Diet-induced Generalized Periodontitis in Lewis Rats.

Periodontitis is an important public health concern worldwide. Because rodents from the genus Rattus are resistant to spontaneous periodontitis, experimental periodontitis must be initiated by mechanical procedures and interventions. Due to their exacerbated Th1 response and imbalanced Th17 regulatory T-cell responses, Lewis rats are highly susceptible to inducible inflammatory and autoimmune diseases. We hypothesized that feeding Lewis rats a diet high in sucrose and casein (HSC) would alter the oral microenvironment and induce inflammation and the development of periodontitis lesions without mechanical intervention. A baseline group (BSL, n = 8) was euthanized at age 6 wk. Beginning at 6 wk of age, 2 groups of Lewis rats were fed standard (STD, n = 12) or HSC (n = 20) chow and euthanized at 29 wk of age. We evaluated the degree of periodontitis through histology and µCT of maxillae and mandibles. The HSC-induced inflammatory response of periodontal tissues was assessed by using immunohistochemistry. Gene expression analysis of inflammatory cytokines associated with Th1 and Th17 responses, innate immunity cytokines, and tissue damage in response to bacteria were assessed also. The potential systemic effects of HSC diet were evaluated by assessing body composition and bone densitometry endpoints; serum leptin and insulin concentrations; and gene expression of inflammatory cytokines in the liver. Placing Lewis rats on HSC diet for 24 wk induced a host Th1-immune response in periodontal tissues and mild to moderate, generalized periodontitis characterized by inflammatory cell infiltration (predominantly T cells and macrophages), osteoclast resorption of alveolar bone, and hyperplasia and migration of the gingival epithelium. HSC-fed Lewis rats developed periodontitis without mechanical intervention in the oral cavity and in the absence of any noteworthy metabolic abnormalities. Consequently, the rat model we described here may be a promising approach for modeling mild to moderate periodontitis that is similar in presentation to the human disease.

Attenuated Amiloride-Sensitive Current and Augmented Calcium-Activated Chloride Current in Marsh Rice Rat (Oryzomys palustris) Airways.

Prolonged heat and sea salt aerosols pose a challenge for the mammalian airway, placing the protective airway surface liquid (ASL) at risk for desiccation. Thus, mammals inhabiting salt marshes might have acquired adaptations for ASL regulation. We studied the airways of the rice rat, a rodent that inhabits salt marshes. We discovered negligible Na+ transport through the epithelial sodium channel (ENaC). In contrast, carbachol induced a large Cl- secretory current that was blocked by the calcium-activated chloride channel (CaCC) inhibitor CaCCinhi-A01. Decreased mRNA expression of α, ß, and γ ENaC, and increased mRNA expression of the CaCC transmembrane member 16A, distinguished the rice rat airway. Rice rat airway cultures also secreted fluid in response to carbachol and displayed an exaggerated expansion of the ASL volume when challenged with 3.5% NaCl. These data suggest that the rice rat airway might possess unique ion transport adaptations to facilitate survival in the salt marsh environment.

Zoledronate treatment duration is linked to bisphosphonate-related osteonecrosis of the jaw prevalence in rice rats with generalized periodontitis.

OBJECTIVES: To determine the extent that zoledronate (ZOL) dose and duration is associated with bisphosphonate-related osteonecrosis of the jaw (BRONJ) prevalence in rice rats with generalized periodontitis (PD), characterize structural and tissue-level features of BRONJ-like lesions in this model, and examine the specific anti-resorptive role of ZOL in BRONJ. MATERIALS AND METHODS: Rice rats (n = 228) consumed high sucrose-casein diet to enhance generalized PD. Groups of rats received 0, 8, 20, 50 or 125 µg/kg IV ZOL/4 weeks encompassing osteoporosis and oncology ZOL doses. Rats from each dose group (n = 9-16) were necropsied after 12, 18, 24 and 30 weeks of treatment. BRONJ-like lesion prevalence and tissue-level features were assessed grossly, histopathologically and by MicroCT. ZOL bone turnover effects were assessed by femoral peripheral quantitative computed tomography, serum bone turnover marker ELISAs and osteoclast immunolabelling. RESULTS: Prevalence of BRONJ-like lesions was significantly associated with (a) ZOL treatment duration, but plateaued at the lowest oncologic dose, and (b) there was a similar dose-related plateau in the systemic anti-resorptive effect of ZOL. ZOL and BRONJ-like lesions also altered the structural and tissue-level features of the jaw. CONCLUSION: The relationship between BRONJ-like lesion prevalence and ZOL dose and duration varies depending on the co- or pre-existing oral risk factor. At clinically relevant doses of ZOL, BRONJ-like lesions are associated with anti-resorptive activity.

Comparison of Isoflurane, Ketamine-Dexmedetomidine, and Ketamine-Xylazine for General Anesthesia during Oral Procedures in Rice Rats (Oryzomys palustris).

Rice rats (Oryzomys palustris) are an unconventional laboratory species that has been used to study photoperiodicity, periodontitis, and osteonecrosis of the jaw. Interventional procedures that require anesthesia, including oral procedures, are sometimes necessary in preclinical settings. The use of anesthetics including isoflurane and ketamine combined with α2-adrenoreceptor agonists, such as dexmedetomidine and xylazine, is well-established for laboratory rodents. However, their effects have been studied only modestly in rice rats. The aims of this study were to 1) determine the safety and consistency of 3 common anesthetic modalities in rice rats; 2) compare the physiologic and clinical responses to these anesthetics, and 3) verify the effectiveness of the most successful modality by testing it during an oral procedure (tooth extraction). Isoflurane, intraperitoneal ketamine-dexmedetomidine, and intraperitoneal ketamine-xylazine were evaluated by using a crossover design, in which each rat received all of the anesthetics. Compared with ketamine-dexmedetomidine and ketamine-xylazine, isoflurane inhalation through a nose cone produced more rapid induction, entry to a surgical plane of anesthesia, and initial recovery. In addition, isoflurane produced optimal anesthesia throughout the procedure for most rats. Unlike ketamine-dexmedetomidine and ketamine-xylazine, isoflurane did not alter rectal temperature, SpO2, or respiratory rate during the surgical tolerance period, whereas ketamine-dexmedetomidine and ketamine-xylazine decreased rectal temperature during the last stage of anesthesia and induced cardiorespiratory depression. Furthermore, 2 rats experienced negative outcomes warranting euthanasia: one after receiving ketamine-dexmedetomidine, and the other after ketamine-xylazine anesthesia. In conclusion, isoflurane was the most reliable and effective anesthetic in rice rats and maintained a surgical depth of anesthesia for as long as 30 min, thus supporting successful tooth extractions.

Prevalence of Food Impaction-Induced Periodontitis in Conventionally Housed Marsh Rice Rats (Oryzomys palustris).

Marsh rice rats (Oryzomys palustris) fed a pelleted diet high in sucrose and casein have been used as a model for moderate to severe periodontitis. Here we characterize the prevalence, location, and histopathologic features of food-impaction lesions (FIL), a unique type of oral event, in rice rats fed standard pelleted rodent chow from weaning until 34 wk of age. Healthy female rats (n = 90; age, 4 wk) were weaned into groups (n = 10 to 24) and were euthanized at 4, 16, 22, 28, or 34 wk of age. At necropsy, high-resolution photographs of the 4 jaw quadrants were examined by 3 independent observers to determine the presence, number, and location of FIL. In addition, gross periodontitis was scored (scale, 0 to 4), and the hemimaxillar surface area containing FIL was measured. Serial sections of decalcified jaws were assessed histologically. The prevalence of FIL increased with age, and was 0% (baseline), 59.1%, 69.6%, 81.8% and 80.0% in rats at age 4, 16, 22, 28, and 34 wk, respectively. FIL were predominantly located (93.9%) in the maxillary palatal surfaces of the interproximal area between molars 2 and 3 and did not affect mandibular surfaces. The percentage of the hemimaxillar surface area occupied by FIL was 6.83%, 4.82%, 2.88%, and 6.52% in rats at age 16, 22, 28, and 34 wk, respectively. Histopathologic changes in FIL varied from localized gingivitis to larger, localized periodontitis-like lesions. These data indicate that FIL are common in rice rats fed standard rodent chow, are slight to mild in severity, and are localized to specific regions in the oral cavity, thus suggesting they may be a suitable model for local maxillary periodontitis when fed standard rodent chow.

Quercetin Partially Preserves Development of Osteoblast Phenotype in Fetal Rat Calvaria Cells in an Oxidative Stress Environment.

Studies are needed to improve understanding of the osteoblast antioxidant response, and the balance between oxidative homeostasis and osteoblast differentiation. The flavonol quercetin aglycone (QRC) up-regulates the osteoblast antioxidant response in vitro without suppressing osteoblast phenotype, suggesting that QRC may preserve osteoblast phenotypic development in cells subsequently exposed to oxidative stress, which suppresses osteoblast differentiation. The aims of this study were to assess the extent that QRC pretreatment preserved development of the osteoblast phenotype in cells subsequently cultured with hydrogen peroxide, an oxidative stressor, and to characterize alterations in the osteoblast antioxidant response and in key antioxidant signaling pathways. We hypothesized that pretreatment with QRC would preserve phenotypic development after hydrogen peroxide treatment, suppress the hydrogen peroxide-induced antioxidant response, and that the antioxidant response would involve alterations in Nrf2 and ERK1/2 signaling. Results showed that treating fetal rat calvarial osteoblasts for 4 days (D5-9) with 300 µM hydrogen peroxide resulted in fewer alkaline phosphatase-positive cells and mineralized nodules, altered cell morphology, and significantly lower osteoblast phenotypic gene expression (P < 0.05). This suppression was partially blocked when cells were pretreated 12 h with 20 µM QRC. Hydrogen peroxide also produced sustained up-regulation of heme oxygenase-1 (HO-1) and γ-glutamate cysteine ligase catalytic subunit (GCLC), which was partially blocked in hydrogen peroxide-treated cells that first received QRC pretreatment. The alterations in the antioxidant stress response coincided with alterations in phosphorylated ERK1/2, but not Nrf2. These results suggest that QRC suppresses hydrogen peroxide-induced activation of the antioxidant response, and partially preserves osteoblast phenotypic development. J. Cell. Physiol. 231: 2779-2788, 2016. © 2016 Wiley Periodicals, Inc.

Role of Polymer Architecture on the Activity of Polymer-Protein Conjugates for the Treatment of Accelerated Bone Loss Disorders.

Polymers of similar molecular weights and chemical constitution but varying in their macromolecular architectures were conjugated to osteoprotegerin (OPG) to determine the effect of polymer topology on protein activity in vitro and in vivo. OPG is a protein that inhibits bone resorption by preventing the formation of mature osteoclasts from the osteoclast precursor cell. Accelerated bone loss disorders, such as osteoporosis, rheumatoid arthritis, and metastatic bone disease, occur as a result of increased osteoclastogenesis, leading to the severe weakening of the bone. OPG has shown promise as a treatment in bone disorders; however, it is rapidly cleared from circulation through rapid liver uptake, and frequent, high doses of the protein are necessary to achieve a therapeutic benefit. We aimed to improve the effectiveness of OPG by creating OPG-polymer bioconjugates, employing reversible addition-fragmentation chain transfer polymerization to create well-defined polymers with branching densities varying from linear, loosely branched to densely branched. Polymers with each of these architectures were conjugated to OPG using a "grafting-to" approach, and the bioconjugates were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The OPG-polymer bioconjugates showed retention of activity in vitro against osteoclasts, and each bioconjugate was shown to be nontoxic. Preliminary in vivo studies further supported the nontoxic characteristics of the bioconjugates, and measurement of the bone mineral density in rats 7 days post-treatment via peripheral quantitative computed tomography suggested a slight increase in bone mineral density after administration of the loosely branched OPG-polymer bioconjugate.

Quercetin Metabolites Up-Regulate the Antioxidant Response in Osteoblasts Isolated From Fetal Rat Calvaria.

Oxidative stress contributes to osteoporosis by suppressing differentiation of osteoblasts, suggesting the osteoblast antioxidant response may be a viable strategy for osteoporosis prevention. Quercetin, an antioxidant flavonol, up-regulates the antioxidant response in many cell types, but studies are needed to understand the effects of quercetin plasma metabolites on the osteoblast antioxidant response. The first specific aim was to examine antioxidant response genes and proteins in osteoblasts exposed to plasma quercetin metabolites. The second specific aim was to identify potential signaling pathways in the osteoblast antioxidant response that mediate the effect of quercetin, specifically Nrf2, ERK1/2, and NFκB p65. Osteoblasts isolated from fetal rat calvaria were treated with doses up to 20 µM of three different quercetin metabolites found in blood plasma after consumption of quercetin-rich foods or supplements: quercetin aglycone (QRC), isorhamnetin (ISO), or quercetin 3-O-glucuronide (Q3G). Alternatively, some cells received a 2:1:1 mixture of all three metabolites (10 µM Q3G: 5 µM ISO: 5 µM QRC) to evaluate synergistic effects. Antioxidant response genes and proteins known to be up-regulated by quercetin were analyzed along with Nrf2, ERK1/2, and NFκB proteins. Both QRC and ISO, but not Q3G, up-regulated heme oxygenase-1 (HO-1) and γ-glutamate cysteine ligase catalytic subunit (GCLC) at the mRNA and protein level. Synergistic effects of metabolites were not observed. Up-regulation of HO-1 and GCLC was associated with suppression of phosphorylated ERK1/2 and NFκB, but no alterations in Nrf2 protein levels were observed. This study shows that the antioxidant response of osteoblasts is differentially stimulated by quercetin metabolites.

Iron chelator deferoxamine alters iron-regulatory genes and proteins and suppresses osteoblast phenotype in fetal rat calvaria cells.

There are few studies describing the extent to which low iron status affects osteoblastogenesis, despite evidence that iron deficiency produces adverse effects on bone density. The purpose of this study was to evaluate alterations in intracellular iron status by measuring iron-regulated gene and protein expression and to describe development of osteoblast phenotype in primary cells treated with iron chelator deferoxamine (DFOM) during differentiation. Using the well-described fetal rat calvaria model, cells were incubated with 0-8 microM DFOM throughout differentiation (confluence to day (D) 21), or only during early differentiation (confluence to D13-15) or late differentiation (D13-15 to D21). Changes in intracellular iron status were determined by measuring alterations in gene and protein expression of transferrin receptor and ferritin light chain and heavy chain. Development of osteoblast phenotype was monitored by measuring expression of genes that are known to be up-regulated during differentiation, analyzing the percentage of mineralized surface area, and counting the number of multi-layered bone nodules at the end of culture. Results indicate that treatment throughout differentiation with 8 microM DFOM alters iron-regulated genes and proteins by mid-differentiation (D13-15) in a pattern consistent with iron deficiency with concomitant down-regulation of osteoblast phenotype genes, especially osteocalcin. Additionally, alkaline phosphatase staining was lower and there was about 70% less mineralized surface area (p<0.05) by D21 in wells treated throughout differentiation with 8 microM DFOM compared to control. Down-regulation of osteocalcin and alkaline phosphatase mRNA (p<0.05) and suppressed mineralization (p<0.05) was also evident at D21 in cells treated only during early differentiation. In contrast, treatment during late differentiation did not alter osteoblastic outcomes by D21. In conclusion, it appears that iron is required for normal osteoblast phenotype development, and that early rather than late differentiation events may be more sensitive to iron availability.

Iron overload alters iron-regulatory genes and proteins, down-regulates osteoblastic phenotype, and is associated with apoptosis in fetal rat calvaria cultures.

Iron overload has been implicated in decreased bone mineral density. However, the effect of iron overload on osteoblast lineage cells remains poorly understood. The purpose of this study was to examine osteoblast differentiation, function, and apoptosis in iron-loaded cells from fetal rat calvaria. Cells were incubated with media supplemented with 0-10 microM ferrous sulfate (FeSO(4)) during differentiation (days 6-20). Intracellular iron status was assessed by measuring iron content in cell layers and changes in transferrin receptor (TrfR) and ferritin gene and protein expression. Osteoblast differentiation and function were evaluated by measuring osteoblast phenotypic gene markers and capacity of cultures to form mineralized bone nodules. Apoptotic hallmarks were evaluated by microscopy. A 2.3-fold increase in media iron concentration resulted in saturable accumulation of iron in the cell layer 20-fold higher than control (p<0.05) by mid-differentiation (day 15, D15). Iron accumulation resulted in rapid and sustained down-regulation of TrfR gene and protein levels (within 24 h) and up-regulation of light and heavy chain ferritin protein levels at late differentiation (day 20, D20). Concurrently, osteoblast phenotype gene markers were suppressed by D15 and a decreased number of mineralized nodules at D20 were observed. Apoptotic events were observed within 24 h of iron loading. These results provide evidence that iron overload alters iron metabolism and suppresses differentiation and function of cells in the osteoblast lineage associated with increased apoptosis.