Role of Primary Cilia in Bone and Cartilage.

The primary cilium is a nonmotile microtubule-based organelle in most vertebrate cell types. The primary cilium plays a critical role in tissue development and homeostasis by sensing and transducing various signaling pathways. Ciliary proteins such as intraflagellar transport (IFT) proteins as well as ciliary motor proteins, kinesin and dynein, comprise a bidirectional intraflagellar transport system needed for cilia formation and function. Mutations in ciliary proteins that lead to loss or dysfunction of primary cilia cause ciliopathies such as Jeune syndrome and Ellis-van Creveld syndrome and cause abnormalities in tooth development. These diseases exhibit severe skeletal and craniofacial dysplasia, highlighting the significance of primary cilia in skeletal development. Cilia are necessary for the propagation of hedgehog, transforming growth factor ß, platelet-derived growth factor, and fibroblast growth factor signaling during osteogenesis and chondrogenesis. Ablation of ciliary proteins such as IFT80 or IFT20 blocks cilia formation, which inhibits osteoblast differentiation, osteoblast polarity, and alignment and reduces bone formation. Similarly, cilia facilitate chondrocyte differentiation and production of a cartilage matrix. Cilia also play a key role in mechanosensing and are needed for increased bone formation in response to mechanical forces.

Cigarette Smoke Exposure Inhibits Osteoclast Apoptosis via the mtROS Pathway.

It is widely known that smoking is a risk factor for bone loss and plays a key role in osteopenia. Despite this well-known association, the mechanisms by which smoking affects bone have not been definitively established. Since smoking increases bone loss and potentially affects bone resorption in response to mechanical force, we investigated the impact of cigarette smoke on osteoclast numbers and underlying mechanisms in a mouse model of orthodontic tooth movement (OTM). The experimental group was exposed to once-daily cigarette smoke while the control group was not, and tooth movement distance and osteoclast numbers were assessed. In addition, the effect of cigarette smoke extract (CSE) on osteoclast precursor proliferation and osteoclast apoptosis was assessed in vitro. We found that cigarette smoke exposure enhanced bone remodeling stimulated by mechanical force and increased osteoclast numbers in vivo. Also, CSE increased the number of osteoclasts by inhibiting osteoclast apoptosis via the mitochondrial reactive oxygen species/cytochrome C/caspase 3 pathway in vitro. Moreover, exposure of mice to cigarette smoke affected bone marrow cells, leading to increased formation of osteoclasts in vitro. This study identifies a previously unknown mechanism of how smoking has a detrimental impact on bone.

The Oral Microbiota Is Modified by Systemic Diseases.

Periodontal diseases are initiated by bacteria that accumulate in a biofilm on the tooth surface and affect the adjacent periodontal tissue. Systemic diseases such as diabetes, rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE) increase susceptibility to destructive periodontal diseases. In human studies and in animal models, these diseases have been shown to enhance inflammation in the periodontium and increase the risk or severity of periodontitis. All 3 systemic diseases are linked to a decrease in bacterial taxa associated with health and an increase in taxa associated with disease. Although there is controversy regarding the specific oral bacterial changes associated with each disease, it has been reported that diabetes increases the levels of Capnocytophaga, Porphyromonas, and Pseudomonas, while Prevotella and Selenomonas are increased in RA and Selenomonas, Leptotrichia, and Prevotella in SLE. In an animal model, diabetes increased the pathogenicity of the oral microbiome, as shown by increased inflammation, osteoclastogenesis, and periodontal bone loss when transferred to normal germ-free hosts. Moreover, in diabetic animals, the increased pathogenicity could be substantially reversed by inhibition of IL-17, indicating that host inflammation altered the microbial pathogenicity. Increased IL-17 has also been shown in SLE, RA, and leukocyte adhesion deficiency and may contribute to oral microbial changes in these diseases. Successful RA treatment with anti-inflammatory drugs partially reverses the oral microbial dysbiosis. Together, these data demonstrate that systemic diseases characterized by enhanced inflammation disturb the oral microbiota and point to IL-17 as key mediator in this process.

Diabetes Activates Periodontal Ligament Fibroblasts via NF-κB In Vivo.

Diabetes mellitus increases periodontitis and pathogenicity of the oral microbiome. To further understand mechanisms through which diabetes affects periodontitis, we examined its impact on periodontal ligament fibroblasts in vivo and in vitro. Periodontitis was induced by inoculation of Porphyromonas gingivalis and Fusobacterium nucleatum in normoglycemic and diabetic mice. Diabetes, induced by multiple low-dose injections of streptozotocin increased osteoclast numbers and recruitment of neutrophils to the periodontal ligament, which could be accounted for by increased CXC motif chemokine 2 (CXCL2) and receptor activator of nuclear factor kappa B ligand (RANKL) expression by these cells. Diabetes also stimulated a significant increase in nuclear factor kappa B (NF-κB) expression and activation in periodontal ligament (PDL) fibroblasts. Surprisingly, we found that PDL fibroblasts express a 2.3-kb regulatory unit of Col1α1 (collagen type 1, alpha 1) promoter typical of osteoblasts. Diabetes-enhanced CXCL2 and RANKL expression in PDL fibroblasts was rescued in transgenic mice with lineage-specific NF-κB inhibition controlled by this regulatory element. In vitro, high glucose increased NF-κB transcriptional activity, NF-κB nuclear localization, and RANKL expression in PDL fibroblasts, which was reduced by NF-κB inhibition. Thus, diabetes induces changes in PDL fibroblast gene expression that can enhance neutrophil recruitment and bone resorption, which may be explained by high glucose-induced NF-κB activation. Furthermore, PDL fibroblasts express a regulatory element in vivo that is typical of committed osteoblasts.

The function of dendritic cells in modulating the host response.

Dendritic cells (DCs) are antigen-presenting cells that capture, process, and present antigens to lymphocytes to initiate and regulate the adaptive immune response. DCs detect bacteria in skin and mucosa and migrate into regional lymph nodes, where they stimulate antigen-specific T and B lymphocyte activation and proliferation. DCs direct CD4 T cells to differentiate to T-cell subsets such as T helper cells types 1, 2, and 17, and regulatory T cells. The periodontium is chronically exposed to oral bacteria that stimulate an inflammatory response to induce gingivitis or periodontitis. DCs play both protective and destructive roles through activation of the acquired immune response and are also reported to be a source of osteoclast precursors that promote bone resorption. FOXO1, a member of the forkhead box O family of transcription factors, plays a significant role in the activation of DCs. The function of DCs in periodontal inflammation has been investigated in a mouse model by lineage-specific deletion of FOXO1 in these cells. Deletion of FOXO1 reduces DC protective function and enhances susceptibility to periodontitis. The kinase Akt, phosphorylates FOXO1 to inhibit FOXO activity. Hence the Akt-FOXO1 axis may play a key role in regulating DCs to have a significant impact on periodontal disease.

Establishment of oral bacterial communities in germ-free mice and the influence of recipient age.

The acquisition of the oral microbiome is a complex process. We examined how the timing of microbial exposure alters bacterial colonization of the tooth surface. Germ-free mice were conventionalized by exposure to specific pathogen-free (SPF) mice to acquire a commensal microbiome over three distinct 4-week periods, 0-4 weeks of age (Conv0-4w), 4-8 weeks (Conv4-8w), or 8-12 weeks (Conv8-12w). Bacterial DNA was extracted from the tooth surface and analyzed by 16S rDNA sequencing. Total bacteria and inflammatory cytokine expression in gingiva were determined by quantitative real-time polymerase chain reaction. After co-housing with SPF mice, Conv0-4w and Conv4-8w mice had low bacterial diversity, whereas Conv8-12w mice had high bacterial diversity that was similar to that of SPF donor mice, as determined by both operational taxonomic units and the Shannon Index. Cluster analysis with unweighted Unifrac distance also supported these trends. This was surprising as the amount of maturation time, 4 weeks, was equal in all conventionalized mice and tooth eruption was largely completed by 4 weeks. This suggests that host factors that occur after tooth eruption have a significant effect on the microbial tooth colonization.

Effect of Aging on Periodontal Inflammation, Microbial Colonization, and Disease Susceptibility.

Periodontitis is a chronic inflammatory disease induced by a biofilm that forms on the tooth surface. Increased periodontal disease is associated with aging. We investigated the effect of aging on challenge by oral pathogens, examining the host response, colonization, and osteoclast numbers in aged versus young mice. We also compared the results with mice with lineage-specific deletion of the transcription factor FOXO1, which reduces dendritic cell (DC) function. Periodontitis was induced by oral inoculation of Porphyromonas gingivalis and Fusobacterium nucleatum in young (4 to 5 mo) and aged (14 to 15 mo) mice. Aged mice as well as mice with reduced DC function had decreased numbers of DCs in lymph nodes, indicative of a diminished host response. In vitro studies suggest that reduced DC numbers in lymph nodes of aged mice may involve the effect of advanced glycation end products on DC migration. Surprisingly, aged mice but not mice with genetically altered DC function had greater production of antibody to P. gingivalis, greater IL-12 expression, and more plasma cells in lymph nodes following oral inoculation as compared with young mice. The greater adaptive immune response in aged versus young mice was linked to enhanced levels of P. gingivalis and reduced bacterial diversity. Thus, reduced bacterial diversity in aged mice may contribute to increased P. gingivalis colonization following inoculation and increased periodontal disease susceptibility, reflected by higher TNF levels and osteoclast numbers in the periodontium of aged versus young mice.

Clopidogrel Enhances Mesenchymal Stem Cell Proliferation Following Periodontitis.

Bone formation is dependent on the differentiation of osteoblasts from mesenchymal stem cells (MSCs). In addition to serving as progenitors, MSCs reduce inflammation and produce factors that stimulate tissue formation. Upon injury, MSCs migrate to the periodontium, where they contribute to regeneration. We examined the effect of clopidogrel and aspirin on MSCs following induction of periodontitis in rats by placement of ligatures. We showed that after the removal of ligatures, which induces resolution of periodontal inflammation, clopidogrel had a significant effect on reducing the inflammatory infiltrate. It also increased the number of osteoblasts and MSCs. Mechanistically, the latter was linked to increased proliferation of MSCs in vivo and in vitro. When given prior to inducing periodontitis, clopidogrel had little effect on MSC or osteoblasts numbers. Applying aspirin before or after induction of periodontitis did not have a significant effect on the parameters measured. These results suggest that clopidogrel may have a positive effect on MSCs in conditions where a reparative process has been initiated.

Gene expression dynamics during diabetic periodontitis.

Diabetes impairs the resolution of periodontal inflammation. We explored pathways altered by inflammation in the diabetic periodontium by using ligatures to induce periodontitis in type-2 diabetic Goto-Kakizaki rats. Ligatures were removed after 7 days, and rats were then treated with TNF inhibitor (pegsunercept) or vehicle alone and euthanized 4 days later. RNA was extracted from periodontal tissue, examined by mRNA profiling, and further analyzed by functional criteria. We found that 1,754 genes were significantly up-regulated and 1,243 were down-regulated by pegsunercept (p < 0.05). Functional analysis revealed up-regulation of neuron-associated and retina-associated gene clusters as well as those related to cell activity and signaling. Others were down-regulated by TNF inhibition and included genes associated with host defense, apoptosis, cell signaling and activity, and coagulation/hemostasis/complement. For selected genes, findings with microarray and rt-PCR agreed. PPAR-α was investigated further by immunohistochemistry due to its anti-inflammatory function and was found to be up-regulated in the gingiva during the resolution of periodontal inflammation and suppressed by diabetes. The results indicate that diabetes-enhanced inflammation both up- and down-regulates genes involved in cellular activity and cell signaling, while it predominantly up-regulates genes involved in the host response, apoptosis, and coagulation/homeostasis/complement and down-regulates mRNA levels of neuron, retina, and energy/metabolism-associated genes.

Interaction of oral bacteria with gingival epithelial cell multilayers.

Primary gingival epithelial cells were cultured in multilayers as a model for the study of interactions with oral bacteria associated with health and periodontal disease. Multilayers maintained at an air-liquid interface in low-calcium medium displayed differentiation and cytokeratin properties characteristic of junctional epithelium. Multilayers were infected with fluorescently labeled Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum or Streptococcus gordonii, and bacterial association was determined by confocal microscopy and quantitative image analysis. Porphyromonas gingivalis invaded intracellularly and spread from cell to cell; A. actinomycetemcomitans and F. nucleatum remained extracellular and showed intercellular movement through the multilayer; whereas S. gordonii remained extracellular and predominantly associated with the superficial cell layer. None of the bacterial species disrupted barrier function as measured by transepithelial electrical resistance. P. gingivalis did not elicit secretion of proinflammatory cytokines. However, A. actinomycetemcomitans and S. gordonii induced interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), IL-6 and IL-8 secretion; and F. nucleatum stimulated production of IL-1ß and TNF-α. Aggregatibacter actinomycetemcomitans, F. nucleatum and S. gordonii, but not P. gingivalis, increased levels of apoptosis after 24 h infection. The results indicate that the organisms with pathogenic potential were able to traverse the epithelium, whereas the commensal bacteria did not. In addition, distinct host responses characterized the interaction between the junctional epithelium and oral bacteria.

Inflammation and uncoupling as mechanisms of periodontal bone loss.

Periodontal disease is characterized by both inflammation and bone loss. Advances in research in both these areas have led to a new appreciation of not only each field but also the intimate relationship between inflammation and bone loss. This relationship has resulted in a new field of science called osteoimmunology and provides a context for better understanding the pathogenesis of periodontal disease. In this review, we discuss several aspects of the immuno-inflammatory host response that ultimately results in loss of alveolar bone. A proposal is made that periodontal inflammation not only stimulates osteoclastogenesis but also interferes with the uncoupling of bone formation and bone resorption, consistent with a pathologic process. Furthermore, arguments based on experimental animal models suggest a critical role of the spatial and temporal aspects of inflammation in the periodontium. A review of these findings leads to a new paradigm to help explain more fully the impact of inflammation on alveolar bone in periodontal disease so that it includes the effects of inflammation on uncoupling of bone formation from resorption.

Altered fibroblast proliferation and apoptosis in diabetic gingival wounds.

Although it is known that diabetes impairs oral wound healing, relatively little is known about the cellular parameters affected, particularly in connective tissue. This study investigated the hypothesis that diabetes impairs connective tissue formation in healing gingiva, and that impaired healing is associated with factors that decrease fibroblast numbers. Full-thickness wounds were created in the palatal gingiva of type 1 and type 2 diabetic and normoglycemic mice. Five days after wounding, diabetic mice had less epithelial wound coverage, less new connective tissue formation, and reduced fibroblast density (p < 0.05). This occurred with increased numbers of caspase-3- and TUNEL-positive fibroblasts, decreased fibroblast proliferation, increased nuclear translocation of the pro-apoptotic transcription factor FOXO1, and increased numbers of polymorphonuclear leukocytes, all of which were significant (p < 0.05). The results suggest that diabetes may decrease fibroblast numbers through increased apoptosis and reduced proliferation, both of which may be mediated through increased activation of FOXO1.

Impaired wound healing in mouse models of diabetes is mediated by TNF-alpha dysregulation and associated with enhanced activation of forkhead box O1 (FOXO1).

AIMS/HYPOTHESIS: The role of TNF-alpha in impaired wound healing in diabetes was examined by focusing on fibroblasts. METHODS: Small excisional wounds were created in the db/db mice model of type 2 diabetes and normoglycaemic littermates, and in a streptozotocin-induced type 1 diabetes mouse model and control mice. Fibroblast apoptosis was measured by the TUNEL assay, proliferation by detection of proliferating cell nuclear antigen, and forkhead box O1 (FOXO1) activity by DNA binding and nuclear translocation. TNF-alpha was specifically inhibited by pegsunercept. RESULTS: Diabetic wounds had increased TNF-alpha, fibroblast apoptosis, caspase-3/7 activity and activation of the pro-apoptotic transcription factor FOXO1, and decreased proliferating cell nuclear antigen positive fibroblasts (p < 0.05). TNF-alpha inhibition improved healing in the diabetic mice and increased fibroblast density. This may be explained by a decrease in fibroblast apoptosis and increased proliferation when TNF-alpha was blocked (p < 0.05). Although decreased fibroblast proliferation and enhanced FOXO1 activity were investigated in type 2 diabetes, they may also be implicated in type 1 diabetes. In vitro, TNF-alpha enhanced mRNA levels of gene sets related to apoptosis and Akt and p53 but not mitochondrial or cell-cycle pathways. FOXO1 small interfering RNA reduced gene sets that regulate apoptosis, Akt, mitochondrial and cell-cycle pathways. TNF-alpha also increased genes involved in inflammation, cytokine, Toll-like receptor and nuclear factor-kB pathways, which were significantly reduced by FOXO1 knockdown. CONCLUSIONS/INTERPRETATION: These studies indicate that TNF-alpha dysregulation in diabetic wounds impairs healing, which may involve enhanced fibroblast apoptosis and decreased proliferation. In vitro, TNF-alpha induced gene sets through FOXO1 that regulate a number of pathways that could influence inflammation and apoptosis.

BMP2 is essential for post natal osteogenesis but not for recruitment of osteogenic stem cells.

The effects of BMP2 on bone marrow stromal cell differentiation and bone formation after bone marrow ablation were determined using C57 BL/6J (B6) mice. Inhibition of BMP2 expression with lentiviral BMP2 shRNA prevented both mineralized nodule formation in vitro and bone formation in vivo, and blocked the expression of Runx2 and osterix, transcriptional determinants of terminal osteogenic differentiation. No effect was observed on the expression of Sox9, a transcription factor, which is the one of the first transcriptional determinant to be expressed in committed chondroprogenitor and osteoprogenitor cells. In vitro studies showed that exogenously added BMP7 rescued the expression of osterix and enhanced the expression of Sox9, but had no effect on the expression of Runx2, while it only partially recovered the development of mineral deposition in the cultures. On the other hand, the exogenous addition of BMP2 rescued both Runx2 and osterix expression, did not enhance the expression of Sox9, but fully recovered the inhibition of mineral deposition in the cultures. Using antibodies against CD146 and Sox9, immunohistological examination of the cell populations found in the medullary space three days after bone marrow ablation, showed qualitatively equal numbers of cells expressing these skeletal progenitor and stem cell markers in control and BMP2 shRNA treated animals. Fluorescence Activated Cell Sorting (FACS) analysis of the cells found with the marrow cavities at three days after marrow ablation using CD146 antibody showed near equal numbers of immunopositive cells in both control and shRNA treated animals. In summary, the differences observed in vitro for BMP2 and BMP7 on osteogenic gene expression and mineralization suggest that they have differing effects on bone cell differentiation. These results further demonstrate that in vivo BMP2 is a central morphogenetic regulator of post natal osteoprogenitor differentiation, but does not affect recruitment of progenitors to the osteoblastic lineage.

Molecular mechanisms controlling bone formation during fracture healing and distraction osteogenesis.

Fracture healing and distraction osteogenesis have important applications in orthopedic, maxillofacial, and periodontal treatment. In this review, the cellular and molecular mechanisms that regulate fracture repair are contrasted with bone regeneration that occurs during distraction osteogenesis. While both processes have many common features, unique differences are observed in the temporal appearance and expression of specific molecular factors that regulate each. The relative importance of inflammatory cytokines in normal and diabetic healing, the transforming growth factor beta superfamily of bone morphogenetic mediators, and the process of angiogenesis are discussed as they relate to bone repair. A complete summary of biological activities and functions of various bioactive factors may be found at COPE (Cytokines & Cells Online Pathfinder Encyclopedia), http://www.copewithcytokines.de/cope.cgi.

Inhibition of experimental periodontitis by a topical boron-based antimicrobial.

AN0128 is a boron-containing compound with antibacterial and anti-inflammatory properties. To test its potential effectiveness in treating periodontal disease, we induced experimental periodontitis in the rat by placing ligatures and assessed the impact of AN0128 and positive and negative controls by micro-CT and histologic measurements. The formation of an inflammatory infiltrate was measured in hematoxylin-and-eosin-stained sections. Daily application of AN0128 (1%) compared with controls reduced bone loss by 38 to 44% (P < 0.05), while vehicle alone had no effect (P > 0.05). The reduction in bone loss with AN0128 was similar to that achieved with a NSAID, ketorolac, and Total toothpaste containing triclosan. AN0128 also reduced the level of gingival inflammation 42% compared with the ligature only (P < 0.05), whereas vehicle alone had no effect (P > 0.05). The results indicate that AN0128 significantly reduces the formation of an inflammatory infiltrate and reduces bone loss, measured histologically and by micro-CT.

Apoptosis in gingival overgrowth tissues.

Variations in the balance between cell proliferation and apoptosis could contribute to the etiology of gingival overgrowth. The aim of this study was to test the hypothesis that, in fibrotic gingival lesions, fibroblast proliferation is stimulated and apoptosis is decreased. Apoptotic index, caspase 3 expression, the proliferative index, FOXO1 expression, and histological inflammation were measured in situ. Analysis of data showed that apoptosis decreased in all forms of gingival overgrowth examined (p < 0.05), and inflammation caused a small but significant increase compared with non-inflamed tissues (p < 0.05). The greatest decrease of apoptosis occurred in the most fibrotic tissues. Cell proliferation was elevated in all forms of gingival overgrowth tested, independent of inflammation (p < 0.05). To identify potential mechanisms of transcriptional regulation of apoptosis, we assessed FOXO1 and caspase 3 expression levels and found them to correlate well with diminished apoptosis. Analysis of data suggests that increased fibroblast proliferation and a simultaneous decrease in apoptosis contribute to gingival overgrowth.

Diabetes enhances periodontal bone loss through enhanced resorption and diminished bone formation.

Using a ligature-induced model in type-2 Zucker diabetic fatty (ZDF) rat and normoglycemic littermates, we investigated whether diabetes primarily affects periodontitis by enhancing bone loss or by limiting osseous repair. Diabetes increased the intensity and duration of the inflammatory infiltrate (P < 0.05). The formation of osteoclasts and percent eroded bone after 7 days of ligature placement was similar, while four days after removal of ligatures, the type 2 diabetic group had significantly higher osteoclast numbers and activity (P < 0.05). The amount of new bone formation following resorption was 2.4- to 2.9-fold higher in normoglycemic vs. diabetic rats (P < 0.05). Diabetes also increased apoptosis and decreased the number of bone-lining cells, osteoblasts, and periodontal ligament fibroblasts (P < 0.05). Thus, diabetes caused a more persistent inflammatory response, greater loss of attachment and more alveolar bone resorption, and impaired new bone formation. The latter may be affected by increased apoptosis of bone-lining and PDL cells.

Diabetes-enhanced inflammation and apoptosis--impact on periodontal pathology.

Diabetes, particularly type 2 diabetes, is a looming health issue with many ramifications. Because diabetes alters the cellular microenvironment in many different types of tissues, it causes myriad untoward effects, collectively referred to as 'diabetic complications'. Two cellular processes affected by diabetes are inflammation and apoptosis. This review discusses how diabetes-enhanced inflammation and apoptosis may affect the oral environment. In particular, dysregulation of tumor necrosis factor and the formation of advanced glycation products, both of which occur at higher levels in diabetic humans and animal models, potentiate inflammatory responses and induce apoptosis of matrix-producing cells. The enhanced loss of fibroblasts and osteoblasts through apoptosis in diabetics could contribute to limited repair of injured tissue, particularly when combined with other known deficits in diabetic wound-healing. These findings may shed light on diabetes-enhanced risk of periodontal diseases.