Expression of bone matrix mRNAs by primary and cloned cultures of the regenerative phenotype of human periodontal fibroblasts

The successful regeneration of periodontal tissues is dependent, in part, on the ability of cells to reconstitute the mineralized tissues of cementum and bone. The aim of the present study was to characterize regeneration-associated cells in terms of their ability to express mineralized tissue macromolecules. Following guided tissue regeneration, cell cultures were established from regenerating tissue, periodontal ligament, and gingiva. Additionally, these cells were transfected, and single-cell-derived clones were established. Following treatment with platelet-derived growth factor-BB and insulin-derived growth factor-1, the presence of mRNA for alkaline phosphatase, osteocalcin, bone sialoprotein, osteopontin, and bone morphogenetic proteins-2 and -4 was assessed. The three cell types expressed similar mRNA levels for alkaline phosphatase, bone morphogenetic protein-2, and bone morphogenetic protein-4, whereas the expression of osteopontin, osteocalcin, and bone sialoprotein was greater in the periodontal ligament and regenerating tissue fibroblasts compared with the gingival fibroblasts. The two growth factors did not affect the expression of any of the genes. This study has identified markers that correlate with the known ability of periodontal ligament and regenerating tissue-derived fibroblasts to facilitate regeneration of the mineralized tissues of the periodontium

Salivary small extracellular vesicles associated miRNAs in periodontal status-a pilot study

This pilot study aims to investigate whether salivary small extracellular vesicle (sEV)-associated microRNAs could act as potential biomarkers for periodontal disease status. Twenty-nine participants (10 who were healthy, nine with gingivitis, 10 with stage III/IV periodontitis) were recruited and unstimulated whole saliva samples were collected. Salivary sEVs were isolated using the size-exclusion chromatography (SEC) method and characterised by morphology, EV-protein and size distribution using transmission electron microscopy (TEM), Western Blot and Nanoparticle Tracking Analysis (NTA), respectively. Ten mature microRNAs (miRNAs) in salivary sEVs and saliva were evaluated using RT-qPCR. The discriminatory power of miRNAs as biomarkers in gingivitis and periodontitis versus healthy controls was evaluated by Receiver Operating Characteristics (ROC) curves. Salivary sEVs were comparable to sEVs morphology, mode, size distribution and particle concentration in healthy, gingivitis and periodontitis patients. Compared to miRNAs in whole saliva, three significantly increased miRNAs (hsa-miR-140-5p, hsa-miR-146a-5p and hsa-miR-628-5p) were only detected in sEVs in periodontitis when compared to that of healthy controls, with a good discriminatory power (area under the curve (AUC) = 0.96) for periodontitis diagnosis. Our study demonstrated that salivary sEVs are a non-invasive source of miRNAs for periodontitis diagnosis. Three miRNAs that are selectively enriched in sEVs, but not whole saliva, could be potential biomarkers for periodontal disease status.Pingping Han, Peter Mark Bartold, Carlos Salomon and Saso Ivanovsk

Periodontal and dental pulp cell-derived small extracellular vesicles: a review of the current status

Extracellular vesicles (EVs) are membrane-bound lipid particles that are secreted by all cell types and function as cell-to-cell communicators through their cargos of protein, nucleic acid, lipids, and metabolites, which are derived from their parent cells. There is limited information on the isolation and the emerging therapeutic role of periodontal and dental pulp cell-derived small EVs (sEVs, <200 nm, or exosome). In this review, we discuss the biogenesis of three EV subtypes (sEVs, microvesicles and apoptotic bodies) and the emerging role of sEVs from periodontal ligament (stem) cells, gingival fibroblasts (or gingival mesenchymal stem cells) and dental pulp cells, and their therapeutic potential in vitro and in vivo. A review of the relevant methodology found that precipitation-based kits and ultracentrifugation are the two most common methods to isolate periodontal (dental pulp) cell sEVs. Periodontal (and pulp) cell sEVs range in size, from 40 nm to 2 μm, due to a lack of standardized isolation protocols. Nevertheless, our review found that these EVs possess anti-inflammatory, osteo/odontogenic, angiogenic and immunomodulatory functions in vitro and in vivo, via reported EV cargos of EV–miRNAs, EV–circRNAs, EV–mRNAs and EV–lncRNAs. This review highlights the considerable therapeutic potential of periodontal and dental pulp cell-derived sEVs in various regenerative applications. View Full-TextShu Hua, Peter Mark Bartold, Karan Gulati, Corey Stephen Moran, Sašo Ivanovski and Pingping Ha

COX-1 and COX-2 in Human Periodontal Disease States

Cyclooxygenase (COX) catalyses the conversion of arachidonic acid into prostanoids and related compounds which have been implicated in periodontal bone loss. Therefore, the aim of this study was to quantify COX-1 and COX-2 expression in gingival tissue derived from healthy/gingivitis and periodontitis sites

Platelet-derived Growth Factor Stimulates Hyaluronate but not Proteoglycan Synthesis by Human Gingival Fibroblasts in vitro

The effect of PDGF-BB on human gingival fibroblasts was monitored in an in vitro system. PDGF was found to be mitogenic for these cells, although it required the presence of low concentrations of fetal calf serum to be active. Proteoglycan and hyaluronate synthesis was analyzed by labeling newly synthesized macromolecules with [35S]-sulfate or [3H]-glucosamine, respectively. Identification of specific glycosaminoglycans was achieved by selective enzymatic or chemical degradations. It was found that cells cultured in the presence of PDGF showed no discernible differences in proteoglycan synthesis relative to the control cultures. There were no alterations in amounts of proteoglycans synthesized, types of sulfated glycosaminoglycans synthesized, or relative hydrodynamic sizes of the proteoglycans. In contrast to the proteoglycans, hyaluronate synthesis was significantly increased in the presence of PDGF. The increase in [3H]-glucosamine incorporation into newly synthesized hyaluronate correlated with an increase in the activity of the enzyme hyaluronate synthetase but could not be accounted for entirely by changes in the specific activity of sugar nucleotide precursors, which did alter slightly under differing culture conditions. It is concluded from these results that PDGF stimulates gingival fibroblasts to proliferate and is associated with a differential effect on proteoglycan and hyaluronate synthesis. These observations may correlate with the observed early events associated with wound healing and repair

Turnover in periodontal connective tissues: dynamic homeostasis of cells, collagen and ground substances.

The connective tissues of the periodontium are composed of two soft tissues and two hard tissues—each of which has unique features. This review considers the constituents of normal, healthy periodontal connective tissues together with an appraisal of the changes in the connective tissue matrices of the periodontium which occur during the development of periodontitis. Recent developments in this field have paved the way for new and exciting vistas in periodontal diagnosis and regeneration which, ultimately, are two important goals in periodontal therapy. Copyrigh

Information generation and processing systems that regulate periodontal structure and function

Article first published online: 11 AUG 2013The periodontium is a very dynamic organ that responds rapidly to mechanical and chemical stimuli. It is very complex in that it is composed of two hard tissues (cementum and bone) and two soft connective tissues (periodontal ligament and gingiva). Together these tissues are defined by the molecules expressed by the resident periodontal cells in each compartment and this determines not only the structure and function of the periodontium but also how it responds to infection and inflammation. The biological activity of these molecules is tightly regulated in time and space to preserve tissue homeostasis, influence inflammatory responses and participate in tissue regeneration. In this issue of Periodontology 2000 we explore new experimental approaches and data sets which help to understand the molecules and cells that regulate tissue form and structure in health, disease and regeneration.P. Mark Bartold and Christopher A. McCulloc

Localization of chondroitin sulphate and dermatan sulphate in human dental pulps--an immunohistochemical study.

The distribution of dermatan sulphate and chondroitin sulphate in human dental pulps has been assessed using monoclonal antibodies and immunoperoxidase localization techniques. The pulpal tissues were reacted with specific antibodies following pretreatment of the sections with chondroitinase ACII or chondroitinase ABC. Both the 4‐ and 6‐sulphated isomers of chondroitin sulphate were detected in the tissues studied. Very little derrmatan sulphate could be detected. These glycosaminoglycans appeared throughout the pulpal connective tissues with a particularly strong localization to the region adjacent to the odontoblastic and predentine layers. Such distribution strongly implicates chondroitin sulphate in the mineralization process of human dentine

Periodontitis: a host-mediated disruption of microbial homeostasis. Unlearning learned concepts

Article first published online: 11 APR 2013New concepts evolve when existing ones fail to address known factors adequately or are invalidated by new evidence. For decades periodontitis has been considered to be caused by specific bacteria or groups of bacteria and, accordingly, treatment protocols have largely been based on anti-infective therapies. However, close inspection of current data leads one to question whether these bacteria are the cause or the result of periodontitis. Good evidence is emerging to suggest that it is indeed the host response to oral bacteria that leads to the tissue changes noted in gingivitis. These changes lead to an altered subgingival environment that favors the emergence of 'periodontal pathogens' and the subsequent development of periodontitis if the genetic and external environmental conditions are favorable for disease development. Thus, it seems that it is indeed the initial early host-inflammatory and immune responses occurring during the development of gingivitis, and not specific bacteria or their so-called virulence factors, which determine whether periodontitis develops and progresses. In this review we consider these concepts and their potential to change the way in which we view and manage the inflammatory periodontal diseases.P. Mark Bartold and Thomas E. Van Dyk

An appraisal of the role of specific bacteria in the initial pathogenesis of periodontitis

BACKGROUND:Historically, inflammatory periodontal diseases (gingivitis and periodontitis) have been recognized as being primarily of bacterial origin. Bacteria are necessary for disease development, but the presence of specific bacteria does not guarantee progression to periodontitis. Periodontitis is a multifactorial disease; specific bacteria are associated with disease, but may not be the target of treatment. Gingivitis and periodontitis are inflammatory conditions associated with bacterial overgrowth. AIM:To analyse evidence for established thought that specific bacteria directly participate in the pathogenesis of periodontitis and question the long-held tenet that penetration of the periodontal connective tissues by bacteria and their products is a significant phase in the initial development of periodontitis. METHODS:The literature was searched for studies on initiation of gingivitis and periodontitis by specific pathogens. The search results were insufficient for a systematic review and have been summarized in a commentary instead. RESULTS:There is very little evidence in the literature to support the commonly held concept that specific bacteria initiate periodontitis. CONCLUSION:We present evidence for a paradigm supporting the central role of inflammation, rather than specific microbiota, in the early pathogenesis of periodontitis, and discuss whether controlling the inflammation can influence the character and composition of the periodontal infection.Peter Mark Bartold, Thomas E. Van Dyk