphocyte cell death rate. Adding IL-1β to the co-culture models resulted in substantial CD4+ T lymphocyte response alterations, whereas adding TNF resulted in only moderate effects. The most changes in CD4+ T lymphocyte parameters upon the addition of IL-1β or TNF-α in a direct co-culture model without insert were qualitatively different from those observed in two other models. Additionally, the co-culture models caused variability in the immunomediator gene expression in untreated and cytokine-triggered hPDL-MSCs. Conclusion These results suggest that both paracrine and cell-to-cell contact mechanisms contribute to the reciprocal interaction between hPDL-MSCs and CD4+ T lymphocytes. The inflammatory environment affects each of these mechanisms, which depends on the type of cytokines used for the activation of MSCs’ immunomodulatory activities. This fact should be considered by comparing the outcomes of the different models.

Mesenchymal stromal cells (MSCs) are multipotent, progenitor cells that reside in tissues across the human body, including the periodontal ligament (PDL) and gingiva. They are a promising therapeutic tool for various degenerative and inflammatory diseases. However, different heterogeneity levels caused by tissue-to-tissue and donor-to-donor variability, and even intercellular differences within a given MSCs population, restrict their therapeutic potential. There are considerable efforts to decipher these heterogeneity levels using different "omics" approaches, including single-cell transcriptomics. Previous studies applied this approach to compare MSCs isolated from various tissues of different individuals, but distinguishing between donor-to-donor and tissue-to-tissue variability is still challenging. In this study, MSCs were isolated from the PDL and gingiva of 5 periodontally healthy individuals and cultured in vitro. A total of 3,844 transcriptomes were generated using single-cell mRNA sequencing. Clustering across the 2 different tissues per donor identified PDL- and gingiva-specific and tissue-spanning MSCs subpopulations with unique upregulated gene sets. Gene/pathway enrichment and protein-protein interaction (PPI) network analysis revealed differences restricted to several cellular processes between tissue-specific subpopulations, indicating a limited tissue-of-origin variability in MSCs. Gene expression, pathway enrichment, and PPI network analysis across all donors' PDL- or gingiva-specific subpopulations showed significant but limited donor-to-donor differences. In conclusion, this study demonstrates tissue- and donor-specific variabilities in the transcriptome level of PDL- and gingiva-derived MSCs, which seem restricted to specific cellular processes. Identifying tissue-specific and tissue-spanning subpopulations highlights the intercellular differences in dental tissue-derived MSCs. It could be reasonable to control MSCs at a single-cell level to ensure their properties before transplantation.

The differentiation ability of human periodontal ligament mesenchymal stromal cells (hPDL-MSCs) in vivo is limited; therefore, some studies considered strategies involving their pre-differentiation in vitro. However, it is not known how the differentiation of hPDL-MSCs influences their immunomodulatory properties. This study investigated how osteogenic differentiation of hPDL-MSCs affects their ability to suppress CD4+ T-lymphocyte proliferation. hPDL-MSCs were cultured for 21 days in osteogenic differentiation or standard culture media. Allogeneic CD4+ T lymphocytes were co-cultured with undifferentiated and differentiated cells in the presence or absence of interferon (IFN)-γ, interleukin (IL)-1β or tumor necrosis factor (TNF)-α, and their proliferation and apoptosis were measured. Additionally, the effects of these cytokines on the expression of immunomodulatory or pro-inflammatory factors were investigated. Our data show that osteogenic differentiation of hPDL-MSCs reduced their ability to suppress the proliferation of CD4+ T lymphocytes in the presence of IFN-γ and enhanced this ability in the presence of IL-1β. These changes were accompanied by a slightly decreased proportion of apoptotic CD4+ in the presence of IFN-γ. The osteogenic differentiation was accompanied by decreases and increases in the activity of indoleamine-2,3-dioxygenase in the presence of IFN-γ and IL-1β, respectively. The basal production of interleukin-8 by hPDL-MSCs was substantially increased upon osteogenic differentiation. In conclusion, this study suggests that pre-differentiation strategies in vitro may impact the immunomodulatory properties of hPDL-MSCs and subsequently affect their therapeutic effectiveness in vivo. These findings provide important insights for the development of MSC-based therapies.

The aesthetic constancy and functional stability of periodontium largely depend on the presence of healthy mucogingival tissue. Soft tissue management is crucial to the success of periodontal surgery. Recently, synthetic substitute materials have been proposed to be used for soft tissue augmentation, but the tissue compatibility of these materials needs to be further investigated. This study aims to assess the in vitro responses of human gingival mesenchymal stromal cells (hG-MSCs) cultured on a Gelatin/Polycaprolactone prototype (GPP) and volume-stable collagen matrix (VSCM). hG-MSCs were cultured onto the GPP, VSCM, or plastic for 3, 7, and 14 days. The proliferation and/or viability were measured by cell counting kit-8 assay and resazurin-based toxicity assay. Cell morphology and adhesion were evaluated by microscopy. The gene expression of collagen type I, alpha1 (COL1A1), α-smooth muscle actin (α-SMA), fibroblast growth factor (FGF-2), vascular endothelial growth factor A (VEGF-A), transforming growth factor beta-1 (TGF-β1), focal adhesion kinase (FAK), integrin beta-1 (ITG-β1), and interleukin 8 (IL-8) was investigated by RT-qPCR. The levels of VEGF-A, TGF-β1, and IL-8 proteins in conditioned media were tested by ELISA. GPP improved both cell proliferation and viability compared to VSCM. The cells grown on GPP exhibited a distinct morphology and attachment performance. COL1A1, α-SMA, VEGF-A, FGF-2, and FAK were positively modulated in hG-MSCs on GPP at different investigation times. GPP increased the gene expression of TGF-β1 but had no effect on protein production. The level of ITG-β1 had no significant changes in cells seeded on GPP at 7 days. At 3 days, notable differences in VEGF-A, TGF-β1, and α-SMA expression levels were observed between cells seeded on GPP and those on VSCM. Meanwhile, GPP showed higher COL1A1 expression compared to VSCM after 14 days, whereas VSCM demonstrated a more significant upregulation in the production of IL-8. Taken together, our data suggest that GPP electrospun nanofibers have great potential as substitutes for soft tissue regeneration in successful periodontal surgery.

The transient worsening of oral health sometimes accompanies orthodontic treatment (OT), and the extent of this effect might depend on whether the patients are treated with traditional brackets or clear aligners. Saliva is an important tool for monitoring oral health and influences the functional properties of various oral cells. This study aimed to compare the effects of saliva from patients undergoing OT with Invisalign aligners and brackets on human gingival fibroblasts and oral epithelial cells in vitro. Unstimulated saliva was collected from 15 patients treated with Invisalign and 16 patients treated with brackets before and 3 and 6 months after therapy began. The saliva was used to stimulate primary human gingival fibroblasts and the oral epithelial Ca9-22 cell line, and the resulting cell response was investigated. Saliva did not exhibit any toxic effect on investigated cells, as shown by the proliferation/viability assay with the MTT method. In human gingival fibroblasts, saliva increased gene expression of various proinflammatory mediators, such as interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1, assessed by qPCR. In epithelial cells, saliva increased the production of IL-8 measured by ELISA and decreased gene expression of various proteins involved in the barrier function. During the therapy, the saliva-induced production of IL-8 tended to be decreased, and the saliva-induced decrease in the expression of barrier protein was partially improved. No difference between aligners and brackets was observed in either cell type. Saliva affects the functional properties of oral cells, but this effect is not influenced by the type of OT.

Introduction Human periodontal ligament-derived mesenchymal stromal cells (hPDL-MSCs) exhibit a tight bi-directional interaction with CD4 + T lymphocytes. The hPDL-MSCs’ immunomodulatory abilities are drastically enhanced by pro-inflammatory cytokines via boosting the expression of various immunomediators. 25-hydroxyvitamin D 3 (25(OH)D 3 ), the major metabolite of vitamin D3 in the blood, affects both hPDL-MSCs and CD4 + T lymphocytes, but its influence on their interaction is unknown. Methods Therefore, primary hPDL-MSCs were stimulated in vitro with tumor necrosis factor (TNF)-α a or interleukin (IL)-1β in the absence and presence of 25(OH)D 3 followed by an indirect co-culture with phytohemagglutinin-activated CD4 + T lymphocytes. The CD4 + T lymphocyte proliferation, viability, and cytokine secretion were analyzed. Additionally, the expression of various immunomediators in hPDL-MSCs was investigated, and their implication was verified by using pharmacological inhibitors. Results 25(OH)D 3 significantly counteracted the suppressive effects of IL-1β-treated hPDL-MSCs on CD4 + T lymphocyte proliferation, whereas no effects were observed in the presence of TNF-α. Additionally, 25(OH)D 3 significantly increased the percentage of viable CD4 + T lymphocytes via TNF-α- or IL-1β-treated hPDL-MSCs. It also caused a significant decrease in interferon-γ, IL-17A, and transforming growth factor-β productions, which were triggered by TNF-α-treated hPDL-MSCs. 25(OH)D 3 significantly decreased the production of various immunomediators in hPDL-MSCs. Inhibition of two of them, prostaglandin E2 and indoleamine-2,3-dioxygenase-1, partially abolished some of the hPDL-MSCs-mediated effects of 25(OH)D 3 on CD4 + T lymphocytes. Conclusion These data indicate that 25(OH)D 3 influences the immunomodulatory activities of hPDL-MSCs. This modulatory potential seems to have high plasticity depending on the local cytokine conditions and may be involved in regulating periodontal tissue inflammatory processes.

Orthodontic tooth movement (OTM) is induced by applying active mechanical forces, causing a local non-infectious inflammatory response in the periodontal ligament (PDL). As a prerequisite for OTM, the inflammation status is associated with increased levels of various cytokines and involves the interaction between immune cells and periodontal ligament stem cells (hPDLSCs). It is well established that hPDLSCs respond to orthodontic forces in several ways, such as by secreting multiple inflammatory factors. Another essential feature of hPDLSCs is their immunomodulatory activities, which are executed through cytokine (e.g., TNF-α and IL-1β)-induced production of various soluble immunomediators (e.g., indoleamine-2,3-dioxygenase-1, tumor necrosis factor-inducible gene 6 protein, prostaglandin E2) and direct cell-to-cell contact (e.g., programmed cell death ligand 1, programmed cell death ligand 2). It is well known that these immunomodulatory abilities are essential for local periodontal tissue homeostasis and regeneration. So far, only a handful of studies provides first hints that hPDLSCs change immunological processes during OTM via their immunomodulatory activities. These studies demonstrate the pro-inflammatory aspect of immunomodulation by hPDLSCs. However, no studies exist which investigate cytokine and cell-to-cell contact mediated immunomodulatory activities of hPDLSCs. In this perspective article, we will discuss the potential role of the immunomodulatory potential of hPDLSCs in establishing and resolving the OTM-associated non-infectious inflammation and hence its potential impact on periodontal tissue homeostasis during OTM.

AbstractObjectivesPolymorphonuclear leucocytes (PMNs) constitute the first line of host defence and are crucial in maintaining periodontal health. Their survival and function are modulated by mesenchymal stromal cells (MSCs) from different origin. Gingival MSCs (GMSCs) play an important role in maintaining oral health and in the initial inflammatory response. The present study aimed to investigate the effects of GMSCs on PMNs apoptosis and reactive oxygen species (ROS) production.MethodsPMNs were either directly incubated with untreated, interleukin (IL)‐1β‐ or tumour necrosis factor (TNF)‐α‐treated GMSCs or stimulated with their conditioned media. Resulting ROS production was evaluated by dichlorofluorescin diacetate staining, whereas PMNs apoptosis was assessed by Annexin V staining, followed by flow cytometry analysis.ResultsWhile conditioned media of untreated and TNF‐α‐treated GMSCs did not affect apoptosis of PMNs, it was significantly delayed by conditioned media of GMSCs treated with IL‐1β. In direct co‐culture, GMSCs exerted anti‐apoptotic effects on PMNs independently of the previous stimulation. However, the strongest impact was observed by IL‐1β‐treated GMSCs. ROS production of PMNs was not influenced by GMSCs or their conditioned media.ConclusionThis study demonstrates for the first time the immunomodulatory properties of GMSCs towards PMNs, revealing that IL‐1β enhances anti‐apoptotic effects of GMSCs.