Expression of angiogenic markers in jawbones and femur in a rat model treated with zoledronic acid.

OBJECTIVES: This study aimed to investigate the gene expression of angiogenic marker in surgically treated jawbones and femur on a rat model administrated with zoledronic acid. RESULTS: No soft tissue fenestration or bone exposure was found in femur. Delayed soft tissue healing was found in both ZA group (3 in mandible, 4 in maxilla) and control group (1 in mandible, 2 in maxilla), while exposed bone was found only in the ZA group (1 in maxilla, 2 in mandible). RT-PCR analysis demonstrated no significant difference in gene expression of angiogenetic markers between ZA-treated and control groups in femur and mandible. In the maxilla, the expression of VEGFA and VEGFR-2 in medium-term ZA group was significantly down-regulated compared with that in the control. The ZA treatment does not change significantly the expression of the angiogenic factors in femur and mandible, but significantly downregulates the expression in maxilla in this rat model. The angiogenesis inhibition may contribute to the development of MRONJ but does not play a key role.

Effects of Soft Tissue Closure on Medication-Related Osteonecrosis of the Jaw in a Rabbit Model with Tooth Extraction: A Pilot Study.

OBJECTIVES: The study investigated the effect of soft tissue closure after tooth extraction on the prevention of medication-related osteonecrosis of the jaw in a rabbit model. MATERIALS AND METHODS: Twenty female New Zealand white rabbits were randomly assigned into the experimental group administrated with zoledronic acid (ZA) and control groups treated with saline. Bilateral lower premolar extraction was performed 4 weeks after ZA/saline administration. Immediately after extraction, the wound on the right mandible was closed by suture while the other side was left open. Animals were sacrificed 4 weeks and 8 weeks after tooth extraction. Fluorochrome labeling solutions were injected subcutaneously to evaluate the bone growth rates. The mandibles were harvested and subjected for microcomputed tomography, confocal microscope, and histomorphological examinations. RESULTS: All extraction sites healed well without any signs of infection. Trabecular thickness (Tb.Th) was significantly higher in the ZA-treated group than in the control group at both week 4 and week 8, while no significant difference was detected in the rest of the assessed parameters. The bone growth rate in mandibles showed gradual reduction in the ZA-treated group. Histological analysis showed that at week 8, the animals in the ZA-treated group had significantly higher incidence of osteonecrosis than that in the control group, while no significance was revealed between the sutured and nonsutured side. CONCLUSIONS: ZA treatment significantly reduces bone growth rates but does not reveal a significant effect on bone mineral density and bone microarchitecture. Soft tissue closure of the extraction socket does not reduce the incidence of ONJ in the ZA-treated rabbit model.

Comparison of Vascular Morphometry in Jawbones and Long Bones: Micro-CT Study in a Rat Model Treated with Zoledronic Acid.

The study was aimed at investigating the effect of zoledronic acid on vascular morphometry in jawbones and long bones on a rat model. Twenty-four skeletal mature Sprague-Dawley female rats were administered oncologic dose of zoledronic acid (ZA) or normal saline for 4 weeks and then subjected to tooth extraction on the mandible and maxilla and a bone defect creation on the femur. After the surgical procedures, ZA or saline treatment was continued until sacrifice at week 2, week 4, and week 8 postoperatively. Vascular perfusion with MICROFIL was performed on all the animals. Micro-CT analysis demonstrated a tendency of decreased vessel density and vessel number in ZA-treated groups but no statistical difference. In conclusion, the neovessel formation is suppressed but not significantly by ZA treatment, indicating that angiogenesis inhibition may contribute to the development of MRONJ but does not play a key role.

Effects of Bisphosphonates on Osseointegration of Dental Implants in Rabbit Model.

This study is to investigate the effect of bisphosphonates on the osseointegration of dental implants in a rabbit model. Twenty female New Zealand White rabbits were equally assigned into control and experiment groups which received saline or zoledronic acid treatment 4 weeks prior to surgery. Titanium dental implant was placed on the calvarial bone. Zoledronic acid or saline treatment continued after surgery for 4 weeks (short-term subgroup) or 8 weeks (long-term subgroup) until sacrifice. Three different fluorochrome labeling solutions were administrated for assessing bone growth rates. Samples of the calvarial bone and mandible were subjected to microcomputed tomography (micro-CT), confocal microscope, and histology analysis. Zoledronic acid treatment significantly reduced bone growth rates in the calvarial bone, but had no significant influence in bone mineral density and trabecular microarchitecture. Significantly lower bone-to-implant contact ratios were found in zoledronic acid-treated animals compared to controls at week 4 but not at week 8. Oncologic dose zoledronic acid suppresses the bone growth rates of the calvarial bone; ZA may have an adverse effect on osseointegration of dental implant in short term, but this effect tends to diminish in long term.

Effect of Periapical Diseases in Development of MRONJ in Immunocompromised Mouse Model.

OBJECTIVES: This study aimed to assess the effect of zoledronic acid on an immunocompromised mice model with periapical disease. MATERIALS AND METHODS: Thirty C57BL/6N mice were randomly divided into three groups (N = 10). All animals were subjected to bilateral ovariectomy (OVX) and then treated with saline (Veh), zoledronic acid (ZA), or concomitant zoledronic acid and dexamethasone (ZA/Dx) for 12 weeks. Eight weeks after starting drug administration, pulpal exposure was conducted on the lower left first molar. Four weeks after pulpal exposure, all mice were sacrificed and the mandibles were collected for radiological and histological examinations. RESULTS: Microcomputed tomography (µ-CT) examination showed significantly reduced periapical bone resorption in the ZA/Dx group and decreased periodontal bone resorption in both ZA and ZA/Dx groups. Higher bone mineral density (BMD) and strengthened microstructure were found in ZA and ZA/Dx groups. More empty lacunae were found in ZA and ZA/Dx groups. CONCLUSIONS: Apical periodontitis aggravates MRONJ under immunocompromised circumstances. Concurrent use of ZA and steroids inhibits alveolar bone resorption but increases the risk of developing MRONJ.

Skeletal Site-Specific Response of Jawbones and Long Bones to Surgical Interventions in Rats Treated with Zoledronic Acid.

Bisphosphonates (BPs) have been extensively used for management of bone diseases with pathologically high resorption. Despite the great clinical benefits, a severe complication known as medication-related osteonecrosis of the jaw (MRONJ) has been reported. It is found that most of the reported MRONJ cases were limited in the jawbones/craniofacial bones instead of long bones. The present study aims to investigate the differential bone response to surgical procedures between jawbones and long bones exposed to BPs. Forty-eight skeletal mature Sprague Dawley female rats were administered oncologic dose of zoledronic acid (ZA) or normal saline for 4 weeks and then subjected to tooth extraction on the mandible and maxilla, and a bone defect creation on the femur. After surgical procedures, ZA or saline treatment were continued until sacrifice at week 2, week 4, and week 8, post-operatively. The samples were subjected to micro-computerized tomography (micro-CT) and histological assessment. Osteonecrosis was only found in jawbones in ZA-treated rats. ZA-treated rats showed significantly higher bone mineral density with greater bone volume in all surgical sites than that in the controls. The length of exposure of ZA did not seem to affect trabecular microstructure, and it only showed higher bone volume and BMD with longer healing time which is expected in the healing process.

Differences between epithelial and mesenchymal human tongue cancer cell lines in experimental metastasis.

Distant metastasis represents the outcome with the worst prognosis for various types of malignant tumors, but little is known regarding the impact of interacting epithelial and mesenchymal phenotypic cancer cells within its etiopathogenesis. In a novel animal model, 48 male athymic Balb/c nude mice underwent subcutaneous and intravenous injection of human tongue cancer cell lines of green fluorescent mesenchymal and red fluorescent epithelial phenotypes, in order to visualize and monitor eventual phenotypic interaction in lung metastasis as well as experimental metastasis in in vivo, ex vivo and histopathological analyses. While the epithelial, but not the mesenchymal, phenotypic human tongue cancer cell line led to direct metastasis in the lungs when injected intravenously, neither of them, even when injected in combination, were able to establish distant metastasis. The results of the present study provide evidence regarding the role of epithelial phenotypic cancer cells in the release of experimental metastasis following tail vein injection in male athymic Balb/c nude mice, in addition to proving fluorescent human tongue cancer cells may be reliably detected under a fluorescence microscope even 8 weeks after the two injection types.

Role of Periapical Diseases in Medication-Related Osteonecrosis of the Jaws.

OBJECTIVE: The present study aimed to investigate the role of periapical diseases in inducing medication-related osteonecrosis of the jaws (MRONJ) using an ovariectomized (OVX) mice model. MATERIALS AND METHODS: Twenty C57BL/6N female mice were randomly assigned to two groups. All mice were subjected to bilateral ovariectomy and then treated with oncologic dose of zoledronic acid (ZA) or vehicle for twelve weeks. Eight weeks after commence of drug administration, a pulpal exposure (PE) operation was performed on the first right lower molar to induce periapical periodontitis; the contralateral non-PE tooth was used as control. All animals were sacrificed four weeks after pulpal exposure, and the mandibles were harvested for radiological and histomorphometrical analysis. RESULTS: Micro computed tomography (µ-CT) examination demonstrated that periapical diseases significantly increased alveolar bone resorption, and the resorption was greatly attenuated by ZA treatment. Concurrent ZA therapy significantly increased bone density and histological osteocyte necrosis in the presence of periapical lesions. CONCLUSION: ZA treatment reduced bone absorption resulting from periapical disease but increased the risk of developing MRONJ in the ovariectomized mouse model.

Tumorigenicity and Validity of Fluorescence Labelled Mesenchymal and Epithelial Human Oral Cancer Cell Lines in Nude Mice.

Tumorigenicity and metastatic activity can be visually monitored in cancer cells that were labelled with stable fluorescence. The aim was to establish and validate local and distant spread of subcutaneously previously injected fluorescence transduced human tongue cancer cell lines of epithelial and mesenchymal phenotype in nude mice. A total of 32 four-week-old male athymic Balb/c nude mice were randomly allocated into 4 groups (n = 8). A single dose of 0.3 mL PBS containing 1 × 107 of four different cancer cell-lines (UM1, UM1-GFP, UM2, and UM2-RFP) was injected subcutaneously into the right side of their posterolateral back. Validity assessment of the labelled cancer cells' tumorigenicity was assessed by physical examination, imaging, and histology four weeks after the injection. The tumor take rate of cancer cells was similar in animals injected with either parental or transduced cancer cells. Transduced cancer cells in mice were easily detectable in vivo and after cryosection using fluorescent imaging. UM1 cells showed increased tumor take rate and mean tumor volume, presenting with disorganized histopathological patterns. Fluorescence labelled epithelial and mesenchymal human tongue cancer cell lines do not change in tumorigenicity or cell phenotype after injection in vivo.

Lysophosphatidic acid upregulates connective tissue growth factor expression in osteoblasts through the GPCR/PKC and PKA pathways.

Lysophosphatidic acid (LPA) is an efficient, bioactive phospholipid involved in various biological processes. In this study, LPA-induced connective tissue growth factor (CTGF/CCN2) expression and the underlying mechanisms were investigated using the MC3T3-E1 cell line. The MC3T3-E1 cells were stimulated with an inhibitor of LPA receptors, an activator and inhibitor of protein kinase C (PKC) and protein kinase A (PKA) for indicated periods of time. RT-qPCR and western blot analyses were used to measure the expression levels of CCN2. Immunofluorescence staining was used to observe the translocation of PKC. The mRNA expression level of CCN2 was increased following stimulation of the cells with LPA; LPA transiently induced the mRNA expression of CCN2; maximum expression levels were observed 2 h following stimulation with LPA. This increase was accompanied by CCN2 protein synthesis. LPA receptor1/3 was inhibited by Ki16425, a specific inhibitor of LPA1/3; as a result, the LPA-induced increase in CCN2 expression was abrogated. LPA also induced the membrane translocation of PKC and enhanced PKC activity in the osteoblasts. Pre-treatment of the osteoblasts with staurosporine prevented the increase in CCN2 expression by induced by LPA, and the activation of PKC by phorbol 12-myristate 13-acetate (PMA) enhanced CCN2 expression, indicating that the PKC pathway is involved in the LPA-induced increase in CCN2 expression. The interference of PKA signaling also led to the induction of CCN2 expresion by LPA. These data indicate that LPA increases CCN2 expression through the activation of PKC and PKA. Thus, the regulatory functions of the PKC and PKA pathways are implicated in the LPA-induced increase in CCN2 expression.