Potential of iPSC-Derived Mesenchymal Stromal Cells for Treating Periodontal Disease.

Mesenchymal stromal cell-like populations have been derived from mouse-induced pluripotent stem cells (miPSC-MSC) with the capability for tissue regeneration. In this study, murine iPSC underwent differentiation towards an MSC-like immunophenotype. Stable miPSC-MSC cultures expressed the MSC-associated markers, CD73, CD105, and Sca-1, but lacked expression of the pluripotency marker, SSEA1, and hematopoietic markers, CD34 and CD45. Functionally, miPSC-MSC exhibited the potential for trilineage differentiation into osteoblasts, adipocytes, and chondrocytes and the capacity to suppress the proliferation of mitogen-activated splenocytes. The efficacy of miPSC-MSC was assessed in an acute inflammation model following systemic or local delivery into mice with subcutaneous implants containing heat-inactivated P. gingivalis. Histological analysis revealed less inflammatory cellular infiltrate within the sponges in mice treated with miPSC-MSC cells delivered locally rather than systemically. Assessment of proinflammatory cytokines in mouse spleens found that CXCL1 transcripts and protein were reduced in mice treated with miPSC-MSC. In a periodontitis model, mice subjected to oral inoculation with P. gingivalis revealed less bone tissue destruction and inflammation within the jaws when treated with miPSC-MSC compared to PBS alone. Our results demonstrated that miPSC-MSC derived from iPSC have the capacity to control acute and chronic inflammatory responses associated with the destruction of periodontal tissue. Therefore, miPSC-MSC present a promising novel source of stromal cells which could be used in the treatment of periodontal disease and other inflammatory systemic diseases such as rheumatoid arthritis.

The effect of triclosan on posttranslational modification of proteins through citrullination and carbamylation.

OBJECTIVES: The aim of this study was evaluate the effect of triclosan on citrullination and carbamylation, two important protein posttranslational modifications associated with inflammatory conditions such as periodontitis and rheumatoid arthritis. MATERIALS AND METHODS: A range of triclosan concentrations were incubated in the presence of appropriate substrates used for the generation of either citrullinated or carbamylated proteins. The effect of triclosan on protein citrullination and carbamylation in macrophages was also assessed. RESULTS: Citrullination and carbamylation were both significantly decreased by triclosan at concentrations six times lower than the 0.3% triclosan approved by the FDA to use in mouthwash and toothpaste. When macrophages were exposed to triclosan, carbamylation was significantly deceased (p = 0.01), and while citrullination also decreased, this reduction was not statistically significant (p = 0.06). CONCLUSION: Triclosan reduced the generation of protein citrullination and carbamylation in vitro. CLINICAL RELEVANCE: Triclosan may be useful as an adjunct therapy in the management of inflammatory periodontal diseases and help to reduce posttranslational protein modification citrullination and carbamylation) in these tissues.

Gingival tissue, an extrasynovial source of malondialdehyde-acetaldehyde adducts, citrullinated and carbamylated proteins.

BACKGROUND AND OBJECTIVE: Postranslational modification of proteins can lead to the production of autoantibodies and loss of immune tolerance. This process has been hypothesised to be a critical factor in the pathogenesis of rheumatoid arthritis. The objective of this study was to demonstrate that inflamed human gingival tissue provides an extrasynovial source of malondialdehyde-acetaldehyde adducts, citrullinated and carbamylated proteins all of which are considered to be linked to the development of rheumatoid arthritis. Identification of such modified proteins in inflamed gingiva may explain, in part, how inflammation of the periodontal tissues may influence the development of rheumatoid arthritis. MATERIAL AND METHODS: Gingival biopsies of healthy, mild and moderate periodontitis were triple stained with antibodies against malondialdehyde-acetaldehyde adducts, citrullinated and carbamylated proteins. RESULTS: Assessment of healthy gingival tissue revealed negligible staining for carbamylated, malondialdehyde-acetaldehyde (MAA), or citrullinated proteins. Mild periodontitis was positive for all three modifications. Furthermore, there was an increase in staining intensity for carbamylated, citrullinated and MAA-modified proteins in moderate periodontitis. Negative staining results were observed for the isotype controls. CONCLUSION: This study provides evidence for the presence of citrullinated, carbamylated and MAA adduct modified proteins in inflamed periodontal tissues. The potential for these proteins to play a role in autoimmunity in a multi-system inflammatory syndromic disease model now needs to be determined.

Histone deacetylases (HDAC) in physiological and pathological bone remodelling.

Histone deacetylases (HDACs)2 play important roles in the epigenetic regulation of gene expression in cells and are emerging therapeutic targets for treating a wide range of diseases. HDAC inhibitors (HDACi)3 that act on multiple HDAC enzymes have been used clinically to treat a number of solid and hematological malignancies. HDACi are also currently being studied for their efficacy in non-malignant diseases, including pathologic bone loss, but this has necessitated a better understanding of the roles of individual HDAC enzymes, particularly the eleven zinc-containing isozymes. Selective isozyme-specific inhibitors currently being developed against class I HDACs (1, 2, 3 and 8) and class II HDACs (4, 5, 6, 7, 9 and 10) will be valuable tools for elucidating the roles played by individual HDACs in different physiological and pathological settings. Isozyme-specific HDACi promise to have greater efficacy and reduced side effects, as required for treating chronic disease over extended periods of time. This article reviews the current understanding of roles for individual HDAC isozymes and effects of HDACi on bone cells, (osteoblasts, osteoclasts and osteocytes), in relation to bone remodelling in conditions characterised by pathological bone loss, including periodontitis, rheumatoid arthritis and myeloma bone disease.

Periodontal therapy and glycaemic control among individuals with type 2 diabetes: reflections from the PerioCardio study.

OBJECTIVES: Diabetes mellitus and periodontal disease are highly prevalent among Indigenous Australian adults. Untreated periodontitis impacts glycaemic control in people with diabetes. The aim of this study was to report on the effect of periodontal therapy on glycaemic control among people with obesity. METHODS: This subgroup analysis is limited to 62 participants with diabetes from the original 273 Aboriginal Australian adults enrolled into the PerioCardio study. Intervention participants received full-mouth non-surgical periodontal scaling during a single, untimed session while controls were untreated. Endpoints of interest included change in glycated haemoglobin (HbA1c), C-reactive protein (CRP) and periodontal status at 3 months post-intervention. RESULTS: There were more females randomized to the treatment group (n = 17) than control (n = 10) while the control group had a higher overall body mass index (BMI) [mean (SD)] 33.1 (9.7 kg m-2 ) versus 29.9 (6.0 kg m-2 ). A greater proportion of males were followed up at 3 months compared to females, P = 0.05. Periodontal therapy did not significantly reduce HbA1c: ancova difference in means 0.22 mmol mol-1 (95% CI -6.25 to 6.69), CRP: ancova difference in means 0.64 (95% CI -1.08, 2.37) or periodontal status at 3 months. CONCLUSIONS: Non-surgical periodontal therapy did not significantly reduce glycated haemoglobin in participants with type 2 diabetes. Reasons are likely to be multifactorial and may be influenced by persistent periodontal inflammation at the follow-up appointments. Alternatively, the BMI of study participants may impact glycaemic control via alternative mechanisms involving the interplay between inflammation and adiposity meaning HbA1c may not be amenable to periodontal therapy in these individuals.

Omega-3 fatty acids as an adjunct for periodontal therapy-a review.

OBJECTIVES: The aim of this article is to present an overview of omega-3 fatty acids, their anti-inflammatory properties and potential use as an adjunct for periodontal therapy. MATERIALS AND METHODS: A general literature search was conducted to provide an overview of omega-3 fatty acids, their metabolism and anti-inflammatory properties. A more specific literature search of PubMed and EMBASE was conducted to identify articles dealing studies investigating the effects of omega-3 fatty acids in the treatment of periodontitis in animals and humans and included cross-sectional, longitudinal and intervention designs. RESULTS: To date, there is good emerging evidence that dietary supplementation with fish oil may be of some benefit and this is enhanced if combined with aspirin. All clinical intervention studies to date have been on small sample sizes, and this indicates there is need for larger and more robust clinical trials to verify these initial findings. CONCLUSIONS: Dietary supplementation with fish oil could be a cost-effective adjunctive therapy to the management of periodontal disease. CLINICAL RELEVANCE: The host modulatory properties of omega-3 fatty acids warrant further assessment of their use as an adjunct in the management of periodontitis.

Tissue engineered periodontal products.

Attainment of periodontal regeneration is a significant clinical goal in the management of advanced periodontal defects arising from periodontitis. Over the past 30 years numerous techniques and materials have been introduced and evaluated clinically and have included guided tissue regeneration, bone grafting materials, growth and other biological factors and gene therapy. With the exception of gene therapy, all have undergone evaluation in humans. All of the products have shown efficacy in promoting periodontal regeneration in animal models but the results in humans remain variable and equivocal concerning attaining complete biological regeneration of damaged periodontal structures. In the early 2000s, the concept of tissue engineering was proposed as a new paradigm for periodontal regeneration based on molecular and cell biology. At this time, tissue engineering was a new and emerging field. Now, 14 years later we revisit the concept of tissue engineering for the periodontium and assess how far we have come, where we are currently situated and what needs to be done in the future to make this concept a reality. In this review, we cover some of the precursor products, which led to our current position in periodontal tissue engineering. The basic concepts of tissue engineering with special emphasis on periodontal tissue engineering products is discussed including the use of mesenchymal stem cells in bioscaffolds and the emerging field of cell sheet technology. Finally, we look into the future to consider what CAD/CAM technology and nanotechnology will have to offer.

The effects of tumour necrosis factor-α on bone cells involved in periodontal alveolar bone loss; osteoclasts, osteoblasts and osteocytes.

Periodontitis is the most common bone loss pathology in adults and if left untreated is responsible for premature tooth loss. Cytokines, such as tumour necrosis factor-α (TNFα), involved in the chronic inflammatory response within the periodontal gingiva, significantly influence the normal bone remodelling processes. In this review, the effects of TNFα on bone metabolism in periodontitis are evaluated in relation to its direct and indirect actions on bone cells including osteoclasts, osteoblasts and osteocytes. Evidence published to date suggests a potent catabolic role for TNFα through the stimulation of osteoclastic bone resorption as well as the suppression of osteoblastic bone formation and osteocytic survival. However, the extent and timing of TNFα exposure in vitro and in vivo greatly influences its effect on skeletal cells, with contradictory anabolic activity observed with TNFα in a number of studies. None the less, it is evident that managing the chronic inflammatory response in addition to the deregulated bone metabolism is required to improve periodontal and inflammatory bone loss treatments‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬.

Class I and II histone deacetylase expression in human chronic periodontitis gingival tissue.

BACKGROUND AND OBJECTIVE: Histone deacetylase inhibitors (HDACi) are being considered to treat chronic inflammatory diseases at low doses. Currently HDACi that are more specific are being developed to target particular HDACs; therefore, this study aimed to determine levels and distribution of class I and II HDAC in human gingival samples obtained from patients with chronic periodontitis. MATERIAL AND METHODS: Gingival biopsies were obtained from patients with and without (mild inflammation, no bone loss) periodontitis. Total RNA was isolated for real-time quantitative polymerase chain reaction to determine expression of HDACs 1-10. Immunohistochemistry was used to determine protein distribution of HDACs 1, 5, 8 and 9. Factor VIII, CD3 and tartrate resistant acid phosphatase (TRAP) were detected in serial sections to identify blood vessels, lymphocytes, pre-osteoclasts and osteoclasts cells respectively. Tumour necrosis factor α (TNF-α) expression was also assessed. RESULTS: mRNA for HDAC 1, 5, 8 and 9 were significantly upregulated in chronic periodontitis gingival tissues compared to non-periodontitis samples (p < 0.05). Significantly higher HDAC 1 protein expression was observed in chronic periodontitis samples (p < 0.05), and was associated with CD3, TRAP and TNF-α-positive cells. HDAC 1, 5, 8 and 9 were expressed strongly by the factor VIII-positive microvasculature in the chronic periodontitis gingival tissues. CONCLUSIONS: HDAC 1, 5, 8 and 9 expression was higher in gingival tissues from patients with chronic periodontitis compared to non-periodontitis samples. Results suggest that these HDACs could therefore be targeted with specific acting HDACi.

Cementum and Periodontal Ligament Regeneration.

The unique anatomy and composition of the periodontium make periodontal tissue healing and regeneration a complex process. Periodontal regeneration aims to recapitulate the crucial stages of wound healing associated with periodontal development in order to restore lost tissues to their original form and function and for regeneration to occur, healing events must progress in an ordered and programmed sequence both temporally and spatially, replicating key developmental events. A number of procedures have been employed to promote true and predictable regeneration of the periodontium. Principally, the approaches are based on the use of graft materials to compensate for the bone loss incurred as a result of periodontal disease, use of barrier membranes for guided tissue regeneration and use of bioactive molecules. More recently, the concept of tissue engineering has been integrated into research and applications of regenerative dentistry, including periodontics, to aim to manage damaged and lost oral tissues, through reconstruction and regeneration of the periodontium and alleviate the shortcomings of more conventional therapeutic options. The essential components for generating effective cellular based therapeutic strategies include a population of multi-potential progenitor cells, presence of signalling molecules/inductive morphogenic signals and a conductive extracellular matrix scaffold or appropriate delivery system. Mesenchymal stem cells are considered suitable candidates for cell-based tissue engineering strategies owing to their extensive expansion rate and potential to differentiate into cells of multiple organs and systems. Mesenchymal stem cells derived from multiple tissue sources have been investigated in pre-clinical animal studies and clinical settings for the treatment and regeneration of the periodontium.

The association between rheumatoid arthritis and periodontitis.

The relationship between rheumatoid arthritis and poor oral health has been recognised for many decades. The association between periodontal infection and the risk of developing RA has been the subject of epidemiological, clinical and basic science research in recent times. Converging and reproducible evidence now makes a clear case for the role of specific periodontal infective pathogens in initiating, amplifying and perpetuating rheumatoid arthritis. The unique enzymatic properties of the periodontal pathogen Porphyromonas gingivalis and its contribution to the burden of citrullinated peptides is now well established. The impact of localized infection such as periodontitis in shaping specific anti-citrullinated peptide immune responses highlights a key area for treatment, prevention and risk assessment in rheumatoid arthritis.

Induced Pluripotent Stem Cells: A New Frontier for Stem Cells in Dentistry.

Induced pluripotent stem cells (iPSCs) are the newest member of a growing list of stem cell populations that hold great potential for use in cell-based treatment approaches in the dental field. This review summarizes the dental tissues that have successfully been utilized to generate iPSC lines, as well as the potential uses of iPSCs for tissue regeneration in different dental applications. While iPSCs display great promise in a number of dental applications, there are safety concerns with these cells that need to be addressed before they can be used in clinical settings. This review outlines some of the apprehensions to the use of iPSCs clinically, and it details approaches that are being employed to ensure the safety and efficacy of these cells. One of the major approaches being investigated is the differentiation of iPSCs prior to use in patients. iPSCs have successfully been differentiated into a wide range of cells and tissue types. This review focuses on 2 differentiation approaches-the differentiation of iPSCs into mesenchymal stem cells and the differentiation of iPSCs into osteoprogenitor cells. Both these resulting populations of cells are particularly relevant to the dental field.

Is there a link between carbamylation and citrullination in periodontal disease and rheumatoid arthritis?

The remarkable similarity in inflammatory response and pathology of periodontal disease and rheumatoid arthritis has been recognized for several decades. However, how these two disease may be interrelated has been less clear. During the pathogenesis of rheumatoid arthritis there is a preclinical immunological phase which precedes the clinical manifestation of rheumatoid arthritis. During this phase serum autoantibodies appear many years before the clinical signs and symptoms of rheumatoid arthritis become apparent. To date, the two best studied autoantibodies have been rheumatoid factor and anti-citrullinated protein antibodies (ACPA). Of these the production of ACPA has been considered very important due to their high predictive value in future manifestation of rheumatoid arthritis. Citrullination is a common post-translational modification of proteins based on the enzymatic conversion of arginine into citrulline. Extra-articular citrullination and production of ACPA, as a priming immunological experience, is well documented in many tissues including the inflamed gingival tissues associated with periodontal disease. More recently, carbamylation of proteins has also been implicated in the pathogenesis of rheumatoid arthritis in a manner similar to citrullination. Carbamylation is a post translational modification of proteins by an enzyme-independent modification of lysine residues against which autoantibodies are subsequently induced. In this article we hypothesise that, like citrullination, carbamylation of proteins and associated antibody production during the gingival inflammation associated with gingivitis and periodontitis may play a role in the pathogenesis of rheumatoid arthritis.

Group C. Initiator paper. Periodontal regeneration--fact or fiction?

Numerous techniques have been tried and tested to regenerate tissues lost to periodontal disease. While there has been some success to date, more work is required to move this to a reliable and clinically predictable procedure. Much of the future success for such treatments will rely largely on our understanding of the biology of both developmental and regenerative processes. Nonetheless, despite the noble goal of periodontal regeneration, the relevance of re-creation of a connective tissue attachment has been questioned. Since formation of a long junctional epithelial attachment to the tooth following a variety of periodontal treatment procedures has been shown to be no more susceptible to further breakdown than a non-diseased site, the question arises as to what purpose do we seek the ultimate outcome of periodontal regeneration? The answer lies in the "fact and fiction" of periodontal regeneration. There is no doubt that the regenerative procedures that have been developed can be shown to be biologically successful at the histological level. Furthermore, the results of periodontal regeneration (particularly guided tissue regeneration) have been stable over the long term (at least up to 10 years). However, the techniques currently under use which show the greatest promise (guided tissue regeneration and growth factors) are still clinically unpredictable because of their highly technique-sensitive nature. In addition, whether the slight clinical improvements offered by these procedures over routine open flap debridement procedures are of cost or patient benefit with regards to improved periodontal health and retention of teeth remains to be established. The next phase in regenerative technologies will undoubtedly involve a deeper understanding of the molecular signaling (both intra- and extra-cellular) and cellular differentiation processes involved in the regenerative processes. So in answer to the question of whether periodontal regeneration is fact or fiction, the answer clearly is that it is both. However, with more work it will become established fact with little fiction and the desired clinical endpoint of predictable regeneration of the periodontal tissues damaged by inflammation to their original form and function will be achieved.

Azithromycin suppresses P. gingivalis LPS-induced pro-inflammatory cytokine and chemokine production by human gingival fibroblasts in vitro.

OBJECTIVE: Azithromycin is a macrolide antibiotic that appears to have both antibacterial and anti-inflammatory properties. This study aimed to investigate the effect of azithromycin on the production of pro-inflammatory cytokines and chemokines by human gingival fibroblasts (HGF) in vitro. MATERIALS AND METHODS: The effects of azithromycin (0.1 to 10 µg/mL) on the production of interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemoattractant protein 1 (MCP-1), and growth-regulated oncogene (GRO) by human gingival fibroblasts cultured in the presence or absence of Porphyromonas gingivalis lipopolysaccharide (LPS) was studied. Cytokine and chemokine protein levels in the culture supernatant were assessed using a Luminex® multiplex immunoassay. RESULTS: P. gingivalis LPS induced cytokine/chemokine (IL-6, IL-8, MCP-1, and GRO) protein production in HGFs, and this effect was suppressed by azithromycin at all concentrations tested. CONCLUSIONS: This study demonstrates that azithromycin suppresses P. gingivalis LPS-induced cytokine/chemokine protein production in HGF, which may explain some of the clinical benefits observed with the adjunctive use of azithromycin in the treatment of periodontitis. CLINICAL RELEVANCE: The current study examines the anti-inflammatory properties of azithromycin which may make it useful as an adjunct treatment to periodontitis. Specifically, we used azithromycin to modulate the production of pro-inflammatory cytokines by gingival fibroblasts known to be important in periodontal inflammation.

Surface scratch assessment of titanium implant abutments and cementum following instrumentation with metal curettes.

OBJECTIVES: This study was conducted to compare the surface scratch resistance of titanium implant abutments and cementum to evaluate the impact of scaling with metal curettes on both surfaces. MATERIALS AND METHODS: A standard in vitro force of 14 N was used to assess the scratch width and depth created by curettes on extracted human tooth roots and titanium implant abutments. Scratch width and depth were analysed using a stereomicroscope and non-contact surface profilometry. RESULTS: The mean force applied during test scaling procedures by experienced volunteer operators was 14 N. Mechanical scaling using this force in vitro of cementum produced a mean scratch width of 59.4 ± 1.9 µm, N = 20, and scratch depth of 0.86 ± 0.03 µm, N = 20, compared to the titanium abutments' mean scratch width of 30.8 ± 1.9 µm, N = 6, and scratch depth of 0.34 ± 0.02 µm, N = 6. These differences were statistically significant (P < 0.05). CONCLUSIONS: Although there a number of factors in the clinical situation which are not easily reproducible in vitro, this proof-of-principle in vitro study is the first to confirm quantitatively that titanium abutments had a significantly greater scratch resistance than cementum when metal curettes were used on these surfaces. This information should be considered, especially if there is a preferred choice of metal instruments for effective dental prophylactic procedures for the maintenance of titanium dental implants. CLINICAL RELEVANCE: Clinical dogma suggests that titanium implant abutment surfaces should not be instrumented with metal instruments due to scratching of the surface. However, since cementum is softer than titanium, the logic of this tenet seems flawed. This study demonstrated for the first time that titanium abutments undergo less scratch damage during scaling with metal curettes than does cementum. Metal curettes may be used on titanium abutments with as much confidence as for root planing on natural teeth.

Establishing and managing a periodontal biobank for research: the sharing of experience.

Periodontal bio-repositories, which allow banking of clinically validated human data and biological samples, provide an opportunity to derive biomarkers for periodontal diagnosis, prognosis and therapeutic activities which are expected to improve patient management. This article presents the establishing of the Malaysian Periodontal Database and Biobank System (MPDBS) which was initiated in 2011 with the aim to facilitate periodontal research. Partnerships were established with collaborating centres. Policies on specimen access, authorship and acknowledgement policies were agreed upon by all participating centres before the initiation of the periodontal biobank. Ethical approval for the collection of samples and data were obtained from institutional ethics review boards. A broad-based approach for informed consent was used, which covered areas related to quality of life impacts, genetics and molecular aspects of periodontal disease. Sample collection and processing was performed using a standardized protocol. Biobanking resources such as equipment and freezers were shared with the Malaysian Oral Cancer Database and Tissue Bank System (MOCDTBS). In the development of the MPDBS, challenges that were previously faced by the MOCDTBS were considered. Future challenges in terms of ethical and legal issues will be faced when international collaborations necessitate the transportation of specimens across borders.

Periodontal ligament-derived cells for periodontal regeneration in animal models: a systematic review.

BACKGROUND AND OBJECTIVE: Implantation of periodontal ligament stem cells is emerging as a potential periodontal regenerative procedure. This systematic review considers the evidence from animal models investigating the use of periodontal ligament stem cells for successful periodontal regeneration. MATERIAL AND METHODS: PubMed, Embase, MEDLINE and Google Scholar were searched to December 2013 for quantitative studies examining the outcome of implanting periodontal ligament stem cells into experimental periodontal defects in animals. Inclusion criteria were: implantation of periodontal ligament stem cells into surgically created periodontal defects for periodontal regeneration; animal models only; source of cells either human or animal; and published in English. We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. RESULTS: From the literature search, 43 studies met the inclusion criteria. A wide variety of surgical defects were created in four species of animal (dog, rat, pig and sheep). Owing to wide variability in defect type, cell source and cell scaffold, no meta-analysis was possible. Outcome measures included new bone, new cementum and new connective tissue formation. In 70.5% of the results, statistically significant improvements of these measures was recorded. CONCLUSION: These results are notable in that they indicate that irrespective of the defect type and animal model used, periodontal ligament stem cell implantation can be expected to result in a beneficial outcome for periodontal regeneration. It is recommended that there is sufficient evidence from preclinical animal studies to warrant moving to human studies to examine the efficacy, safety, feasibility (autologous vs. allogeneic transplantation) and delivery of periodontal ligament stem cells for periodontal regeneration.