The Impact of mRNA Technology in Regenerative Therapy: Lessons for Oral Tissue Regeneration.

Oral tissue regeneration following chronic diseases and injuries is limited by the natural endogenous wound-healing process. Current regenerative approaches implement exogenous systems, including stem cells, scaffolds, growth factors, and plasmid DNA/viral vectors, that induce variable clinical outcomes. An innovative approach that is safe, effective, and inexpensive is needed. The lipid nanoparticle-encapsulated nucleoside-modified messenger RNA (mRNA) platform has proven to be a successful vaccine modality against coronavirus disease 2019, demonstrating safety and high efficacy in humans. The same fundamental technology platform could be applied to facilitate the development of mRNA-based regenerative therapy. While the platform has not yet been studied in the field of oral tissue regeneration, mRNA therapeutics encoding growth factors have been evaluated and demonstrated promising findings in various models of soft and hard tissue regeneration such as myocardial infarction, diabetic wound healing, and calvarial and femoral bone defects. Because restoration of both soft and hard tissues is crucial to oral tissue physiology, this new therapeutic modality may help to overcome challenges associated with the reconstruction of the unique and complex architecture of oral tissues. This review discusses mRNA therapeutics with an emphasis on findings and lessons in different regenerative animal models, and it speculates how we can apply mRNA-based platforms for oral tissue regeneration.

Cigarette smoke extract modulates human beta-defensin-2 and interleukin-8 expression in human gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Human gingival epithelial cells (HGECs) are continually exposed to oral bacteria and to other harmful agents. Their responses to stimuli are critical in maintaining periodontal homeostasis. The aim of this study was to investigate the modulating effect of cigarette smoke extract (CSE) on the innate immune responses of HGECs. MATERIAL AND METHODS: Toll-like receptor (TLR) expression of HGECs was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The effect of CSE or nicotine on the expression of the antimicrobial peptide human beta-defensin-2 (hBD-2) and the pro-inflammatory cytokine interleukin (IL)-8 in stimulated HGEC cultures was evaluated by RT-PCR and enzyme-linked immunosorbent assay. RESULTS: The HGECs expressed mRNA of TLRs 1, 2, 3, 5, 6, 9, 10, and minimally of TLR4, but not of TLRs 7 or 8. Stimulation of HGECs with highly purified TLR2, 3 or 5 ligands led to expression of hBD-2 and of IL-8. Enhancement of hBD-2 and IL-8 was observed in HGECs after combined stimulation with Porphyromonas gingivalis lipopolysaccharide (TLR2 ligand) and tumour necrosis factor-alpha, compared with stimulation using either agent alone. After CSE exposure, hBD-2 expression was markedly reduced in stimulated HGEC cultures, whereas IL-8 expression was markedly increased. These effects were also observed, but were markedly attenuated, upon nicotine treatment. CONCLUSION: Human gingival epithelial cells play a critical role in orchestrating the innate immune responses of periodontal tissue via TLR signalling. Our results represent the first demonstration that CSE can modulate HGEC function by suppressing hBD-2 and enhancing IL-8 production, and this may be, in part, a possible mechanism which promotes periodontal disease.

Effects of IL-17 on human gingival fibroblasts.

Interleukin (IL)-17 is present in inflammatory periodontal lesions, thus suggesting a role in mediating inflammation. We tested the hypothesis that IL-17, especially when combined with interferon (IFN)-gamma, may modulate the responses of human gingival fibroblasts (HGFs). IL-17 induced IL-8 and minimal intercellular adhesion molecule (ICAM)-1 expression. It had no effect on expression of HLA-DR, CD40, or the immune-suppressive enzyme indoleamine 2,3-dioxygenase (IDO). The effects of IL-17 on HGFs were compared with those of IFN-gamma. Unlike IL-17, IFN-gamma augmented the expression of HLA-DR, ICAM-1, and IDO, but not IL-8. Thus, IL-17 and IFN-gamma induce different HGF responses when administered separately. Interestingly, when IL-17 and IFN-gamma were combined, marked enhancement of ICAM-1, IL-8, and IDO expression by HGFs was observed. These findings suggest that IL-17, especially when combined with IFN-gamma, could play an important role in immune modulation through stimulation of HGFs in periodontal disease.

Monocyte activation by Porphyromonas gingivalis LPS in aggressive periodontitis with the use of whole-blood cultures.

In this study, we re-visited the issue of hyper-responsiveness of monocytes to bacterial lipopolysaccharide (LPS) in aggressive periodontitis patients. We used whole-blood cultures to compare monocyte activation by Porphyromonas gingivalis LPS between Thai subjects with generalized aggressive periodontitis and those without periodontitis. Upon stimulation with P. gingivalis LPS, expression of co-stimulatory molecules on monocytes and expression of CD69 on NK and gamma delta T-cells were analyzed by flow cytometry, and the production of interleukin-1 beta and prostaglandin E(2) was monitored by ELISA. LPS stimulation resulted in a dose-dependent up-regulation of CD40, CD80, and CD86 on monocytes, and up-regulation of CD69 on NK cells and gamma delta T-cells in both the periodontitis and non-periodontitis groups. The levels of activation markers and the mediator production after LPS stimulation were quite similar for both groups. In conclusion, we did not observe hyper-responsiveness of monocytes to P. gingivalis LPS challenge in Thai patients with aggressive periodontitis.

LPS-stimulated human gingival fibroblasts inhibit the differentiation of monocytes into osteoclasts through the production of osteoprotegerin.

Periodontitis is an inflammatory bone disease caused by Gram-negative anaerobic bacteria, but the precise mechanism of bone destruction remains unknown. Activated T lymphocytes secrete receptor activator of NF-kappaB ligand (RANKL) and support the differentiation of monocytes into mature osteoclasts. The purpose of this study was to examine the expression of RANKL and its inhibitor, osteoprotegerin (OPG), in inflamed gingival tissue and to clarify the role of human gingival fibroblasts (HGFs) in osteoclastogenesis regulated by RANKL. HGFs and gingival mononuclear cells (GMCs) were obtained from chronic periodontitis patients during routine periodontal surgery. Expression of OPG and RANKL mRNA in gingival tissue and HGFs was examined with RT-PCR. OPG production was measured using ELISA. Expression of RANKL, CD4, CD8 and CD69 on GMCs was determined by flow-cytometry using RANK-Fc fusion protein and the respective monoclonal antibodies. Osteoclastogenesis by RANKL was assayed by counting the number of tartarate-resistant acid phosphatase (TRAP)-positive cells after culturing human peripheral blood monocytes with recombinant human RANKL and macrophage-colony stimulating factor (M-CSF) for 10 days. OPG and RANKL mRNA were expressed in 80% (16/20) and 25% (5/20) of periodontitis lesions, respectively. OPG, but not RANKL, mRNA was expressed within HGFs. OPG mRNA expression and production by HGFs was augmented by LPS stimulation. All GMC samples expressed CD69, and two of five GMC samples expressed RANKL. The culture supernatant of LPS-stimulated gingival fibroblasts significantly reduced the number of TRAP positive cells generated by culturing monocytes with RANKL and M-CSF. The present study suggests that LPS-stimulated HGFs inhibit monocyte differentiation into osteoclasts through the production of OPG.

The immune modulation of B-cell responses by Porphyromonas ginginvalis and interleukin-10.

BACKGROUND: Polyclonal B-cell activation induced by periodontopathic bacteria has been cited as being important for elevated numbers of B cells, but the role of bacteria in the pathogenesis of periodontal disease remains unknown. In this study, we used an in vitro model to investigate the activation of immune cells by the periodontopathic bacterium Porphyromonas gingivalis in healthy subjects. METHODS: Peripheral blood mononuclear cells (PBMC) or purified subsets of lymphocytes were stimulated with sonicated extracts of P. gingivalis for 24 hours. Cells were harvested and monitored for expression of CD69 by flow cytometry. Cytokine production (IL-10, IL-12, and IL-15) in P. gingivalis-stimulated PBMC cultures was measured by ELISA. To identify IL-10 producer cells, a cell depletion experiment was used and confirmed by the ability of the purified cell population to produce IL-10. To evaluate the effect of P. gingivalis and IL-10, the proliferative response of purified B cells was assessed by [3H] thymidine uptake. RESULTS: PBMC cultured with P. gingivalis led to a large number of activated B and natural killer (NK) cells as monitored by CD69 expression. When positively sorted cells were used, the bacterium itself could directly activate only B cells but not NK cells, alphabeta, and gammadelta T cells. Measurement of B-cell regulatory cytokine production in P. gingivalis-stimulated PBMC cultures revealed a large amount of IL-10 but no detectable IL-12 or IL-15; the major producing cells were monocytes, not B cells or alphabeta T cells. When IL-10 was added to B cells in the presence of bacteria, significantly increased B-cell proliferative responses were observed. CONCLUSIONS: These results suggest that P. gingivalis, both directly and indirectly via macrophage IL-10, may play an important role in polyclonal B-cell activation associated with periodontal disease.

Individual diversities in interferon gamma production by human peripheral blood mononuclear cells stimulated with periodontopathic bacteria.

Polarization of type 1 (Th1) or type 2 (Th2) immune responses determines the prognosis of many infectious diseases. Interferon gamma (IFN-gamma) and IL-4 are key cytokines for the development of type 1 and type 2 immune responses, respectively. The aim of this study was to examine individual diversities in the polarization of type 1 and type 2 responses against periodontopathic bacteria. Peripheral blood mononuclear cells (PBMCs) from adult periodontitis (AP) patients and healthy (H) subjects were stimulated with Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Bacteroides forsythus with or without polymyxin-B, CTLA-4 Ig and anti-IL-12 antibody. IFN-gamma, IL-4 and IL-12 in the culture supernatant were measured. IFN-gamma and IL-4 producing cells were also examined using a multiparameter flow cytometric assay. Bone resorption rate in AP patients was calculated using Schei's method, and the probing pocket depth was also measured. PBMCs from AP patients and H subjects produced IFN-gamma and IL-12, whereas the production of IL-4 was rarely observed. Among the bacteria tested, A. actinomycetemcomitans was the most potent inducer of IFN-gamma and IL-12, and the reaction was inhibited by polymyxin-B. IFN-gamma was found to be produced by T cells in the PBMCs, and the production was significantly reduced by CTLA-4 Ig and anti-IL-12 neutralizing antibody. The amount of IFN-gamma produced by the PBMCs of AP patients and H subjects varied among individuals, and was significantly correlated with the amount of IL-12 produced in a particular individual. The production of IFN-gamma was not related with periodontal condition which was evaluated using bone resorption and pocket depth. These results suggest that polarization of type 1 response against periodontopathic bacteria is dependent on the production of IL-12 by monocytes, and that IL-12 stimulates IFN-gamma production. However, individual diversities of IFN-gamma production might not be directly related to the severity of periodontitis.

The establishment of human T cell lines reactive with specific periodontal bacteria.

Human T cell lines (TCLs) were obtained by stimulation of peripheral blood mononuclear cells (PBMCs) with the 2 periodontopathic bacteria, Porphyromonas gingivalis FDC-381 and Fusobacterium nucleatum FDC-263. After the first round of stimulation and rest, the cells responded specifically to the bacteria originally used to establish each line. Throughout the culture period, the responsiveness of each of the TCLs to their specific bacteria increased. Phenotypic analysis of the TCLs revealed heterogeneity of cell types. In both TCLs approximately 80% of the cells were T cells, all of which bore the alpha beta T cell receptor. The P. gingivalis-reactive TCL (PG-TCL) showed approximately equal proportions of CD4+ and CD8+ cells, whereas the F. nucleatum-reactive TCL (FN-TCL) was predominantly CD4+. The expression of CD25, HLA-DR, CD45RA and CD29 on these CD4+ cells varied throughout the culture period of 45 days. These results demonstrate that it is possible to establish long-term T cell lines reacting to specific periodontopathic bacteria.

Effect of initial treatment of chronic inflammatory periodontal disease on the frequency of peripheral blood T-lymphocytes specific to periodontopathic bacteria.

Limit dilution analysis (LDA) was used to determine the effect of initial treatment of chronic inflammatory periodontal disease on the frequency of periodontopathic bacteria-specific T-cells in peripheral blood. Eleven marginal gingivitis (MG) and 8 adult periodontitis (AP) subjects took part in the study. The proliferative T-lymphocyte precursor (PTL-P) frequencies to Porphyromonas gingivalis and Actinomyces viscosus were determined using LDA and Poisson statistics both before and after treatment. Tetanus toxoid was used as a control antigen. Treatment resulted in a significant reduction in clinical disease parameters in both groups. The median peak PTL-P frequency for P. gingivalis was significantly higher in the AP group compared with the MG group before treatment. This was not the case after treatment nor with A. viscosus. In the MG group the median peak PTL-P frequency with both P. gingivalis and A. viscosus declined as a result of treatment. Although this decline was not statistically significant it may indicate an antigen-specific response in this group. In the AP group the median peak PTL-P frequency with P. gingivalis before treatment was 83.76 x 10(-6) (approximately 1 in 12,000) and after treatment it was 36.17 x 10(-6) (approximately 1 in 28,000). Dose-response relationships showed at each concentration of organisms/well this trend for a decline in PTL-P frequency after treatment, suggesting that any increased responsiveness to this organism in this group may be largely antigen-specific. However, there was no difference in this group in the median peak PTL-P frequency with A. viscosus before and after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Limit dilution analysis of peripheral blood T lymphocytes specific to periodontopathic bacteria.

Limit dilution analysis (LDA) was used to determine the presence and frequency of periodontopathic-bacteria-specific T cells in the peripheral blood of patients with chronic inflammatory periodontal disease. Twelve adult periodontitis (AP), 13 marginal gingivitis (MG) and 12 healthy control subjects took part in the study. Bacteroides gingivalis and Actinomyces viscosus were used as test organisms, while tetanus toxoid was used as the control antigen. The median PTL-p frequencies to B. gingivalis were 46.33 x 10(-6), 45.33 x 10(-6) and 58.83 x 10(-6) in the control, gingivitis and AP groups respectively, while the median PTL-p frequencies to A. viscosus were 13.8 x 10(-6), 17.33 x 10(-6) and 11.5 x 10(-6), again in the control, gingivitis and AP groups. There were no statistically significant differences between the groups. All subjects displayed 'single-hit' kinetics with the control tetanus toxoid antigen and, with three exceptions, 'single-hit' kinetics was also found with the two test organisms. One control subject displayed a 'saw-tooth' curve with A. viscosus and a 'suppressor' curve with B. gingivalis, while two MG subjects had a 'saw-tooth' curve with B. gingivalis. These complex curves suggest that, in some subjects, more than one limiting cell type may exist in the cultures. Nevertheless, the results of the present study illustrate that lymphocytes specific to periodontopathic bacteria exist in the peripheral blood of both diseased and non-diseased subjects.

The proportion of suppressor-inducer T-lymphocytes is reduced in recurrent aphthous stomatitis.

A flow cytometric analysis of peripheral blood lymphocytes was undertaken in recurrent aphthous stomatitis patients. The project aimed at detecting differences within lymphocyte subsets using type-specific monoclonal antibodies. Peripheral blood samples were taken from RAS patients in both active and remission phases of the disease and from a group of healthy control subjects. There were no statistical differences between the active and remission phases within any of the lymphocyte subsets examined. There was, however, a significant difference between the RAS group and the control group. RAS patients have depressed CD4+ cell numbers and elevated CD8+ cell numbers. The CD4:CD8 ratio is also depressed. A dissection of the CD4+ subset shows raised numbers of CD4+, 4B4+ lymphocytes and depressed numbers of CD4+, 2H4+ lymphocytes. Previous studies have shown disruption of peripheral blood lymphocyte numbers in Behçet's syndrome. A similar pattern has now been shown in uncomplicated cases of minor RAS.