Journal of Dental Research

Calcium phosphate cements (CPCs) have excellent biocompatibility and osteoconductivity for dental, craniofacial, and orthopedic applications. This article reviews recent developments in stem cell delivery via CPC for bone regeneration. This includes: (1) biofunctionalization of the CPC scaffold, (2) co-culturing of osteoblasts/endothelial cells and prevascularization of CPC, (3) seeding of CPC with different stem cell species, (4) human umbilical cord mesenchymal stem cell (hUCMSC) and bone marrow MSC (hBMSC) seeding on CPC for bone regeneration, and (5) human embryonic stem cell (hESC) and induced pluripotent stem cell (hiPSC) seeding with CPC for bone regeneration. Cells exhibited good attachment/proliferation in CPC scaffolds. Stem-cell-CPC constructs generated more new bone and blood vessels in vivo than did the CPC control without cells. hUCMSCs, hESC-MSCs, and hiPSC-MSCs in CPC generated new bone and blood vessels similar to those of hBMSCs; hence, they were viable cell sources for bone engineering. CPC with hESC-MSCs and hiPSC-MSCs generated new bone two- to three-fold that of the CPC control. Therefore, this article demonstrates that: (1) CPC scaffolds are suitable for delivering cells; (2) hUCMSCs, hESCs, and hiPSCs are promising alternatives to hBMSCs, which require invasive procedures to harvest with limited cell quantity; and (3) stem-cell-CPC constructs are highly promising for bone regeneration in dental, craniofacial, and orthopedic applications.

The first genome-wide association study of dental caries focused on primary teeth in children aged 3 to 12 yr and nominated several novel genes: ACTN2, EDARADD, EPHA7, LPO, MPPED2, MTR, and ZMPSTE24. Here we interrogated 156 single-nucleotide polymorphisms (SNPs) within these candidate genes for evidence of association with dental caries experience in 13 race- and age-stratified samples from 6 independent studies (n = 3600). Analysis was performed separately for each sample, and results were combined across samples via meta-analysis. MPPED2 was significantly associated with caries via meta-analysis across the 5 childhood samples, with 4 SNPs showing significant associations after gene-wise adjustment for multiple comparisons (p < .0026). These results corroborate the previous genome-wide association study, although the functional role of MPPED2 in caries etiology remains unknown. ACTN2 also showed significant association via meta-analysis across childhood samples (p = .0014). Moreover, in adults, genetic association was observed for ACTN2 SNPs in individual samples (p < .0025), but no single SNP was significant via meta-analysis across all 8 adult samples. Given its compelling biological role in organizing ameloblasts during amelogenesis, this study strengthens the hypothesis that ACTN2 influences caries risk. Results for the other candidate genes neither proved nor precluded their associations with dental caries.

Objective: Nearly all state Medicaid programs reimburse nondental primary care providers (PCPs) for providing preventive oral health services to young children; yet, little is known about how treatment outcomes compare with children visiting dentists. This study compared the association between the provider of preventive services (PCP, dentist, or both) with Medicaid-enrolled children before their third birthday and subsequent dental caries-related treatment (CRT) and CRT payment. Methods: We conducted a retrospective study of young children enrolled in North Carolina Medicaid during 2000 to 2006. The annual number of CRT and CRT payments per child between the ages of 3 and 5 yr were estimated with a zero-inflated negative binomial regression and a hurdle model, respectively. Models were adjusted for relevant child- and county-level characteristics and used propensity score weighting to address observed confounding. Results: We examined 41,453 children with > 1 preventive oral health visit from a PCP, dentist, or both before their third birthday. Unadjusted annual mean CRT and payments were lowest among children who had only PCP visits (CRT = 0.87, payment = $172) and higher among children with only dentist visits (CRT = 1.48, payment = $234) and both PCP and dentist visits (CRT = 1.52, payment = $273). Adjusted results indicated that children who had dentist visits (with or without PCP visits) had significantly more CRT and higher CRT payments per year during the ages of 3 and 4 yr than children who had only PCP visits. However, these differences attenuated each year after age 3 yr. Conclusions: Because of children’s increased opportunity to receive multiple visits in medical offices during well-child visits, preventive oral health services provided by PCPs may lead to a greater reduction in CRT than dentist visits alone. This study supports guidelines and reimbursement policies that allow preventive dental visits based on individual needs.

Vitamin D deficiency and oral diseases (periodontitis, caries, and tooth loss) are highly prevalent in Germany. Previous studies suggested that vitamin D might be a modifiable and protective factor for periodontitis, caries, and tooth loss. However, prospective studies investigating such associations are limited. We explored the association between the concentration of serum 25-hydroxy vitamin D (25OHD) and incidence of tooth loss, progression of clinical attachment loss (CAL) ≥ 3 mm, and progression of restorative and caries status in a population-based longitudinal study. We analyzed data from 1,904 participants from the Study of Health in Pomerania with a five-year follow-up. Generalized estimating equation models were applied to evaluate tooth-specific associations between serum 25OHD and incidence of tooth loss, progression of CAL ≥ 3 mm, and progression of restorative and caries status. Age, sex, education, smoking status, alcohol drinking, waist circumference, dental visit frequency, reasons of dental visit, vitamin D or calcium supplements, and season of blood draw were considered as confounders. Serum 25OHD was inversely associated with incidence of tooth loss. A significant dose-response relationship (p = .0022) was observed across the quintiles of serum 25OHD. After adjusting for multiple confounders, each 10-µg/L increase of serum 25OHD was associated with a 13% decreased risk of tooth loss (risk ratio: 0.87; 95% confidence interval: 0.79, 0.96). The association was attenuated for changes of CAL ≥ 3 mm when adjusting for multiple confounders. No significant association was found between serum 25OHD and caries progression. Vitamin D might be a protective factor for tooth loss. The effect might partially be mediated by its effect on periodontitis.

Objective: Evaluate the validity, reliability, and factorial invariance of the complete Portuguese version of the Oral Health Impact Profile (OHIP) and its short version (OHIP-14). Methods: A total of 1,162 adults enrolled in the Faculty of Dentistry of Araraquara/UNESP participated in the study; 73.1% were women; and the mean age was 40.7 ± 16.3 yr. We conducted a confirmatory factor analysis, where χ²/df, comparative fit index, goodness of fit index, and root mean square error of approximation were used as indices of goodness of fit. The convergent validity was judged from the average variance extracted and the composite reliability, and the internal consistency was estimated by Cronbach standardized alpha. The stability of the models was evaluated by multigroup analysis in independent samples (test and validation) and between users and nonusers of dental prosthesis. Results: We found best-fitting models for the OHIP-14 and among dental prosthesis users. The convergent validity was below adequate values for the factors “functional limitation” and “physical pain” for the complete version and for the factors “functional limitation” and “psychological discomfort” for the OHIP-14. Values of composite reliability and internal consistency were below adequate in the OHIP-14 for the factors “functional limitation” and “psychological discomfort.” We detected strong invariance between test and validation samples of the full version and weak invariance for OHIP-14. The models for users and nonusers of dental prosthesis were not invariant for both versions. Conclusion: The reduced version of the OHIP was parsimonious, reliable, and valid to capture the construct “impact of oral health on quality of life,” which was more pronounced in prosthesis users.

Evidence from biological and human studies strongly supports a role for MMP and TIMP genes as candidate genes for non-syndromic cleft lip with or without cleft palate (NSCL/P). We previously showed the association of promoter polymorphisms in MMP3 (rs3025058 and rs522616) and TIMP2 (rs8179096) with NSCL/P. In this study, we examined the functional significance of these polymorphisms. A specific DNA-protein complex for MMP3 rs522616 A was detected, and this allele by itself showed greater promoter activity than the G allele. However, the effect of rs522616 was ultimately regulated by the rs3025058 allele on the background. For TIMP2 rs8179096, the T allele showed a 2.5-fold increase in promoter activity when compared with allele C, whereas both C and T alleles were found to bind to nuclear factor kappa B. Our results provide new evidence that promoter polymorphisms in MMP3 and TIMP2 are functional and may affect gene transcription with possible effects on craniofacial development leading to NSCL/P.

BET proteins are a group of epigenetic regulators controlling transcription through reading acetylated histone tails and recruiting transcription complexes. They are considered as potential therapeutic targets in many distinct diseases. A novel synthetic bromodomain and extraterminal domain (BET) inhibitor, JQ1, was proved to suppress oncogene transcription and inflammatory responses. The present study was aimed to investigate the effects of JQ1 on inflammatory response and bone destruction in experimental periodontitis. We found that JQ1 significantly suppressed lipopolysaccharide (LPS)-stimulated inflammatory cytokine transcription, including interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α), as well as receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast markers, such as c-Fos, nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), tartrate-resistant acid phosphatase (TRAP) and cathepsin K in vitro. JQ1 also inhibited toll-like receptors 2/4 (TLR2/4) expression and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) phosphorylation and nuclear translocation. Chromatin immunoprecipitation and quantitative polymerase chain reaction (ChIP-qPCR) revealed that JQ1 neutralized BRD4 enrichment at several gene promoter regions, including NF-κB, TNF-α, c-Fos, and NFATc1. In a murine periodontitis model, systemic administration of JQ1 significantly inhibited inflammatory cytokine expression in diseased gingival tissues. Alveolar bone loss was alleviated in JQ1-treated mice because of reduced osteoclasts in periodontal tissues. These unprecedented results suggest the BET inhibitor JQ1 as a prospective new approach for treating periodontitis.

The Proteoglycan 4 (Prg4) product lubricin plays essential roles in boundary lubrication and movement in limb synovial joints, but its roles in temporomandibular joint (TMJ) are unclear. Thus, we characterized the TMJ phenotype in wild-type and Prg4^–/– mouse littermates over age. As early as 2 weeks of age, mutant mice exhibited hyperplasia in the glenoid fossa articular cartilage, articular disc, and synovial membrane. By 1 month of age, there were fewer condylar superficial tenascin-C/Col1-positive cells and more numerous apoptotic condylar apical cells, while chondroprogenitors displayed higher mitotic activity, and Sox9-, Col2-, and ColX-expressing chondrocyte zones were significantly expanded. Mutant subchondral bone contained numerous Catepsin K- expressing osteoclasts at the chondro-osseous junction, increased invasive marrow cavities, and suboptimal subchondral bone. Mutant glenoid fossa, disc, synovial cells, and condyles displayed higher Hyaluronan synthase 2 expression. Mutant discs also lost their characteristic concave shape, exhibited ectopic chondrocyte differentiation, and occasionally adhered to condylar surfaces. A fibrinoid substance of unclear origin often covered the condylar surface. By 6 months of age, mutant condyles displayed osteoarthritic degradation with apical/mid-zone separation. In sum, lubricin exerts multiple essential direct and indirect roles to preserve TMJ structural and cellular integrity over post-natal life.

Porcine dentin sialophosphoprotein (DSPP) is the most abundant non-collagenous protein in dentin. It is processed by proteases into 3 independent proteins: dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP). We fractionated DPP and DSP along with TGF-β activity by ion exchange (IE) chromatography from developing pig molars and measured their alkaline phosphatase (ALP)-stimulating activity in human periodontal (HPDL) cells with or without TGF-β receptor inhibitor. We then purified TGF-β-unbound or -bound DPP and DSP by reverse-phase high-performance liquid chromatography (RP-HPLC) using the ALP-HPDL system. The TGF-β isoform bound to DPP and DSP was identified as being TGF-β1 by both ELISA and LC-MS/MS analysis. We incubated carrier-free human recombinant TGF-β1 (CF-hTGF-β1) with TGF-β-unbound DPP or DSP and characterized the binding on IE-HPLC using the ALP-HPDL system. When only CF-hTGF-β1 was incubated, approximately 3.6% of the ALP-stimulating activity remained. DPP and DSP rescued the loss of TGF-β1 activity. Approximately 19% and 10% of the ALP stimulating activities were retained by the binding of TGF-β to DPP and DSP, respectively. The type I collagen infrequently bound to CF-hTGF-β1. We conclude that both DPP and DSP help retain TGF-β1 activity in porcine dentin.

The precise regulation of odontoblast differentiation and osteoclastogenic cytokine expression in human dental pulp cells (HDPCs) is crucial for the pathology of bacteria-related pulpitis. Although the up-regulation of nucleotide-binding oligomerization domain-containing protein 2 (NOD2) has been reported in inflamed human dental pulps, the role of NOD2 in the differentiation of HDPCs remains unclear. Here, we show the involvement of NOD2 in odontoblast differentiation together with osteoclastogenic cytokine expression in HDPCs. Treatment with muramyl dipeptide (MDP), a known NOD2-agonist, significantly inhibited odontoblast differentiation of HDPCs, as revealed by reduced ALP activity, osteoblast/odontoblast marker expression, and mineralized nodule formation. Importantly, the forced down-regulation of NOD2 by small interfering RNA (siRNA) recovered MDP-down-regulated odontoblast differentiation. MDP-elicited suppression of odontoblast differentiation resulted from the increased expression of MKP-1 protein and the subsequent decline of MAPKs phosphorylation, which is a prerequisite for odontoblast differentiation. Furthermore, we found that MDP treatment elevated the expression of osteoclastogenic cytokines in HDPCs, which was also reversed by NOD2 silencing. Analysis of these data, taken together, suggests that the regulation of NOD2 expression upon MDP challenge might serve as an intrinsic mechanism that underlies the hindered dentin formation and accelerated dentin resorption in bacterial infection-mediated pulpitis.

Abbreviations: HDPCs, human dental pulp cells; NOD2, nucleotide-binding oligomerization domain-containing protein 2; MDP, muramyl dipeptide; siRNA, small interfering RNA; MAPKs, mitogen-activated protein kinases; MKP-1, MAPK phosphatase-1; BMMs, bone-marrow-derived macrophages; and CM, conditioned medium.

Glechoma hederacea (GH), commonly known as ground-ivy or gill-over-the-ground, has been extensively used in folk remedies for relieving symptoms of inflammatory disorders. However, the molecular mechanisms underlying the therapeutic action of GH are poorly understood. Here, we demonstrate that GH constituents inhibit osteoclastogenesis by abrogating receptor activator of nuclear κ-B ligand (RANKL)-induced free cytosolic Ca²⁺ ([Ca²⁺]_i) oscillations. To evaluate the effect of GH on osteoclastogenesis, we assessed the formation of multi-nucleated cells (MNCs), enzymatic activity of tartrate-resistant acidic phosphatase (TRAP), expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), and [Ca²⁺]_i alterations in response to treatment with GH ethanol extract (GHE) in primarily cultured bone marrow–derived macrophages (BMMs). Treatment of RANKL-stimulated or non-stimulated BMMs with GHE markedly suppressed MNC formation, TRAP activity, and NFATc1 expression in a dose-dependent manner. Additionally, GHE treatment induced a large transient elevation in [Ca²⁺]_i while suppressing RANKL-induced [Ca²⁺]_i oscillations, which are essential for NFATc1 activation. GHE-evoked increase in [Ca²⁺]_i was dependent on extracellular Ca²⁺ and was inhibited by 1,4-dihydropyridine (DHP), inhibitor of voltage-gated Ca²⁺ channels (VGCCs), but was independent of store-operated Ca²⁺ channels. Notably, after transient [Ca²⁺] elevation, treatment with GHE desensitized the VGCCs, resulting in an abrogation of RANKL-induced [Ca²⁺]_i oscillations and MNC formation. These findings demonstrate that treatment of BMMs with GHE suppresses RANKL-mediated osteoclastogenesis by activating and then desensitizing DHP-sensitive VGCCs, suggesting potential applications of GH in the treatment of bone disorders, such as periodontitis, osteoporosis, and rheumatoid arthritis.

Aging may negatively affect gingival wound-healing. However, little is known about the mechanisms underlying this phenomenon. The present study examined the cellular responses associated with gingival wound-healing in aging. Primary cultures of human gingival fibroblasts were obtained from healthy young and aged donors for the analysis of cell proliferation, cell invasion, myofibroblastic differentiation, and collagen gel remodeling. Serum from young and old rats was used to stimulate cell migration. Gingival repair was evaluated in Sprague-Dawley rats of different ages. Data were analyzed by the Mann-Whitney and Kruskal-Wallis tests, with a p value of .05. Fibroblasts from aged donors showed a significant decrease in cell proliferation, migration, Rac activation, and collagen remodeling when compared with young fibroblasts. Serum from young rats induced higher cell migration when compared with serum from old rats. After TGF-beta1 stimulation, both young and old fibroblasts demonstrated increased levels of alpha-SMA. However, alpha-SMA was incorporated into actin stress fibers in young but not in old fibroblasts. After 7 days of repair, a significant delay in gingival wound-healing was observed in old rats. The present study suggests that cell migration, myofibroblastic differentiation, collagen gel remodeling, and proliferation are decreased in aged fibroblasts. In addition, altered cell migration in wound-healing may be attributable not only to cellular defects but also to changes in serum factors associated with the senescence process.

Although the reported percentage of bone-implant contact is far lower than 100%, the cause of such low levels of bone formation has rarely been investigated. This study tested the negative biological effect of hydrocarbon deposition onto titanium surfaces, which has been reported to be inevitable. Osteogenic MC3T3-E1 cells were cultured on titanium disks on which the carbon concentration was experimentally regulated to achieve carbon/titanium (C/Ti) ratios of 0.3, 0.7, and 1.0. Initial cellular activities such as cell attachment and cell spreading were concentration-dependently suppressed by the amount of carbon on the titanium surface. The osteoblastic functions of alkaline phosphatase activity and calcium mineralization were also reduced by more than 40% on the C/Ti (1.0) surface. These results indicate that osteoblast activity is influenced by the degree of hydrocarbon contamination on titanium implants and suggest that hydrocarbon decomposition before implant placement may increase the biocompatibility of titanium.

Whole body vibration (WBV) stimulation has a beneficial effect on the recovery of osteoporotic bone. We aimed to investigate the immediate effect of WBV on lipopolysaccharide (LPS)–mediated inflammatory bone loss by varying the exposure timing. Balb/C mice were divided into the following groups: control, LPS (L), and LPS with vibration (LV). The L and LV groups received LPS (5 mg/kg) by 2 intraperitoneal injections on days 0 and 4. The LV group was exposed to WBV (0.4 g, 45 Hz) either during LPS treatment (LV1) or after cessation of LPS injection (LV2) and then continued WBV treatment for 10 min/d for 3 d. Evaluation based on micro–computed tomography was performed 7 d after the first injection, when the L group showed a significant decrease in bone volume (−25.8%) and bone mineral density (−33.5%) compared with the control group. The LV2 group recovered bone volume (35%) and bone mineral density (19.9%) compared with the L group, whereas the LV1 group showed no improvement. This vibratory signal showed a suppressive effect on the LPS-mediated induction of inflammatory cytokines such as IL-1β or TNF-α in human mesenchymal stem cells in vitro. These findings suggest that immediate exposure to WBV after the conclusion of LPS treatment efficiently reduces trabecular bone loss, but WBV might be less effective during the course of treatment with inflammatory factor.