Osteonecrosis of the Jaws (ONJ), a significant side effect of anti-resorptive or anti-angiogenic medications, is described as exposed bone in the oral cavity that often presents with pain, infection, and often significantly decreases quality of life of affected patients. Largely seen in patients taking anti-resorptives for osteoporosis, bone malignancies, and bone metastases, the pathophysiology of ONJ has remained largely elusive. Additionally, treatment options for the disease have remained empirical, often focused on managing symptomology. Others have recommended surgical resection of the affected region, often leaving large defects that can be devastating. Here, we develop an ONJ animal model in rats, harness its power to investigate the effects of anti-resorptive discontinuation. Additionally, we explore whether other medications with possible anti-resorptive properties can lead to development of ONJ. Finally, we evaluate clinical data to better understand ONJ pathophysiology, as well as ONJ treatment.

Following clinical observations, where tooth extraction occurs only in diseased teeth, such as those with periapical or periodontal disease, we hypothesized that significant periapical disease prior to tooth extraction will lead to ONJ. Thus, we treated rats with high-dose bisphosphonates, and extracted teeth with experimental periapical disease (EPD). All BP-na�ve animals healed uneventfully. Interestingly, only animals treated with BPs with extraction of teeth with EPD developed clinical, radiographic, and histologic signs of ONJ. Our findings here show that pre-existing periapical disease is necessary to induce ONJ in rats treated with BPs.

Using the animal model described above, we investigated with discontinuation of BPs or the RANK-L inhibitor, OPG-Fc (a denosumab surrogate for rodents), is time or drug dependent. First, we observed that discontinuation of BPs prior to tooth extraction did not ameliorate ONJ burden, while discontinuation of OPG-Fc prior to tooth extraction ameliorated ONJ burden. In contrast, discontinuation of BPs or OPG-Fc in rats with established ONJ did not lead to resolution. These findings have direct clinical correlation and can be used for the development of clinical guidelines.

With our understanding of ONJ and its relationship to systemic anti-resorptive use, we investigated whether treatment with romosozumab, a monoclonal antibody against sclerostin used for the treatment of osteoporosis, would lead to ONJ. In clinical studies, romosozumab led to decreased bone resorption markers; thus, while not a purely catabolic agent, it may have the potential to cause ONJ. Using an established model of experimental periodontitis (EP) in ovariectomized rats, we found that treatment with romosozumab did not lead to ONJ.

Driven by these translational findings, we evaluated a cohort of patients with osteopetrosis and pycnodysostosis, inherited genetic diseases that lead to an anti-resorptive like phenotype. In these patients, we observed areas of exposed, non-healing bone that were phenotypically identically to patients with ONJ from anti-resorptive use. Radiographic and histologic findings confirmed findings; in-vitro resorption pit assays revealed an absence of osteoclastic resorptive activity in these patients.

Finally, we retrospectively evaluated a cohort of ONJ patients that had been treated with local wound care. In these patients, we found that over 75% of patients healed with bone sequestration, leaving healed mucosa on the underlying surface. We found that healing time correlated with their wound care abilities, with patients with better wound care exfoliating the necrotic bone prior to those with poor wound care.

The jaw and long bone (LB) are part of bones that make up the skeleton. Although the jaw is, to some extent, analogous to long bone and other bones of the body, they have a distinct developmental origin and mode of ossification. Systemic diseases affect the jaw differently compared to other bones, and the existence of jaw-specific bone pathologies suggests that it employs a different bone homeostatic mechanism. Marrow cells are the functioning orchestrators that maintain the balance between bone formation and bone resorption. The apparent distinction of mandible (MB) and its specific diseases justify that the understanding of MB cell functions and differentiation cannot be concluded from studies of other bone sites. Thus our overall objective is to study MB vs. LB marrow cell characteristics and functions. We, first, established a protocol for rat MB marrow cell isolation, including bone marrow stromal cells (BMSCs) and osteoclast (OC) precursors. Characterization of BMSCs uncovered an enhanced ability of the MB vs. LB BMSCs to induce bone formation both in vitro and in vivo. Taking molecular differences into consideration, we assessed the potential clinical relevance of the MB vs. LB BMSC bone regeneration potential in the critical-sized intramembranous calvarial and endochondral femoral defects. MB BMSCs could regenerate both types of bone and produced better quality bone in intramembranous bone defects. Next, differences in osteoclastogenesis were investigated. Our data demonstrate that although the MB marrow contains an increased number of OC precursors, under parathyroid hormone and 1,25 dihydroxyvitaminD3 stimulation, the LB marrow has a higher osteoclastogenic potential. This appears to be, at least in part, due to the higher RANKL stimulation and OPG inhibition of LB vs. MB BMSCs by these hormones. The differences in the MB vs. LB at the cellular level elucidate the existence of jaw specific diseases, particularly bisphosphonate-related osteonecrosis of the jaw (BRONJ). The jaw intrinsically has reduced osteoclast formation ability and cannot counteract the inhibitory effect of bisphosphonates to reestablish the normal bone resorption process. The increased sensitivity in nature of the jaw to antiresorptive treatments could explain, at least in part, the pathophysiology of BRONJ and its exclusive clinical manifestations in the jaw bone.

Medication related osteonecrosis of the jaws (MRONJ) is a severe adverse effect of antiresorptive and angiogenic medication prescribed to patients with osteoporosis or bone malignancies. Our laboratory has been working on the elucidation of the mechanisms of this disease for the past decade. In this dissertation, we further characterized pathogenesis in rodents and humans and explored the delivery of RANKL as a therapeutic approach.

Tooth extraction was performed on diseased or healthy teeth in mice treated with saline, OPG-Fc (the equivalent of denosumab in rodents) or bisphosphonates. Extraction of healthy teeth did not suffice to induce MRONJ in mice treated with antiresorptives. In contrast, extraction of diseased teeth induced mucosal defects, absence of radiographic healing, periosteal reaction, significant osteonecrosis and bone exposure in mice treated with antiresorptives. Extraction of periodontally diseased teeth in zoledronate (ZA) treated rats resulted in altered socket healing with a significant amount of osteonecrosis. An increased signal of collagen type III, MMP-9, MMP-13 and α-SMA was seen in the sockets of these rats compared to saline treated rats or zoledronate treated rats with extraction of healthy teeth.

We also took advantage of our patient database to investigate the radiographic appearance of stage 0 MRONJ patients. We concluded that sclerosis, cortical erosion, periosteal reaction, sequestration and crater-like defect are more commonly present in stage 0 MRONJ patients as opposed to dental disease patients. Additionally, if a stage 0 MRONJ patient presents with radiographic sequestration at initial visit, they are at a higher risk to progress to frank bone exposure in less than a year.

Next, we explored local RANKL delivery as a potential therapy for MRONJ. RANKL was delivered in the sockets of ZA or saline treated rats in a collagen tape. In the extraction sockets of ZA treated rats with RANKL treatment, there was an improvement in mucosal and radiographic healing, a decrease in osteonecrosis and bone exposure and a simultaneous increase in osteoclast numbers compared to the ZA treated rats that had not received local RANKL treatment.

We also investigated the role of macrophages in MRONJ development. In a model of periodontitis, zoledronate treated mice demonstrated a predominance of M1 macrophages (macs) and an absence of M2 macs compared to the saline treated mice. The above M1 macrophages also expressed MMP-13. Rosiglitazone treatment reversed the M1/M2 ratio in ZA treated mice 2 weeks after periodontal disease was induced and reduced epithelium to crest distance in ZA mice. However, a difference was not detected in the bone exposure incidence, the epithelium to crest distance or the osteonecrotic areas between ZA mice with or without rosiglitazone treatment. Our data provide insight to the pathogenesis of MRONJ, describe the radiographic profile of stage 0 MRONJ patients and suggest that local RANKL delivery may be efficient in minimizing MRONJ incidence and improving socket healing.

Bisphosphonates (BPs) have been used clinically for over 30 years to treat postmenopausal osteoporosis, hypercalcemia of malignancy and metabolic bone diseases. Although the use of BPs have been beneficial for these pathologies, several recent reports have linked BP usage to osteonecrosis of the jaw. A common factor amongst these case reports suggests a significant link of dental disease and bisphosphonate treatment in the development of bisphosphonate related osteonecrosis of the jaw (BRONJ). Since the pathophysiology of BRONJ is unknown, we developed two BRONJ animal models where aggressive periodontal and periapical disease was induced with zoledronic acid (ZA) treatment in the rat and mouse, respectively.

Ligature-induced periodontal disease caused significant alveolar bone loss while ZA treatment attenuated this effect in rats. Significant changes of alveolar bone morphology including sequestration and extensive periosteal bone formation were observed by microCT in the ligated site of BP treated animals. These findings were confirmed histologically, demonstrating 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.

Mandibular tooth drillings causing pulpal exposure in the right first and second mandibular molars caused significant periapical lesion development in the mouse. Periapical lesion were quantified from radiographic images and showed a significant increase in lesion space in vehicle versus ZA treated mice. Significant qualitative differences were seen in the morphology of the alveolar bone in ZA treated mice through microCT analysis. Periosteal bone formation was significantly increased in periapical disease induced ZA treated mice versus control mice. Extensive necrotic bone areas were demonstrated the absence of lacunae in osteocytes. TRAP staining shows no significant difference in the number of osteoclasts between ZA versus vehicle treated animals. However, significant morphologic differences were observed by the detachment osteoclasts and flattening of the ruffled border in ZA treated mice. BRONJ developed in 100% of periapical disease induced ZA treated mice.

Our data suggest that dental disease and BP therapy is sufficient and necessary for BRONJ development. These animal models can serve as tools to understand the pathophysiology of BRONJ.

Objectives: The human craniofacial skeleton demonstrates considerable phenotypic variation. Half of all birth defects involve the skull and face, with the mandible being very commonly affected in a variety of syndromes. Heritability and genes responsible for craniofacial phenotypes are poorly understood and studied due to the complexity of the region and confounding environmental effects. The objective of this project is to assess and identify regions of significant variation in shape of the human mandible by utilizing reliable methods of morphometric analysis, particularly in 3D. The long-term goal of this project is to characterize genetic determinants of craniofacial phenotype. Three specific aims are proposed:

1) Define the parameters for each of the three skeletal growth patterns.

2) Analyze mandibular shapes of the skeletal patterns using morphometric methods to identify differences among the three groups.

3) Verify and establish methodology of salivary DNA collection and extraction for future down-stream applications.

Methods: Pre-treatment CBCTs of forty-five adult patients from the Orthodontic Clinic at the UCLA School of Dentistry were analyzed for measurement of craniofacial traits, 15 cases for each of the three skeletal growth patterns – brachyfacial, mesofacial, and dolichofacial. Inclusion criteria encompassed healthy adult non-growing patients at least 18 years old, male or female. Exclusion criteria comprised syndromic patients, alveolar/palatal cleft patients, individuals with hemimandibular or hemifacial asymmetry, history of orthodontic treatment involving orthopedic movement, and systemic disease that affects bone metabolism. All subjects had a high-resolution head CBCT scan obtained prior to orthodontic treatment as part of their diagnosis and treatment planning. The axial image slices were reconstructed and converted into DICOM format then exported into modified orthodontic analysis software for craniofacial analysis. Each hemimandible was segmented from the whole craniofacial structure, and three-dimensional anatomical landmarks were chosen to best describe its shape. Procrustes alignment of the landmark points was performed for both principal component analysis (PCA) and elliptical Fourier function analysis (EFFA) to identify where the variation in shape occurs between the averages of each of the three groups. Statistical significance was examined with ANOVA and Tukey’s HSD test. Extraction of high-quality genomic DNA of subjects was begun for future genome-wide association studies (GWAS) utilizing a non-invasive method of saliva collection and a DNA isolation kit. The purified DNA has been stored in -80?C for future experiments.

Results: Statistically significant area of variation between the three skeletal growth patterns was found with Procrustes and principal component analysis, and the results were further substantiated with elliptical Fourier function analysis. Additional trends were evident from the elliptical Fourier analysis. Target human gene was amplified and visualized on gels upon collection of saliva and DNA extraction following a troubleshot protocol.

Conclusions: Landmark-based analytical methods as the industry standard in morphometrics are useful in identifying shape differences. However, coupling with outline-based morphometrics is an even more powerful and informative tool in elucidating significant areas of variation in shape. Whole saliva can be effectively utilized for human DNA collection and isolation for future gene studies.

INTRODUCTION: Cortical bone microarchitecture exhibits a level of plasticity which adapts to changes in health status. In periodontal disease, bone loss can occur as a result of bacterial plaque induced inflammation around teeth. As we learn more about the mechanisms driving periodontal bone loss, one aspect that is not well understood are the changes in cortical bone and their role in alveolar bone remodeling. Previous studies utilizing long bones have recognized the importance of cortical bone microarchitecture in understanding disease pathogenesis, and preliminary evidence suggests similarities may exist in jaw bones as well.  OBJECTIVE: The purpose of this �CT study is to identify and characterize transcortical canal porosities in rat jaws, and to evaluate the anatomical changes in response to experimental periodontitis.

MATERIAL AND METHODS: 14 eight-week-old, Wistar Han rats underwent ligature placement, utilizing 4-0 silk sutures ligated around the left maxillary second molars, and 28-gauge stainless-steel wire around the left mandibular first molars. Contralateral molars served as non-ligated control teeth in each animal. Animals were monitored to ensure ligature presence, and randomly assigned to euthanasia 2 or 4-weeks after ligature placement. Maxillae and mandibles were trimmed, placed in 10% formalin for 48-hours, then stored in 70% ethanol for �CT imaging at 15�m and 5�m resolution. CTan imaging software was used to quantify vertical bone loss circumferentially at 15�m resolution. A global threshold was applied to segment transcortical canal spaces at 5�m resolution, in order to quantify number and size of canals.

RESULTS: At 15�m resolution, three-dimensional reconstructions of the buccal cortical bone exhibited increased porosity in the presence of ligature-induced experimental periodontitis, over healthy non-ligature molars. Further analysis of selected binarized slices at 5�m resolution, revealed an increase in number and volumetric percentage of intracortical porosities in experimental periodontitis molars compared to non-ligature controls.

CONCLUSIONS: Our observations demonstrate a complex network of canals exists within the cortical jaw bones of healthy rats. After 2-weeks of experimental periodontitis, an increase in the size and number of transcortical canals was observed, indicating that cortical bone morphology is highly dynamic in response to oral disease. These results enhance our current understanding of bone microstructure and disease driven adaptation. Our ongoing studies are directed at further interrogating the role of intracortical canals in oral inflammatory bone remodeling.

Purpose

Materials and methods

Results

Conclusion

Objectives: Current methodologies in the field of morphometrics still employ the use of 2-dimensional images followed by a landmark-based Procrustes superimposition method to evaluate differences in shape, which can be tedious, subject to operator error, and fail to capture the true nature of shape variation between samples. The primary objective of this study is to address these limitations through the exploration and application of current methodologies used in neuroimaging and brain mapping to the field of morphometrics. By collaborating with Paul Thompson and his team at the Laboratory of Neuroimaging (LONI), we aim to:

1. Generate a 3D surface image of the mouse mandible and create an average surface for a cross sectional sample of parental strains in the Hybrid Mouse Diversity Panel (HMDP), as well as a developing longitudinal sample within a single strain.

2. Apply and modify the latest technologies used in brain mapping research to identify regional shape differences between the mandibles of these samples in 3 dimensions without the use of landmark identification, with the ultimate goal of identifying heritable quantitative traits.

3. Develop a useful, intuitive and visual method for evaluation of shape differences.

Methods: A total of fifty-three mice were studied. Ten mice, equally divided across five parental inbred strains, were obtained from the HMDP, as well as an additional 43 animals at various ages from the C57BL/6 strain. Skulls were subsequently scanned using μCT, then DICOM files transferred into a Beta version of Dolphin Imaging ® 11.7 Software. Hemi-mandibles were segmented from the entire skull and surface meshes were created using the same software. Topological correction of each hemi-mandible was performed before parametric registration and resampling of the surfaces to the same number of mesh points. Once registered to each other, a population average was then created and used as a reference template for shape comparison. Shapes were compared using the independently validated neuroimaging techniques of medial axis and tensor-based morphometrics.

Results: Population averages were created for the mandibles and four specific areas of significant shape change were identified in both the interstrain and intrastrain samples. Visual heat maps were also created to display shape differences.

Conclusions: Tensor-based morphometric evaluation of mandibular shape has far superior visualization and localization potential when compared to the current method using landmark-based analysis, while offering a more reliable solution as it eliminates the need for consistent landmark placement. Coupled with medial thickness computation, followed by additional refinement, this methodology could potentially be applied to a variety of applications concerned with evaluating shape difference in three dimensions.

Objectives: Current analyses of 3D cone beam computed tomography (CBCT) images used in orthodontic diagnosis and treatment planning still rely upon 2D linear and angular measures, along with subjective visual evaluation, to assess complex facial disharmony and other dentofacial characteristics. The overall objective of this investigation is to overcome these limitations by developing and testing protocol for mapping the surface of the human skull in three dimensions using CBCT images. In collaboration with the Laboratory of Neuroimaging (LONI) at UCLA, the specific goals of this project are:

-To apply and modify advanced technology used in brain mapping research to accurately and efficiently map the skull surface in 3D

-To apply and modify existing mathematical functions to find the average of multiple skull surfaces

-And to develop protocol for superimposing a sample skull on the average skull model, yielding a color-coded map of surface deviation and dysmorphology

Methods: Active patient files in the UCLA Section of Orthodontics were reviewed for the presence of a NewTom ® 3G CBCT scan. The search resulted in 67 patient scans that met the inclusion criteria for the study. CBCT files in DICOM format were first uploaded into a Beta version of Dolphin Imaging ® 11.5 Software. The surface was then segmented using a triangular mesh approach, after which the file was exported to MeshLab ® for viewing and to remove undesired nodes. Topology of each skull was corrected using a principal axis star map and smoothing of the extrapolated regions. Shapes were mapped to a sphere using conformal and area preserving maps, and were then registered using a spherical patch mapping approach. Finally an average was created using 7-parameter procrustes alignment.

Results: Size-standardized and non-size standardized average skull models were successfully created for the 67 patient sample. Color-coded displacement maps were generated for a sample patient to demonstrate the potential clinical applicability of this protocol.

Conclusions: The results of this investigation suggest that it is possible to average multiple shapes of highly variable topology such as the human skull. The most immediate application of this research will be rapid and detailed diagnostic imaging analysis for orthodontic and surgical treatment planning, particularly in complex cases involving congenital facial anomalies, facial asymmetry, and trauma. There is also great potential for application to areas outside orthodontics such as anthropometrics and genomics.