development-direct-pulp-capping-model-for-evaluation-pulpal-wound

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

The UCLA School of Dentistry

Genetic polymorphism of cytochrome P450 CYP2C19 influences the efficacy of proton pump inhibitor (PPI) in Helicobacter pylori (H. pylori) eradication therapy. We investigated the difference in the cure rates of H. pylori infection by triple (rabeprazole plus amoxacillin and clarithromycin) therapy in relation to CYP2C19 genotype status.

Efficacy of triple therapy with rabeprazole for Helicobacter pylori infection in relation to CYP2C19 genotype.

The patient population varies in nutritional deficiencies, which may confound the host response to biomaterials. The objective of this study was to evaluate the effect of a common deficiency of vitamin D on implant osseointegration in the rat model.

Vitamin D and bone physiology: demonstration of vitamin D deficiency in an implant osseointegration rat model.

Bisphosphonates (BPs) are chemically stable analogs of pyrophosphate exhibiting strong affinity to bone and have been used for the treatment of diseases characterized by excessive bone resorption. Contrary to the widely accepted BP accumulation model in bone after repeated applications, we report here that an equilibrium-dependent BP-crystalline bone mineral interaction may better explain BP bio-distribution and anti-catabolic bone remodeling and may be relevant to the appearance of osteonecrosis of the jaw (ONJ) in rats. Fluorescent-labeled BP analogs were synthesized and used to evaluate the mode of bone adsorption. After fluorescent-labeled BP adsorbed on crystalline calcium phosphates in vitro, subsequent BP application replaced the previously absorbed BP depending on the dose and the relative binding affinity to hydroxyapatite. The in vivo intravenous zoledronate (ZOL) injection of repeated fractional doses resulted in lower serum CTX and TRAP5b measurements than a single bolus injection in spite of the equivalent cumulative dose. Repeated injections resulted in the distribution of fluorescent-labeled BP on the large area of bone surfaces; whereas the single bolus injection gave rise to the intense BP bio-distribution at selected bone sites such as the alveolar process of jawbones. Necrotic maxillary alveolar bone was predominantly observed in vitamin D deficiency rats treated with bolus ZOL injection. The palatal necrotic bone was characteristically sequestrated by the fistulation of hyperplastic oral epithelium, suggesting the initial development of ONJ-like lesions in rats. Our results suggest that equilibrium-dependent BP-bone interaction may, in part, determine the effectiveness and influence side effects of long-term and repeated applications of BPs.

Equilibrium-dependent bisphosphonate interaction with crystalline bone mineral explains anti-resorptive pharmacokinetics and prevalence of osteonecrosis of the jaw in rats.

Drug-induced osteonecrosis of the jaw (ONJ) is a detrimental intraoral lesion that often occurs after dental-related interventions in patients undergoing treatment with bisphosphonates or denosumab, the neutralizing human anti-receptor activator of NF-κB ligand (RANKL) antibody (Ab). The cause of ONJ by these drugs has been speculated to their direct effects on osteoclasts. However, the extent to which osteoclasts contribute to ONJ pathogenesis remains controversial. Herein, by using a tooth-extraction mouse model with i.v. administration of mouse anti-RANKL Ab or the bisphosphonate zoledronate (ZOL), we show that unresorbed bone due to impaired formation or suppressed functions of osteoclasts, respectively, is associated with ONJ development. After tooth extraction, ONJ-like lesions developed 50% in the anti-RANKL Ab-treated mice and 30% in the ZOL-treated mice. Nonviable and unresorbed bone was found more in anti-RANKL Ab-treated mice compared with mice receiving ZOL. All mice receiving anti-RANKL Ab had an undetectable tartrate-resistant acid phosphatase (TRAP) level in the serum and no TRAP-positive osteoclasts at the extracted sockets, whereas ZOL-treated mice had a decreased TRAP level without altering the numbers of TRAP-positive osteoclasts. Interestingly, the absence of newly formed woven bone in the extracted sockets was evident in ONJ-like lesions from both anti-RANKL Ab- and ZOL-treated mice. Our study suggests that the lack of osteoclasts' bone-resorptive functions by these drugs and suppression of woven bone formation after dental trauma may be associated with ONJ development.

Impaired bone resorption and woven bone formation are associated with development of osteonecrosis of the jaw-like lesions by bisphosphonate and anti-receptor activator of NF-κB ligand antibody in mice.

Osteonecrosis of the jaw (ONJ), an uncommon co-morbidity in patients treated with bisphosphonates (BP), occurs in the segment of jawbone interfacing oral mucosa. This study aimed to investigate a role of oral mucosal barrier γδ T cells in the pathogenesis of ONJ. Female C57Bl/6J (B6) mice received a bolus zoledronate intravenous injection (ZOL, 540 μg/kg), and their maxillary left first molars were extracted 1 week later. ZOL-treated mice (WT ZOL) delayed oral wound healing with patent open wounds 4 weeks after tooth extraction with characteristic oral epithelial hyperplasia. γδ T cells appeared within the tooth extraction site and hyperplastic epithelium in WT ZOL mice. In ZOL-treated γδ T cell null (Tcrd(-/-) ZOL) mice, the tooth extraction open wound progressively closed; however, histological ONJ-like lesions were identified in 75 and 60% of WT ZOL and Tcrd(-/-) ZOL mice, respectively. Although the bone exposure phenotype of ONJ was predominantly observed in WT ZOL mice, Tcrd(-/-) ZOL mice developed the pustule/fistula disease phenotype. We further addressed the role of γδ T cells from human peripheral blood (h-γδ T cells). When co-cultured with ZOL-pretreated human osteoclasts in vitro, h-γδ T cells exhibited rapid expansion and robust IFN-γ secretion. When h-γδ T cells were injected into ZOL-treated immunodeficient (Rag2(-/-) ZOL) mice, the oral epithelial hyperplasia developed. However, Rag2(-/-) ZOL mice did not develop osteonecrosis. The results indicate that γδ T cells are unlikely to influence the core osteonecrosis mechanism; however, they may serve as a critical modifier contributing to the different oral mucosal disease variations of ONJ.

Osteonecrosis of the Jaw Developed in Mice: DISEASE VARIANTS REGULATED BY γδ T CELLS IN ORAL MUCOSAL BARRIER IMMUNITY.

The aim of this study is to establish osteoclasts as key immune effectors capable of activating the function of Natural Killer (NK) cells, and expanding their numbers, and to determine in vivo and in vitro effect of bisphosphonates (BPs) during NK cell interaction with osteoclasts and on systemic and local immune function. The profiles of 27 cytokines, chemokines and growth factors released from osteoclasts were found to be different from dendritic cells and M1 macrophages but resembling to untreated monocytes and M2 macrophages. Nitrogen-containing BPs Zoledronate (ZOL) and Alendronate (ALN), but not non-nitrogen-containing BPs Etidronate (ETI), triggered increased release of pro-inflammatory mediators from osteoclasts while all three BPs decreased pit formation by osteoclasts. ZOL and ALN mediated significant release of IL-6, TNF-` and IL-1β, whereas they inhibited IL-10 secretion by osteoclasts. Treatment of osteoclasts with ZOL inhibited NK cell mediated cytotoxicity whereas it induced significant secretion of cytokines and chemokines. NK cells lysed osteoclasts much more than their precursor cells monocytes, and this correlated with the decreased expression of MHC class I expression on osteoclasts. Intravenous injection of ZOL in mice induced pro-inflammatory microenvironment in bone marrow and demonstrated significant immune activation. By contrast, tooth extraction wound of gingival tissues exhibited profound immune suppressive microenvironment associated with dysregulated wound healing to the effect of ZOL which could potentially be responsible for the pathogenesis of Osteonecrosis of the Jaw (ONJ). Finally, based on the data obtained in this paper we demonstrate that osteoclasts can be used as targets for the expansion of NK cells with superior function for immunotherapy of cancer.

Bisphosphonate-induced differential modulation of immune cell function in gingiva and bone marrow in vivo: role in osteoclast-mediated NK cell activation.

Erratum: Bisphosphonate-induced differential modulation of immune cell function in gingiva and bone marrow in vivo: Role in osteoclast-mediated NK cell activation.

Injury to the barrier tissue initiates a rapid distribution of myeloid immune cells from bone marrow, which guide sound wound healing. Bisphosphonates, a widely used anti-bone resorptive drug with minimal systemic side effects, have been linked to an abnormal wound healing in the oral barrier tissue leading to, in some cases, osteonecrosis of the jaw (ONJ). Here we report that the development of ONJ may involve abnormal phenotypic plasticity of Ly6G+/Gr1+ myeloid cells in the oral barrier tissue undergoing tooth extraction wound healing. A bolus intravenous zoledronate (ZOL) injection to female C57Bl/6 mice followed by maxillary first molar extraction resulted in the development of ONJ-like lesion during the second week of wound healing. The multiplex assay of dissociated oral barrier cells exhibited the secretion of cytokines and chemokines, which was significantly modulated in ZOL mice. Tooth extraction-induced distribution of Ly6G+/Gr1+ cells in the oral barrier tissue increased in ZOL mice at week 2. ONJ-like lesion in ZOL mice contained Ly6G+/Gr1+ cells with abnormal size and morphology as well as different flow cytometric staining intensity. When anti-Ly6G (Gr1) antibody was intraperitoneally injected for 5 days during the second week of tooth extraction, CD11b+GR1(hi) cells in bone marrow and Ly6G+ cells in the oral barrier tissue were depleted, and the development of ONJ-like lesion was significantly attenuated. This study suggests that local modulation of myeloid cell plasticity in the oral barrier tissue may provide the basis for pathogenesis and thus therapeutic as well as preventive strategy of ONJ.

Sil Park

The Shapiro Family Laboratory of Viral Oncology and Aging Research

The UCLA School of Dentistry

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice