Inflammatory and bone remodelling related biomarkers following periodontal transplantation of the tissue engineered biocomplex.

OBJECTIVES: To assess gingival crevicular fluid (GCF) levels of inflammatory and bone remodelling related biomarkers following transplantation of a tissue-engineered biocomplex into intrabony defects at several time-points over 12-months. MATERIALS AND METHODS: Group-A (n = 9) received the Minimal Access Flap (MAF) surgical technique combined with a biocomplex of autologous clinical-grade alveolar bone-marrow mesenchymal stem cells in collagen scaffolds enriched with an autologous fibrin/platelet lysate (aFPL). Group-B (n = 10) received the MAF surgery, with collagen scaffolds enriched with aFPL and Group-C (n = 8) received the MAF surgery alone. GCF was collected from the osseous defects of subjects via paper strips/30 sec at baseline, 6-weeks, 3-, 6-, 9-, 12-months post-surgery. Levels of inflammatory and bone remodelling-related biomarkers in GCF were determined by ELISA. RESULTS: Group-A demonstrated significantly higher GCF levels of BMP-7 at 6-9 months than baseline, with gradually decreasing levels of pro-inflammatory and pro-osteoclastogenic markers (TNF-α, RANKL) over the study-period; and an overall decrease in the RANKL/OPG ratio at 9-12 months than baseline (all p < 0.001). In comparison, only modest interim changes were observed in Groups-B and -C. CONCLUSIONS: At the protein level, the approach of MAF and biocomplex transplantation provided greater tissue regeneration potential as cell-based therapy appeared to modulate inflammation and bone remodelling in residual periodontal defects. CLINICAL RELEVANCE: Transplantation of a tissue engineered construct into periodontal intrabony defects demonstrated a biochemical pattern for inflammatory control and tissue regeneration over 12-months compared to the control treatments. Understanding the biological healing events of stem cell transplantation may facilitate the design of novel treatment strategies. CLINICAL DATABASE REGISTRATION: ClinicalTrials.gov ID: NCT02449005.

Characterization of the soft-tissue wall lining residual periodontal pockets and implications in periodontal wound healing.

AIM: To characterize the soft-tissue wall of remaining periodontal pockets for wound healing-related parameters versus healthy gingival crevices in the same individuals. MATERIALS AND METHODS: Gingival tissues collected from the diseased interface of pockets (GT biopsies) and from healthy gingival crevices (G biopsies) were subjected to RT2-profiler PCR Array for wound healing-related markers and network analysis of differentially expressed genes. Lymphangiogenesis-related gene expression was determined by qRT-PCR. The migration potential of mesenchymal stem cells isolated from GT biopsies (GT-MSCs) and G biopsies (G-MSCs) was evaluated by the scratch- and the transwell migration assays. The total collagen protein content was determined in GT-MSCs and G-MSCs homogenates. RESULTS: Gene-ontology analysis on significantly upregulated genes expressed in GT biopsies revealed enrichment of several genes involved in processes related to matrix remodeling, collagen deposition, and integrin signaling. No significantly expressed genes were seen in G biopsies. Regarding lymphangiogenesis-related genes, GT biopsies demonstrated greater expression for PROX1 than G biopsies (p = 0.05). Lower migration potential (p < 0.001), yet greater production of collagen protein (p = 0.05), was found for GT-MSCs over G-MSCs. CONCLUSION: Differential expression patterns of various molecular pathways in biopsies and cell cultures of diseased versus healthy gingival tissues indicate a potential of the former for tissue remodeling and repair. CLINICAL RELEVANCE: In the course of periodontitis, granulation tissue is formed within a periodontal defect in an attempt to reconstruct the site. Following treatment procedures periodontal granulation tissue remains inflamed but appears to retain healing potential.

Biological Effects of "Inflammageing" on Human Oral Cells: Insights into a Potential Confounder of Age-Related Diseases.

OBJECTIVES: The term "inflammageing" describes the process of inflammation-induced aging that leads living cells to a state of permanent cell cycle arrest due to chronic antigenic irritation. This in vitro study aimed to shed light on the mechanisms of "inflammageing" on human oral cells. METHODS: Primary cultures of human gingival fibroblasts (hGFs) were exposed to variable pro-inflammatory stimuli, including lipopolysaccharide (LPS), Tumor Necrosis Factor-alpha (TNFa), and gingival crevicular fluid (GCF) collected from active periodontal pockets of systemically healthy patients. Inflammageing was studied through two experimental models, employing either late-passage ("aged") cells (p. 10) that were exposed to the pro-inflammatory stimuli or early-passage ("young") cells (p. 1) continuously exposed during a period of several passages (up to p. 10) to the above-mentioned stimuli. Cells were evaluated for the expression of beta-galactosidase activity (histochemical staining), senescence-associated genes (qPCR analysis), and biomarkers related to a Senescence-Associated Secretory Phenotype (SASP), through proteome profile analysis and bioinformatics. RESULTS: A significant increase (p < 0.05) in beta-galactosidase-positive cells was observed after exposure to each pro-inflammatory stimulus. The senescence-associated gene expression included upregulation for CCND1 and downregulation for SUSD6, and STAG1, a profile typical for cellular senescence. Overall, pro-inflammatory priming of late-passage cells caused more pronounced effects in terms of senescence than long-term exposure of early-passage cells to these stimuli. Proteomic analysis showed induction of SASP, evidenced by upregulation of several pro-inflammatory proteins (IL-6, IL-10, IL-16, IP-10, MCP-1, MCP-2, M-CSF, MIP-1a, MIP-1b, TNFb, sTNF-RI, sTNF-RII, TIMP-2) implicated in cellular aging and immune responses. The least potent impact on the induction of SASP was provoked by LPS and the most pronounced by GCF. CONCLUSION: This study demonstrates that long-term exposure of hGFs to various pro-inflammatory signals induced or accelerated cellular senescence with the most pronounced impact noted for the late-passage cells. The outcome of these analyses provides insights into oral chronic inflammation as a potential confounder of age-related diseases.

Microbial-stem cell interactions in periodontal disease.

Periodontitis is initiated by hyper-inflammatory responses in the periodontal tissues that generate dysbiotic ecological changes within the microbial communities. As a result, supportive tissues of the tooth are damaged and periodontal attachment is lost. Gingival recession, formation of periodontal pockets with the presence of bleeding, and often suppuration and/or tooth mobility are evident upon clinical examination. These changes may ultimately lead to tooth loss. Mesenchymal stem cells (MSCs) are implicated in controlling periodontal disease progression and have been shown to play a key role in periodontal tissue homeostasis and regeneration. Evidence shows that MSCs interact with subgingival microorganisms and their by-products and modulate the activity of immune cells by either paracrine mechanisms or direct cell-to-cell contact. The aim of this review is to reveal the interactions that take place between microbes and in particular periodontal pathogens and MSCs in order to understand the factors and mechanisms that modulate the regenerative capacity of periodontal tissues and the ability of the host to defend against putative pathogens. The clinical implications of these interactions in terms of anti-inflammatory and paracrine responses of MSCs, anti-microbial properties and alterations in function including their regenerative potential are critically discussed based on literature findings. In addition, future directions to design periodontal research models and study ex vivo the microbial-stem cell interactions are introduced.

A tissue-engineered biocomplex for periodontal reconstruction. A proof-of-principle randomized clinical study.

AIM: To assess the safety/efficacy of a tissue-engineered biocomplex in periodontal reconstruction. METHODS: Twenty-seven intrabony defects were block-randomized across three treatment groups: Group-A (NA  = 9) received autologous clinical-grade alveolar bone marrow mesenchymal stem cells (a-BMMSCs), seeded into collagen scaffolds, enriched with autologous fibrin/platelet lysate (aFPL). In Group-B (NB  = 10), the collagen scaffold/aFPL devoid of a-BMMSCs filled the osseous defect. Group-C (NC  = 8) received Minimal Access Flap surgery retaining the soft tissue wall of defects identically with Groups-A/-B. Subjects were clinically/radiographically assessed before anaesthesia (baseline) and repeatedly over 12 months. RESULTS: Quality controls were satisfied before biocomplex transplantation. There were no adverse healing events. All approaches led to significant clinical improvements (p < .001) with no inter-group differences. At 12 months, the estimated marginal means for all groups were as follows: 3.0 (95% CI: 1.9-4.1) mm for attachment gain; 3.7 (2.7-4.8) mm for probing pocket depth reduction; 0.7 (0.2-1.3) mm increase in recession. An overall greater mean reduction in the radiographic Cemento-Enamel Junction to Bottom Defect (CEJ-BD) distance was found for Groups-A/-C over Group-B (p < .023). CONCLUSION: Radiographic evidence of bone fill was less pronounced in Group-B, although clinical improvements were similar across groups. All approaches aimed to trigger the innate healing potential of tissues. Cell-based therapy is justified for periodontal reconstruction and remains promising in selected cases.

Interim tissue changes following connective tissue grafting and two-stage implant placement. A randomized clinical trial.

AIM: To determine tissue changes at implants placed either conventionally or in combination with a connective tissue graft (CTG). MATERIALS AND METHODS: Forty-eight partially edentulous subjects were randomized into two treatment Groups, and 46 completed the study. Group-A (NA  = 23) received crestal implant placement. In Group-B (NB  = 23), a CTG harvested from the palate was stabilized over the implant neck. At the time of implant placement (T0), Groups were categorized as having thin mucosa ≤ 2.5 mm at the surgical site (NSubgroup-AI  = 12, NSubgroup-BI  = 11) or thick mucosa > 2.5 mm (NSubgroup-AII  = 11, NSubgroup-BII  = 12). Mucosa thickness, width of keratinized tissue (WKT), crestal bone levels and relative bone thickness were determined at T0 and at the two-stage surgery (T1). RESULTS: At T1, on the alveolar crest, mucosa thickness significantly decreased in the thick mucosa Subgroups (-AII/-BII, both p = 0.001), but increased at thin mucosa grafted sites (p = 0.049). No significant changes were noted in the WKT for either group. Thin mucosa grafted Subgroups (-AI/-BI) demonstrated significant decreases in crestal bone levels (both p ≤ 0.008). Crestal relative bone thickness decreased in all Subgroups (p ≤ 0.027 for significant changes). CONCLUSIONS: Connective tissue grafting resulted in smaller reductions of mucosa thickness on the alveolar crest and appeared to have the greater effects at sites with initially thin mucosa.

Clinical associations between acetylcholine levels and cholinesterase activity in saliva and gingival crevicular fluid and periodontal diseases.

AIM: The oral mucosa possesses a non-neuronal cholinergic system. This study aimed to determine clinical evidence for a role of cholinergic mechanisms in the pathogenesis of periodontal diseases. MATERIALS AND METHODS: Fifty healthy participants, 52 patients with gingivitis and 49 with periodontitis were recruited. Full periodontal parameters were recorded and saliva and gingival crevicular fluid (GCF) collected. Levels of acetylcholine and inflammatory mediators were quantified using commercially available assay kits. Acetylcholinesterase and butyrylcholinesterase activities were measured using a published biochemical assay. RESULTS: Acetylcholine levels are significantly elevated in saliva and GCF, whereas GCF levels of butyrylcholinesterase activity are significantly decreased, in patients with periodontal diseases. Acetylcholine levels in saliva and GCF correlated positively with clinical markers of disease severity and with increased levels of IL-17A and IL-17F. In contrast, butyrylcholinesterase activity levels in GCF showed significant negative correlations with clinical markers of disease severity and IL-17A and IL-17F levels. None of the findings were due to smoking. CONCLUSIONS: Elevated acetylcholine levels and reduced butyrylcholinesterase activity are clinically associated with periodontal diseases and elevated levels of IL-17A and IL-17F. Therefore, non-neuronal cholinergic mechanisms may influence IL-17 biology and the aetiopathogenesis of periodontal diseases and therefore are possible therapeutic targets.

Oral hygiene reinforcement in the simplified periodontal treatment of 1 hour.

AIM: To compare the clinical and microbiological outcome of the 1-h ultrasonic debridement of chronic periodontitis patients (CPP) with and without frequent sessions of oral hygiene reinforcement. METHODS: Clinical measurements and subgingival plaque were collected from 44 CPP at baseline, 3- and 6-months. The control group received a single session of 1-h full-mouth ultrasonic debridement, while oral hygiene instructions (OHI) were reiterated over four visits. In the test group, OHI were limited in the 1-h treatment session. At 3-months, both groups received additional debridement and OHI. The "Checkerboard" DNA-DNA hybridization technique quantified Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola in plaque. RESULTS: At three months, smaller reductions in plaque and bleeding indices, and in P. gingivalis numbers were noted in the test group, while these differences disappeared at six months. After the 3-month re-treatment visit, the test group presented with a greater probing pocket depth (PPD) reduction. Plaque negatively affected PPD in a similar manner after both treatment approaches. CONCLUSIONS: Lack of oral hygiene reinforcement in the 1-h full-mouth debridement resulted in higher plaque and bleeding scores and numbers of P. gingivalis at three months; professional removal of dental biofilm every three months is beneficial in subjects with compromised plaque control.

Nonsurgical mechanical treatment strategies for periodontal disease.

The comparison of the efficacy of surgical and nonsurgical procedures revealed that scaling and root planing alone or in combination with flap procedures are effective methods for the treatment of chronic periodontitis. Also, the consistent message is that in treating deep pockets, open-flap debridement results in greater probing pocket depth reduction and clinical attachment gain than nonsurgical modalities. Nonsurgical modalities in shallower pockets consistently involve less post-therapy recession and are clearly recognized as being more conservative. Research is still needed on the clinical benefit of the granulation tissue removal that is a feature of periodontal surgical therapy and, to a lesser extent, occurs through indirect trauma in nonsurgical therapy.