Effect of metformin on periimplant wound healing in a rat model of type 2 diabetes.

PURPOSE: To investigate the effects of hyperglycemia and metformin (a popular biguanide antidiabetic) on periimplant healing. METHODS: Thirty-six male rats were assigned to 3 groups: (1) nondiabetic Wistar-Kyoto rats (controls), (2) Goto-Kakizaki (GK) spontaneously diabetic rats (GK group), and (3) GK rats were fed metformin (100 mg/kg body weight per day) in their water for 4 weeks (GK + Met group). The right maxillary first molars were extracted and sites were allowed 1 month to heal. Titanium implants (1 × 3 mm) were placed in healed extraction sites. Six rats from each group were analyzed at weeks 1 and 4 by micro computed tomography for bone/implant contact ratio, percent bone volume, trabecular number, and bone mineral density. Blood was also analyzed for glucose, HbA1c, and pyridinoline (PYD). RESULTS: At week 1, glucose levels in the GK-Met rats were high, and all bone parameters were similar to GK rats (lower bone parameters and higher PYD than controls). At week 4, glucose levels in the GK-Met rats and all parameters were similar to controls. CONCLUSIONS: Hyperglycemic GK type 2 diabetic rats showed improved blood glucose and wound healing around oral implants after metformin administration.

Development of a rat model of bisphosphonate-related osteonecrosis of the jaw (BRONJ).

The purpose of this study was to develop a rat model predictive of bisphosphonate-related osteonecrosis of the jaw (BRONJ) after exodontias. Thirty female rats were randomized into 2 groups, control and experimental. The experimental group received 2 intravenous injections of zoledronate (20 µg/kg). The mesial root of the right mandibular first molar was extracted. Rats were euthanized at 0, 4, and 8 weeks. Bone mineral density (BMD), collagen breakdown (pyridinium [PYD]), vascular regeneration (VEGF), and histology were examined. A trend toward higher PYD values was suggested in control vs experimental groups after wounding. Serum VEGF increased significantly after wounding for both control and experimental groups. After 8 weeks, VEGF continued to rise for the experimental group only. In the extraction socket area, BMD was significantly lower after wounding in control vs. zoledronate-treated rats. Histology sections from experimental groups showed bacteria and bone necrosis. Consistent findings of BRONJ features similar to those in humans were observed after zoledronate treatment.

Effect of systemic parathyroid hormone (1-34) and a beta-tricalcium phosphate biomaterial on local bone formation in a critical-size rat calvarial defect model.

OBJECTIVE: The objective of this study was to evaluate local bone formation following systemic administration of parathyroid hormone (1-34) (PTH), a surgically implanted synthetic beta-tricalcium phosphate (beta-TCP) bone biomaterial serving as a matrix to support new bone formation. MATERIALS AND METHODS: Critical-size, 8 mm, calvarial through-and-through osteotomy defects were surgically created in 100 adult male Sprague-Dawley rats. The animals were randomized into five groups of 20 animals each to receive one of the following treatments: PTH (15 microg PTH/kg/day; subcutaneously), PTH/beta-TCP, beta-TCP, or particulate human demineralized freeze-dried bone (DFDB), and sham-surgery controls. Ten animals/group were euthanized at 4 and 8 weeks post-surgery for radiographic and histometric analysis. RESULTS: The histometric analysis showed that systemic PTH significantly enhanced local bone formation, bone fill averaging (+/-SE) 32.2+/-4.0% compared with PTH/beta-TCP (15.7+/-2.4%), beta-TCP (12.5+/-2.3%), DFDB (14.5+/-2.3%), and sham-surgery control (10.0+/-1.5%) at 4 weeks (p<0.014). Systemic PTH showed significantly enhanced bone formation (41.5+/-4.0%) compared with PTH/beta-TCP (22.4+/-3.0%), beta-TCP (21.3+/-4.4%), and with the sham-surgery control (23.8+/-4.2%) at 8 weeks (p<0.025). The DFDB group showed significantly increased bone formation from 4 (14.5+/-2.3%) to 8 weeks (32.0+/-3.2%) (p<0.006). The PTH/beta-TCP and beta-TCP groups both showed limited biomaterials resorption. The radiographic analysis was not diagnostic to distinguish local bone formation from the radiopaque beta-TCP biomaterial. CONCLUSIONS: Systemic administration of PTH significantly stimulates local bone formation. Bone formation was significantly limited by the beta-TCP biomaterial.

Advanced glycation endproducts and rat dental implant osseointegration.

Advanced glycation endproducts (AGEs) are a diverse group of molecular adducts formed in environments high in reducing sugars that accumulate with aging and in diabetes. This study tests the hypothesis that AGEs inhibit the stabile osseointegration of dental implants through tissue interactions that interfere with bone turnover and compromise the biomechanical properties at the bone-implant interface. Maxillary first molars were extracted from 32 rats and allowed to heal for 4 weeks. Titanium implants (1 mm x 3 mm) were placed in the healed sockets of 2 groups of 16 rats consisting of 8 rats injected 3 times/wk for 1 month with AGE (prepared from glucose and lysine) and 8 rats injected with vehicle as a control. AGE injections continued for an additional 14 or 28 days before sacrifice. X-ray images, blood, and tissues were collected to examine bone/implant contact ratio, serum pyridinoline ([PYD] a collagen breakdown marker), osteocalcin ([OSC] a bone formation marker), and for immunohistochemistry with antibodies to AGE and the bone turnover-marker protein matrix metalloproteinase1. Compared with the AGE-treated groups, the controls showed significantly higher bone/implant contact at both 14- and 28-day time points. PYD (P < .05) and OSC (trend) levels from controls showed decreases at 28 days when compared with AGE-treated groups. Immunohistochemistry with AGE-specific and bone turnover marker antibodies showed stronger staining associated with the implant/tissue interface in AGE-treated rats. Our studies indicate an association between AGE and inhibition of bone turnover, suggesting that the formation of AGE in high glycemic conditions, such as diabetes, may contribute to a slower rate of osseointegration that negatively affects implant stability.

Rat model for studying tissue changes induced by the mechanical environment surrounding loaded titanium implants.

PURPOSE: This study presents a new rat oral implant model for assessing histologic changes in the mechanical environment surrounding loaded and unloaded dental implants. MATERIALS AND METHODS: The maxillary left first molar from retired breeder rats was extracted, and the site was allowed to heal for 1 month. A titanium miniscrew implant was then placed into the site and allowed to heal for 21 days. The mandibular left first molars in one group of rats were extracted to create an unloaded condition; in a second group of rats the mandibular left first molars were left in occlusion with the opposing screw head to simulate loading. Radiographs were taken on the day of placement and again at 7 days, 14 days, and 21 days after placement and were used to estimate the bone-implant contact ratio. The rats were sacrificed after 21 days. Peri-implant tissue samples from day 21 were processed for histology and immunohistochemistry with antibodies to osteocalcin and matrix metalloproteinase 13 (MMP-13). Two-dimensional finite element models were created from images of the histologic sections and immunohistochemical samples to observe tissue changes. RESULTS: Areas of high shear stress adjacent to the helical threads of loaded implants were associated with osteocalcin localization and bone formation but only minimal localization of MMP-13. Bone adjacent to unloaded implants showed fibrous tissue and extensive MMP-13 localization surrounding the apical two-thirds of each implant. These results agree with estimated bone-implant contact ratios, which showed a steady decrease in contact ratio for the unloaded implant group but a significantly higher contact ratio in the loaded group between 14 and 21 days. CONCLUSION: The rat oral implant model is useful for studies of the mechanical and physiologic environment affecting osseointegration in loaded and unloaded implants.

Effects of induced premaxillary suture fusion on the craniofacial morphology in growing rats.

OBJECTIVE: Due to premaxillary rapid development and fusion with the maxilla at the fetus stage, the functions of the premaxillary suture still remain unclear. This study was designed to explore the effect of artificial induced premaxillary suture fusion on craniofacial morphology. METHODS: Thirty Sprague Dawley rats were divided into control and experimental groups, with 3 week, 5 week and 8 week subgroups of five animals each. An incision was made in each rat along the premaxillary suture and cyanoacrylate was administered to immobilize the exposed premaxillary suture for experimental rats. No glue was applied to controls. Weights, dental impressions and radiographs were taken before and after surgery until sacrifice and used to determine the differences between groups using the one-way ANOVA test. RESULTS: After immobilizing the premaxillary suture, significant changes in the craniofacial morphology were measured at the different time points. In the experimental groups, local changes occurred at the 3rd week. A global alteration in craniofacial morphology was apparent at the 8th week in the experimental group compared to the control. At each successive time point, craniofacial morphological alterations increased in rats with fused premaxillary sutures. CONCLUSIONS: Induced premaxillary suture fusion can inhibit the growth of the premaxilla and cause extensive craniofacial morphological changes. These findings suggest that premaxillary suture fusion may be related to craniofacial malformation or malocclusion and to the formation of the flattened craniofacial profile in humans.

Effect of ovariectomy and alendronate on implant osseointegration in rat maxillary bone.

Bisphosphonates such as alendronate (ALD), although controversial, are worthy of investigation for the enhancement of implant osseointegration in patients with low bone mass who are already taking bisphosphonates for osteoporosis. These patients may receive additional benefits and be acceptable candidates for dental implants without needing to change their medication regimen and possibly as a result of their medication regimen. The purpose of this study was to compare implant osseointegration in maxillary bone of normal rats with a rat model of postmenopausal estrogen deficiency (ovariectomized [OVX]), with and without ALD. An experimental group of 32 rats was divided in 4 groups: ALD-OVX (n=8 OVX with ALD), OVX (n=8 OVX without ALD), ALD (n=8 normal rats with ALD), and control (n=8 normal rats). All rats received one titanium microscrew implant in the left edentulous region of the maxillary arch. The ALD-OVX and ALD groups received subcutaneous injections of ALD 3 times a week. On the fourth week after ALD administration, an implant was placed in all 32 rats. The maxilla of each rat was radiographed 4 times: at 0, 7, 14, and 28 days. On day 28 after implant placement, all rats were killed, and the peri-implant tissue was embedded in plastic or paraffin for histological examination. The X rays were used for a chronologic calculation of the contact ratio between implant and bone surfaces. Radiographic bone density was determined at 3 points: mesial, apical, and distal. The results show that osseointegration of the implants was impaired in the estrogen-deficient OVX rats compared with the ALD-OVX rats. Fifty percent of the implants were lost at 2 weeks in the OVX group. Radiographic evidence suggested that none of the implants in the OVX group osseointegrated. In the histologic examination more bone was observed around implants from the ALD-OVX and ALD groups than around implants from the OVX group. The OVX group presented a dramatic reduction in implant bone contact at 2 weeks and a significant 13% reduction at 4 weeks vs day of implant (P = .006). The ALD-OVX group presented 50% more bone density than the OVX group (P = .0003). Both ALD groups (ALD and ALD-OVX) had significantly higher radiographic bone density than the other groups (P < .01 for each comparison). In conclusion, osseointegration of implants was enhanced by ALD. Radiographic bone density and contact ratio improved with ALD administration. Implant osseointegration was impaired by estrogen deficiency in the OVX group.

Hematopoietic Stem Cells as a Novel Source of Dental Tissue Cells.

While earlier studies have suggested that cells positive for hematopoietic markers can be found in dental tissues, it has yet to be confirmed. To conclusively demonstrate this, we utilized a unique transgenic model in which all hematopoietic cells are green fluorescent protein+ (GFP+). Pulp, periodontal ligament (PDL) and alveolar bone (AvB) cell culture analysis demonstrated numerous GFP+ cells, which were also CD45+ (indicating hematopoietic origin) and co-expressed markers of cellular populations in pulp (dentin matrix protein-1, dentin sialophosphoprotein, alpha smooth muscle actin [ASMA], osteocalcin), in PDL (periostin, ASMA, vimentin, osteocalcin) and in AvB (Runx-2, bone sialoprotein, alkaline phosphatase, osteocalcin). Transplantation of clonal population derived from a single GFP+ hematopoietic stem cell (HSC), into lethally irradiated recipient mice, demonstrated numerous GFP+ cells within dental tissues of recipient mice, which also stained for markers of cell populations in pulp, PDL and AvB (used above), indicating that transplanted HSCs can differentiate into cells in dental tissues. These hematopoietic-derived cells deposited collagen and can differentiate in osteogenic media, indicating that they are functional. Thus, our studies demonstrate, for the first time, that cells in pulp, PDL and AvB can have a hematopoietic origin, thereby opening new avenues of therapy for dental diseases and injuries.

Green tea polyphenols reduce autoimmune symptoms in a murine model for human Sjogren's syndrome and protect human salivary acinar cells from TNF-alpha-induced cytotoxicity.

Sjogren's syndrome (SS) is a relatively common autoimmune disorder. A key feature of SS is lymphocytic infiltration of the salivary and lacrimal glands, associated with the destruction of secretory functions of these glands. Current treatment of SS targets the symptoms but is unable to reduce or prevent the damage to the glands. We reported previously that the major green tea polyphenol (GTP) epigallocatechin-3-gallate (EGCG) inhibits autoantigen expression in normal human keratinocytes and immortalized normal human salivary acinar cells (Hsu et al. 2005). However, it is not known whether GTPs have this effect in vivo, if they can reduce lymphocytic infiltration, or protect salivary acinar cells from tumor necrosis factor-alpha (TNF-alpha)-induced cytotoxicity. Here, we demonstrate that in the NOD mouse, a model for human SS, oral administration of green tea extract reduced the serum total autoantibody levels and the autoimmune-induced lymphocytic infiltration of the submandibular glands. Further, we show that EGCG protected normal human salivary acinar cells from TNF-alpha-induced cytotoxicity. This protection was associated with specific phosphorylation of p38 MAPK, and inhibitors of the p38 MAPK pathway blocked the protective effect. In conclusion, GTPs may provide a degree of protection against autoimmune-induced tissue damage in SS, mediated in part through activation of MAPK elements.

Infiltration/evaporation-induced shrinkage of demineralized dentin by solvated model adhesives.

During dentin bonding, solvated adhesive comonomers are applied to water-saturated decalcified dentin matrices. When alcohol-solvated hydrophilic or hydrophobic methacrylate monomers are applied, they chemically remove water and cause matrix shrinkage during comonomer infiltration. Evaporation of solvent induces further shrinkage. The purpose of this work was to compare the shrinkage of water-saturated dentin matrices infiltrated with ethanol- or methanol-solvated 2-hydroxyethyl methacrylate (HEMA), 2,2-bis[4(2-hydroxy-3-methacryloyloxy-propyloxy)-phenyl] propane (BisGMA), or triethyleneglycol dimethacrylate (TEGDMA) at 90/10, 70/30, 50/50, and 30/70 mass fraction % alcohol/monomer before and after evaporation of alcohol. Thin (ca 0.2 mm) disks of human mid-coronal dentin were demineralized and placed in a well beneath the contact probe of a linear variable differential transformer (LVDT). The height of the matrix was measured before and after random application of one of the twelve alcohol/monomer mixtures. Matrix height was measured during infiltration and during solvent evaporation. Between trials, residual monomer was extracted using ethanol. These studies were repeated on specimens in which 100% alcohol was used to substitute for water in the matrix. Both studies revealed that matrices shrink 30-50% but that pretreatment of matrices with alcohol prevents BisGMA phase separations from occurring. Wet bonding with ethanol instead of water permits infiltration of relatively hydrophobic alcohol/monomers.

Net expansion of dried demineralized dentin matrix produced by monomer/alcohol saturation and solvent evaporation.

The purpose of this work was to determine if nonaqueous methacrylate monomer/alcohol mixtures could expand dried collapsed demineralized dentin matrix. Thin disks (ca. 200 microm) of human dentin were demineralized and placed in wells beneath contact probes of linear variable differential transformers. The probes were placed on water-saturated expanded matrices to record the shrinkage associated with drying. Monomer mixtures containing hydroxyethyl methacrylate, 2,2-bis[4-(2-hydroxy-3 methacryloyloxy)propoxyphenyl] propane, or triethyleneglycol dimethacrylate were mixed with methanol or ethanol at alcohol/monomer mass fraction % of 90/10, 70/30, 50/50, or 30/70. They were randomly applied to the dried matrices to determine the rate and magnitude of expansion; then shrinkage was recorded during evaporation of the alcohols. The results indicated that matrix expansion was positively correlated with the Hoy's solubility parameters for hydrogen bonding forces (delta(h)) of the monomer/solvent mixtures (p < 0.001). Expansions were more rapid with methanol-containing than with ethanol-containing monomer mixtures. For the test solutions, triethyleneglycol dimethacrylate-containing mixtures produced the slowest rate of matrix expansion and hydroxyethyl methacrylate-containing mixtures the most rapid expansion. When the solvents were evaporated, the matrix shrank in proportion to the solvent content and the delta(h) of the monomer-solvent mixtures. The results indicate that expansion of dried, collapsed dentin matrices requires that the delta(h) of the mixtures be larger than 17 (J/cm(3))(1/2). The greater the delta(h) of the monomer solutions, the greater the rate and extent of expansion.

Mechanical Stretching of Mouse Calvarial Osteoblasts In Vitro Models Changes in MMP-2 and MMP-9 Expression at the Bone-Implant Interface.

Bone to mechanical loading elicits a biological response that has clinical significance for several areas in dental medicine, including orthodontic tooth movement, tempromandibular joint disease, and endosseous dental implant osseointegration. Human orthopedic studies of failed hip implant sites have identified increased mRNA expression of several collagen-degrading matrix metalloproteinases (MMPs), while in vitro experiments have shown increases in MMP secretion after exposure to inflammatory mediators. This investigation evaluates the effects of mechanical deformation on in vitro osteoblasts by assessing changes in MMP gene expression and enzyme activity. We seeded mouse neonatal calvarial osteoblasts onto flexible 6-well plates and subjected to continuous cyclic mechanical stretching. The expression and activity of mRNA for several MMPs (2, 3, 9, and 10) was assessed. When subjected to mechanical stress in culture, only mRNA specific for MMP-9 was significantly increased compared to nonstretched controls (P < .005). Measurement of MMP activity by gelatin zymography demonstrated that none of the MMPs showed increased activity with stretching; however, MMP-2 activity decreased. Our results suggest that in response to stretch, MMP-2 responds rapidly by inhibiting conversion of a MMP-2 to the active form, while a slower up-regulation of MMP-9 may play a role in the long-term remodeling of extracellular matrix in response to continuous mechanical loading. This study suggests that the regulation of metalloproteinases at both the mRNA and protein level are important in the response of bone to mechanical stress.

Role of p21WAF1 in green tea polyphenol-induced growth arrest and apoptosis of oral carcinoma cells.

The cyclin-dependent kinase inhibitor p21WAF1 participates in cell growth, differentiation, and apoptosis. p21WAF1 can be induced by green tea polyphenol EGCG in several cancer cell types, but its role in the oral cancer cell response to EGCG is not known. We found that EGCG upregulates p21WAF1 in an oral carcinoma cell line, OSC2, by cDNA microarray. The current study determined the impact of siRNA-suppressed p21WAF1 and its response to EGCG on cell growth, DNA synthesis and apoptosis by RT-PCR, Western blot, BrdU incorporation, MTT and caspase 3 activity assays. Suppression of p21WAF1 by siRNA resulted in an accelerated cell growth and DNA synthesis, and increased cell viability. However, caspase 3 activity was not significantly inhibited. The evidence showed that p21WAF1 is involved in EGCG-induced growth arrest of OSC2 cells, which may facilitate caspase 3-mediated apoptosis. Thus, expression of functional p21WAF1 may promote phytochemical-mediated growth arrest and apoptosis in oral carcinoma cells.

Removal of pamidronate from bone in rats using systemic and local chelation.

OBJECTIVES: Bisphosphonates become adsorbed on hydroxyapatite crystals in the bone matrix. In case of side-effects, stopping the treatment would not affect the bisphosphonates already deposited in bone. This study tests the feasibility of in-vivo targeted removal of bisphosphonates from bone using chelating agents. DESIGN: 32 Sprague Dawley rats were given an injection of fluorescent pamidronate (OsteoSense EX; 0.16nmol/g). They were treated with either systemic (cadmium) or local [ethylenediaminetetraacetic (EDTA) or citric acid (CA)] chelating agents to induce the removal of the bisphosphonate from bone. We evaluated the decrease in fluorescence in the alveolar bone, femur, tibia, and vertebrae. We also analyzed the systemic effects of treatment. RESULTS: Systemic chelation reduced the pamidronate signal universally. However, the maximum reduction was observed in the alveolar bone and femur (22% and 21%, p values 0.008 and 0.028, respectively). Systemic chelation did not impair calcium homeostasis. The chelation effect was not due to a systemic toxic effect on the liver or kidney. On the other hand local chelation at the extraction site significantly (p=0.011) decreased the pamidronate signal at bony surfaces of the socket. CONCLUSIONS: Systemic and local chelating agents can remove bisphosphonate from bone. This study establishes a new concept for the prevention of side effects of bisphosphonates during high-risk situations.

Correlation of changes in the mandible and retina/choroid vasculature of a rat model of BRONJ.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) causes bones of the mandible and maxilla to become necrotic and protrude into the oral cavity. Compromised blood supply to bone is also a feature of BRONJ. The design of this study was first to use our established technique of molar extraction and IV bisphosphonate injection to produce features of BRONJ in rats that mimic the human disease; second to confirm vascular changes in the mandible and eye using micro-CT of vascular casts, and image analysis of retina/choroid images; and third to show parallel bisphosphonate-induced changes in the structure and markers of the vasculature of the bone and eye. The results of this study show structural changes in the eye and mandible as well as biochemical changes including the up-regulation of VEGF in response to the bisphosphonate-associated ischemia. These changes are not associated with angiogenesis in either the eye or mandible as determined by reduced vascular complexity. These results suggest that observations of direct changes to the vasculature in the retina/choroid structures of the eye in patients taking bisphosphonates could serve as a window to the progression of debilitating changes occurring as a result of bisphosphonate therapy.

In vitro study of the adverse effect of nicotine and physical strain on human gingival fibroblasts as a model of the healing of wounds commonly found in the military.

BACKGROUND: Significant adverse effects on fibroblast growth and metabolism are observed with nicotine. We investigated the synergistic effects of nicotine and cyclical mechanical strain (CMS) on human gingival fibroblasts (HGFs) in a wound-healing model. METHODS: HGFs were isolated and grown in Dulbecco's modified Eagle's medium. Three-millimeter wounds were created on a confluent cell monolayer grown in a media containing 0, 1, 2, or 4 mM nicotine, with or without CMS. The applied deformation regimen remains constant for 6 days. On days 1, 2, 4, and 6, the cells were stained with hematoxylin and eosin Y for the evaluation of wound repopulation. RESULTS: The application of CMS alone demonstrates a biphasic response, with an initial stimulatory effect on wound repopulation (days 1-2) and less repopulation during the later phase (days 4-6). The addition of nicotine clearly demonstrated a time and inverse dose-dependent relationship on wound repopulation, with no effect during the early phase and reduced wound repopulation during the later phase. CONCLUSIONS: Initial treatment of HGF wounds with CMS resulted in faster wound repopulation regardless of nicotine presence. By day 6, wound healing of HGF exposed to both nicotine and CMS is delayed. These findings suggest that CMS and nicotine may affect fibroblasts and delay wound healing at other sites in the body as well.

Histologic Evaluation of Osseous Regeneration Following Combination Therapy With Platelet-Rich Plasma and Bio-Oss in a Rat Calvarial Critical-Size Defect Model.

Platelet-rich plasma (PRP) is an autogenous source of growth factors shown to facilitate human bone growth. Bio-Oss, an osteoconductive xenograft, is used clinically to regenerate periodontal defects, restore dental alveolar ridges, and facilitate sinus-lift procedures. The purpose of this study was to analyze whether a combination of PRP and Bio-Oss would enhance bone regeneration better than either material alone. PRP and/or Bio-Oss were administered in an 8-mm critical-size defect (CSD) rat calvarial model of bone defect between 2 polytetrafluoroethylene membranes to prevent soft tissue incursion. Eight weeks after the induction of the CSD, histologic sections were stained with hematoxylin and eosin stain and analyzed via light microscopy. Qualitative analyses revealed new bone regeneration in all 4 groups. The Bio-Oss and PRP plus Bio-Oss groups demonstrated greater areas of closure in the defects than the control or PRP-only groups because of the space-maintaining ability of Bio-Oss. The groups grafted with Bio-Oss showed close contact with new bone growth throughout the defects, suggesting a stronger graft. The use of PRP alone or in combination with Bio-Oss, however, did not appear to enhance osseous regeneration at 8 weeks. Areas grafted with Bio-Oss demonstrated greater space-maintaining capacity than controls, and PRP was an effective vehicle for placement of the Bio-Oss. However, at 8 weeks this study was unable to demonstrate a significant advantage of using PRP plus Bio-Oss over using Bio-Oss alone.

Vascular alterations in the sprague-dawley rat mandible during intravenous bisphosphonate therapy.

Long-term use of intravenous bisphosphonates, such as zoledronic acid (zoledronate), has been linked to bisphosphonate-related osteonecrosis of the jaw (BRONJ). Invasive dental surgery seems to trigger the bone necrosis in most cases. To determine the effects of zoledronic acid on the vascular structure of the rat mandible. Extracted of the mandibular first molar in rats that received 2 IV injections of zoledronate (20 µg/kg), 4 weeks apart. Zoledronate-treated rats (n = 18) were then compared to a control group of untreated rats (n = 18). At the fourth, eighth, and 12th week after molar extraction, 8 rat mandibles from each group were perfused with 35% radiopaque triphenylbismuth in methyl methacrylate via carotid artery perfusion. Mandibles were harvested and examined by micro-CT to assess the spatial and dimensional changes of the vasculature as a result of zoledronate treatment. The micro-CT analysis showed that zoledronic acid-treated rats had blood vessels that were thicker, less connected, and less ordered than control rats that were not exposed to zoledronic acid. This study demonstrated that treatment with zoledronic acid in rats is associated with vascular changes in alveolar bone. Further studies are underway to explore whether these vascular changes contribute to the pathogenesis of BRONJ.

A Model for Osteonecrosis of the Jaw with Zoledronate Treatment following Repeated Major Trauma.

This study aims to develop a reproducible rat model for post-traumatic bisphosphonate-related osteonecrosis of the jaw (BRONJ). In our previous studies using dental extraction as an inducing factor, only 30%-60% of zoledronate-treated animals fulfilled the definition of clinical BRONJ. We modified the zoledronate regimen and introduced repeated surgical extraction to illicit quantifiable BRONJ in all animals. Eighty retired-breeder female Sprague-Dawley rats were divided between the treatment (i.v. zoledronate; 80 µg/kg/week for 13 weeks) and control (saline) groups. On week 13, the left mandibular first molar was surgically extracted, followed by the second molar a week later. Animals were euthanized at 1-week, 2-weeks, and 8-weeks following extraction. The occurrence and severity of BRONJ were scored in each animal based on gross and MicroCT analysis. Parameters of bone formation and osteoclast functions at the extraction site were compared between groups. All zoledronate-treated animals developed a severe case of BRONJ that fulfilled the clinical definition of the condition in humans. Osteoclast attachment continued to be defective eight weeks after stopping the treatment. There were no signs of kidney or liver toxicity. Our data confirmed that repeated surgical extraction (major trauma) by itself consistently precipitated massive bone necrosis in ZA-treated animals, eliminating the need to induce pre-existing infection or comorbidity. These results will be the basis for further studies examining the in-vivo pathogenesis and prevention of BRONJ.

Taurine in submandibular gland of the rat: effect of muscarinic drugs.

Taurine exerts a number of actions in mammalian cells, including regulation of ion transport and osmoregulation. The production and secretion of saliva involve transepithelial ion transport, thereby making the plasma-like primary saliva hypotonic before secretion. Therefore, it is plausible to suggest modulation of salivary taurine by muscarinic agents that affect salivary gland function. One of the objectives of this study was to determine tissue content and localization of taurine in the submandibular gland of the rat. Further, we determined whether treatment with muscarinic drugs that either increase (e.g., pilocarpine) or decrease (e.g., propantheline) saliva secretion affects the submandibular gland taurine content. The results indicate that the submandibular gland contains an appreciable amount of taurine (8.9 +/- 0.3 micromoles/g wet wt). Further, acute treatment of the rats with either of the muscarinic drugs did not significantly affect tissue taurine content compared to the control group. By contrast, chronic treatment with propantheline, but not pilocarpine, reduced the tissue content of taurine compared to the control rats (p<0.05). Utilizing light microscopic immunohistochemical techniques, intense immunoreactivity was found primarily in the striated ducts of the submandibular gland. Neither pilocarpine nor propantheline treatment led to differential distribution of immunoreactivity in this tissue. In conclusion, the submandibular gland contains an appreciable amount of taurine, primarily in the striated ducts, that can be decreased by chronic muscarinic receptor blockade.