Sotirios Tetradis

Position statement of the American Academy of Oral and Maxillofacial Radiology on selection criteria for the use of radiology in dental implantology with emphasis on cone beam computed tomography

A Position Paper Subcommittee of the American Academy of Oral and Maxillofacial Radiology (AAOMR) reviewed the literature since the original position statement on selection criteria for radiology in dental implantology, published in 2000. All current planar modalities, including intraoral, panoramic, and cephalometric, as well as cone beam computed tomography (CBCT) are discussed, along with radiation dosimetry and anatomy considerations. We provide research-based, consensus-derived clinical guidance for practitioners on the appropriate use of specific imaging modalities in dental implant treatment planning. Specifically, the AAOMR recommends that cross-sectional imaging be used for the assessment of all dental implant sites and that CBCT is the imaging method of choice for gaining this information. This document will be periodically revised to reflect new evidence.

Osteonecrosis of the Jaw in a Patient on Denosumab

Osteonecrosis of the jaws (ONJ) clinically presents as exposed, necrotic bone in the maxilla or mandible of at least eight week duration, with or without the presence of pain, infection, or previous trauma in a patient who has not received radiation to the jaws (1–3). Although necrotic bone exposure has been reported in the jaws of a variety of patients not receiving bisphosphonates (BPs;4–9), the number of BP related ONJ cases has continued to steadily increase since the first report in 2003 (10). To date, a direct causal relationship between BP use and ONJ has not been established (11,12). However, many retrospective and prospective analyses have identified cases of ONJ where BP therapy, especially the more potent intravenous preparations, was the only consistent variable, strongly suggesting that BPs play a significant role in ONJ pathophysiology (13–24). Potential mechanisms underlying bisphosphonate related osteonecrosis of the jaws (BRONJ) pathophysiology have generated great debate in the literature (25,26). It is not surprising that many hypotheses attempt to explain the unique localization of BRONJ exclusively to the jaws, including altered bone remodeling, angiogenesis inhibition, constant microtrauma, soft tissue BP toxicity, and bacterial infection (15,18,25,27–29). Importantly, ONJ incidence correlation with BP potency suggests that inhibition of osteoclast function and differentiation might be a key factor in the pathophysiology of the disease. Currently other inhibitors of osteoclast differentiation and function are entering the pharmacologic armamentarium for the treatment of diseases with increased bone turnover. The association of these new therapies with ONJ is uncertain. We report a case of ONJ in a patient receiving Denosumab, a human RANKL monoclonal antibody currently in clinical trials for the treatment of osteoporosis, primary and metastatic bone cancer, giant cell tumor, and rheumatoid arthritis (30–33).

Oxysterols Regulate Differentiation of Mesenchymal Stem Cells: Pro-Bone and Anti-Fat†

Pluripotent mesenchymal stem cells can undergo lineage‐specific differentiation in adult organisms. However, understanding of the factors and mechanisms that drive this differentiation is limited. We show the novel ability of specific oxysterols to regulate lineage‐specific differentiation of mesenchymal stem cells into osteogenic cells while inhibiting their adipogenic differentiation. Such effects may have important implications for intervention with osteoporosis.

Periodontal disease and bisphosphonates induce osteonecrosis of the jaws in the rat

Bisphosphonates (BPs) are medications used commonly to treat primary and metastatic bone cancer, as well as osteoporosis. Although BPs improve bone mineral density, reduce fracture risk, and reduce hypercalcemia of malignancy, some patients develop BP‐related osteonecrosis of the jaws (BRONJ). This devastating complication is defined as clinically exposed bone in the maxillofacial region for more than 8 weeks. Despite an increasing number of BRONJ cases since first reported, the disease pathophysiology remains largely unknown. Since published studies suggest a significant role for dental disease in the pathophysiology of BRONJ, we developed a BRONJ animal model where aggressive periodontal disease is induced by ligature placement around the crown of the right maxillary first molar in the presence of vehicle (veh) or zoledronic acid (ZA), a potent BP. Ligature placement induced significant alveolar bone loss, which was attenuated by ZA treatment. Osteonecrosis was observed associated with ligature‐induced periodontitis in the ZA‐treated group. This was seen as sequestration and extensive periosteal alveolar bone formation on micro–computed tomography (µCT) in the ligated site of BP‐treated animals. Histologic examination confirmed these findings, seen as necrotic bone with diffuse loss of osteocytes and empty lacunae, rimming of the necrotic bone by squamous epithelium and inflammation, and exposure to the oral cavity. Importantly, the rat lesions were strikingly similar to those of BRONJ patients. Our data suggest that dental disease and potent BP therapy are sufficient for BRONJ development in the rat.

Parathyroid Hormone Regulates the Expression of Fibroblast Growth Factor-2 mRNA and Fibroblast Growth Factor Receptor mRNA in Osteoblastic Cells

We examined the effect of parathyroid hormone (PTH) on basic fibroblast growth factor‐2 (FGF‐2) and FGF receptor (FGFR) expression in osteoblastic MC3T3‐E1 cells and in neonatal mouse calvariae. Treatment of MC3T3‐E1 cells with PTH(1–34) (10–8M) or forskolin (FSK; 10–5M) transiently increased a 7 kb FGF‐2 transcript with a peak at 2 h. The PTH increase in FGF‐2 mRNA was maintained in the presence of cycloheximide. PTH also increased FGFR‐1 mRNA at 2 h and transiently increased FGFR‐2 mRNA at 1 h. FGFR‐3 and FGFR‐4 mRNA transcripts were not detected in MC3T3‐E1 cells. In cells transiently transfected with an 1800‐bp FGF‐2 promoter‐luciferase reporter, PTH and FSK increased luciferase activity at 2 h and 4 h. Immunohistochemistry showed that PTH and FSK increased FGF‐2 protein labeling in the nuclei of MC3T3‐E1 cells. PTH also increased FGF‐2 mRNA, and FGFR‐1 and FGFR‐2 mRNA levels within 30 minutes in neonatal mouse calvarial organ cultures. We conclude that PTH and cAMP stimulate FGF‐2 mRNA abundance in part through a transcriptional mechanism. PTH also regulated FGFR gene expression. We hypothesize that some effects of PTH on bone remodeling may be mediated by regulation of FGF‐2 and FGFR expression in osteoblastic cells.

Reproducible and inexpensive probe preparation for oligonucleotide arrays

We present a new protocol for the preparation of nucleic acids for microarray hybridization. DNA is fragmented quantitatively and reproducibly by using a hydroxyl radical-based reaction, which is initiated by hydrogen peroxide, iron(II)-EDTA and ascorbic acid. Following fragmentation, the nucleic acid fragments are densely biotinylated using a biotinylated psoralen analog plus UVA light and hybridized on microarrays. This non-enzymatic protocol circumvents several practical difficulties associated with DNA preparation for microarrays: the lack of reproducible fragmentation patterns associated with enzymatic methods; the large amount of labeled nucleic acids required by some array designs, which is often combined with a limited amount of starting material; and the high cost associated with currently used biotinylation methods. The method is applicable to any form of nucleic acid, but is particularly useful when applying double-stranded DNA on oligonucleotide arrays. Validation of this protocol is demonstrated by hybridizing PCR products with oligonucleotide-coated microspheres and PCR amplified cDNA with Affymetrix Cancer GeneChip microarrays.

Application of cone beam computed tomography for assessment of the temporomandibular joints

Radiographic examination is essential for the diagnosis and management of temporomandibular joint (TMJ) disorders. The goals of TMJ radiography are to evaluate cortical and trabecular architecture of the bony structures and confirm their integrity, to assess the extent and monitor progression of osseous changes, and to evaluate the response to treatment. Accurate evaluation of the TMJ by conventional radiography is limited by structure superimposition. Cone beam computed tomography (CBCT) provides high-resolution multiplanar images and delivers substantially lower radiation dose, compared with multislice CT. CBCT allows examination of TMJ anatomy without superimposition and distortion to facilitate analysis of bone morphology, joint space and dynamic function in all three dimensions. This article will describe the role of CBCT imaging for the assessment of the TMJ osseous structures and present typical appearances of common pathological conditions of the TMJ.

Osteogenic Potential of Mandibular vs. Long-bone Marrow Stromal Cells

Although fundamentally similar to other bones, the jaws demonstrate discrete responses to developmental, mechanical, and homeostatic regulatory signals. Here, we hypothesized that rat mandible vs. long-bone marrow-derived cells possess different osteogenic potential. We established a protocol for rat mandible and long-bone marrow stromal cell (BMSC) isolation and culture. Mandible BMSC cultures formed more colonies, suggesting an increased CFU-F population. Both mandible and long-bone BMSCs differentiated into osteoblasts. However, mandible BMSCs demonstrated augmented alkaline phosphatase activity, mineralization, and osteoblast gene expression. Importantly, upon implantation into nude mice, mandible BMSCs formed 70% larger bone nodules containing three-fold more mineralized bone compared with long-bone BMSCs. Analysis of these data demonstrates an increased osteogenic potential and augmented capacity of mandible BMSCs to induce bone formation in vitro and in vivo. Our findings support differences in the mechanisms underlying mandible homeostasis and the pathophysiology of diseases unique to the jaws.

Periapical disease and bisphosphonates induce osteonecrosis of the jaws in mice.

Osteonecrosis of the jaw (ONJ) is a well‐recognized complication of antiresorptive medications, such as bisphosphonates (BPs). Although ONJ is most common after tooth extractions in patients receiving high‐dose BPs, many patients do not experience oral trauma. Animal models using tooth extractions and high BP doses recapitulate several clinical, radiographic, and histologic findings of ONJ. We and others have reported on rat models of ONJ using experimental dental disease in the absence of tooth extraction. These models emphasize the importance of dental infection/inflammation for ONJ development. Here, we extend our original report in the rat, and present a mouse model of ONJ in the presence of dental disease. Mice were injected with high dose zoledronic acid and pulpal exposure of mandibular molars was performed to induce periapical disease. After 8 weeks, quantitative and qualitative radiographic and histologic analyses of mouse mandibles were done. Periapical lesions were larger in vehicle‐treated versus BP‐treated mice. Importantly, radiographic features resembling clinical ONJ, including thickening of the lamina dura, periosteal bone deposition, and increased trabecular density, were seen in the drilled site of BP‐treated animals. Histologically, osteonecrosis, periosteal thickening, periosteal bone apposition, epithelial migration, and bone exposure were present in the BP‐treated animals in the presence of periapical disease. No difference in tartrate‐resistant acid phosphatase (TRAP)+ cell numbers was observed, but round, detached, and removed from the bone surface cells were present in BP‐treated animals. Although 88% of the BP‐treated animals showed areas of osteonecrosis in the dental disease site, only 33% developed bone exposure, suggesting that osteonecrosis precedes bone exposure. Our data further emphasize the importance of dental disease in ONJ development, provide qualitative and quantitative measures of ONJ, and present a novel mouse ONJ model in the absence of tooth extraction that should be useful in further exploring ONJ pathophysiological mechanisms.

Adverse Effects of Hyperlipidemia on Bone Regeneration and Strength

Hyperlipidemia increases the risk for generation of lipid oxidation products, which accumulate in the subendothelial spaces of vasculature and bone. Atherogenic high‐fat diets increase serum levels of oxidized lipids, which are known to attenuate osteogenesis in culture and to promote bone loss in mice. In this study, we investigated whether oxidized lipids affect bone regeneration and mechanical strength. Wild‐type (WT) and hyperlipidemic (Ldlr−/−) mice were placed on a high‐fat (HF) diet for 13 weeks. Bilateral cranial defects were introduced on each side of the sagittal suture, and 5 weeks postsurgery on the respective diets, the repair/regeneration of cranial bones and mechanical properties of femoral bones were assessed. MicroCT and histological analyses demonstrated that bone regeneration was significantly impaired by the HF diet in WT and Ldlr−/− mice. In femoral bone, cortical bone volume fraction (bone volume [BV]/tissue volume [TV]) was significantly reduced, whereas cortical porosity was increased by the HF diet in Ldlr−/− but not in WT mice. Femoral bone strength and stiffness, measured by three‐point bending analysis, were significantly reduced by the HF diet in Ldlr−/−, but not in WT mice. Serum analysis showed that the HF diet significantly increased levels of parathyroid hormone, tumor necrosis factor (TNF)‐α, calcium, and phosphorus, whereas it reduced procollagen type I N‐terminal propeptide, a serum marker of bone formation, in Ldlr−/−, but not in WT mice. The serum level of carboxyl‐terminal collagen crosslinks, a marker for bone resorption, was also 1.7‐fold greater in Ldlr−/− mice. These findings suggest that hyperlipidemia induces secondary hyperparathyroidism and impairs bone regeneration and mechanical strength.