Interspecies Comparison of Alveolar Bone Biology, Part I: Morphology and Physiology of Pristine Bone.

INTRODUCTION: Few interspecies comparisons of alveolar bone have been documented, and this knowledge gap raises questions about which animal models most accurately represent human dental conditions or responses to surgical interventions. OBJECTIVES: The objective of this study was to employ state-of-the-art quantitative metrics to directly assess and compare the structural and functional characteristics of alveolar bone among humans, mini pigs, rats, and mice. METHODS: The same anatomic location (i.e., the posterior maxillae) was analyzed in all species via micro-computed tomographic imaging, followed by quantitative analyses, coupled with histology and immunohistochemistry. Bone remodeling was evaluated with alkaline phosphatase activity and tartrate-resistant acid phosphatase staining to identify osteoblast and osteoclast activities. In vivo fluorochrome labeling was used as a means to assess mineral apposition rates. RESULTS: Collectively, these analyses demonstrated that bone volume differed among the species, while bone mineral density was equal. All species showed a similar density of alveolar osteocytes, with a highly conserved pattern of collagen organization. Collagen maturation was equal among mouse, rat, and mini pig. Bone remodeling was a shared feature among the species, with morphologically indistinguishable hemiosteonal appearances, osteocytic perilacunar remodeling, and similar mineral apposition rates in alveolar bone. CONCLUSIONS: Our analyses demonstrated equivalencies among the 4 species in a plurality of the biological features of alveolar bone. Despite contradictory results from older studies, we found no evidence for the superiority of pig models over rodent models in representing human bone biology. KNOWLEDGE TRANSFER STATEMENT: Animal models are extensively used to evaluate bone tissue engineering strategies, yet there are few state-of-the-art studies that rigorously compare and quantify the factors influencing selection of a given animal model. Consequently, there is an urgent need to assess preclinical animal models for their predictive value to dental research. Our article addresses this knowledge gap and, in doing so, provides a foundation for more effective standardization among animal models commonly used in dentistry.

Osteoporotic Changes in the Periodontium Impair Alveolar Bone Healing.

Osteoporosis is associated with decreased bone density and increased bone fragility, but how this disease affects alveolar bone healing is not clear. The objective of this study was to determine the extent to which osteoporosis affects the jaw skeleton and then to evaluate possible mechanisms whereby an osteoporotic phenotype might affect the rate of alveolar bone healing following tooth extraction. Using an ovariectomized mouse model coupled with micro-computed tomographic imaging, histologic, molecular, and cellular assays, we first demonstrated that the appendicular and jaw skeletons both develop osteoporotic phenotypes. Next, we demonstrated that osteoporotic mice exhibit atrophy of the periodontal ligament (PDL) and that this atrophy was accompanied by a reduction in the pool of osteoprogenitor cells in the PDL. The paucity of PDL-derived osteoprogenitor cells in osteoporotic mice was associated with significantly slower extraction socket healing. Collectively, these analyses demonstrate that the jaw skeleton is susceptible to the untoward effects of osteoporosis that manifest as thinner, more porous alveolar bone, PDL thinning, and slower bone repair. These findings have potential clinical significance for older osteopenic patients undergoing reconstructive procedures.

Biomechanics of Immediate Postextraction Implant Osseointegration.

The aim of this study was to gain insights into the biology and mechanics of immediate postextraction implant osseointegration. To mimic clinical practice, murine first molar extraction was followed by osteotomy site preparation, specifically in the palatal root socket. The osteotomy was positioned such that it removed periodontal ligament (PDL) only on the palatal aspect of the socket, leaving the buccal aspect undisturbed. This strategy created 2 distinct peri-implant environments: on the palatal aspect, the implant was in direct contact with bone, while on the buccal aspect, a PDL-filled gap existed between the implant and bone. Finite element modeling showed high strains on the palatal aspect, where bone was compressed by the implant. Osteocyte death and bone resorption predominated on the palatal aspect, leading to the loss of peri-implant bone. On the buccal aspect, where finite element modeling revealed low strains, there was minimal osteocyte death and robust peri-implant bone formation. Initially, the buccal aspect was filled with PDL remnants, which we found directly provided Wnt-responsive cells that were responsible for new bone formation and osseointegration. On the palatal aspect, which was devoid of PDL and Wnt-responsive cells, adding exogenous liposomal WNT3A created an osteogenic environment for rapid peri-implant bone formation. Thus, we conclude that low strain and high Wnt signaling favor osseointegration of immediate postextraction implants. The PDL harbors Wnt-responsive cells that are inherently osteogenic, and if the PDL tissue is healthy, it is reasonable to preserve this tissue during immediate implant placement.

Wnt-Responsive Odontoblasts Secrete New Dentin after Superficial Tooth Injury.

The objective of our experiments was to identify new therapeutic strategies to stimulate dentin formation in an adult tooth. To address this objective, we evaluated dentin production in 2 acute trauma models: one involving a pulp exposure and the other involving a superficial dentin injury. Molecular, cellular, and histologic analyses revealed that in response to a severe injury, where the pulp is exposed to the oral cavity, cell death is rampant and the repair response initiates from surviving pulp cells and, to a lesser extent, surviving odontoblasts. When an injury is superficial, as in the case of a dentin injury model, then disturbances are largely confined to pulp tissue immediately underneath the damaged dentin tubules. We found that the pulp remained vital and innervated; primary odontoblasts upregulated HIF1α; and the rate of mineralization was significantly increased. A tamoxifen-inducible Axin2CreERT2/+; R26R mTmG/+ reporter strain was then used to demonstrate that a population of long-lived Wnt-responsive odontoblasts, which secreted dentin throughout the life of the animal, were responsible for depositing new dentin in response to a superficial injury. Amplifying Wnt signaling in the pulp stimulates dentin secretion, and in the dentin injury model, we show that a liposomal formulation of human WNT3A protein passes through dentinal tubules and is capable of upregulating Wnt signaling in the pulp. These data provide strong proof of concept for a therapeutic pulp-capping material to stimulate Wnt signaling in odontoblasts and thus improve the pulp repair response.

A Wnt-Responsive PDL Population Effectuates Extraction Socket Healing.

Stem cells residing in the periodontal ligament (PDL) support the homeostasis of the periodontium, but their in vivo identity, source(s), and function(s) remain poorly understood. Here, using a lineage-tracing mouse strain, we identified a quiescent Wnt-responsive population in the PDL that became activated in response to tooth extraction. The Wnt-responsive population expanded by proliferation, then migrated from the PDL remnants that remained attached to bundle bone, into the socket. Once there, the Wnt-responsive progeny upregulated osteogenic protein expression, differentiated into osteoblasts, and generated the new bone that healed the socket. Using a liposomal WNT3A protein therapeutic, we showed that a single application at the time of extraction was sufficient to accelerate extraction socket healing 2-fold. Collectively, these data identify a new stem cell population in the intact periodontium that is directly responsible for alveolar bone healing after tooth removal.

A Comparative Assessment of Implant Site Viability in Humans and Rats.

Our long-term objective is to devise methods to improve osteotomy site preparation and, in doing so, facilitate implant osseointegration. As a first step in this process, we developed a standardized oral osteotomy model in ovariectomized rats. There were 2 unique features to this model: first, the rats exhibited an osteopenic phenotype, reminiscent of the bone health that has been reported for the average dental implant patient population. Second, osteotomies were produced in healed tooth extraction sites and therefore represented the placement of most implants in patients. Commercially available drills were then used to produce osteotomies in a patient cohort and in the rat model. Molecular, cellular, and histologic analyses demonstrated a close alignment between the responses of human and rodent alveolar bone to osteotomy site preparation. Most notably in both patients and rats, all drilling tools created a zone of dead and dying osteocytes around the osteotomy. In rat tissues, which could be collected at multiple time points after osteotomy, the fate of the dead alveolar bone was followed. Over the course of a week, osteoclast activity was responsible for resorbing the necrotic bone, which in turn stimulated the deposition of a new bone matrix by osteoblasts. Collectively, these analyses support the use of an ovariectomy surgery rat model to gain insights into the response of human bone to osteotomy site preparation. The data also suggest that reducing the zone of osteocyte death will improve osteotomy site viability, leading to faster new bone formation around implants.

Poleward transport of TPX2 in the mammalian mitotic spindle requires dynein, Eg5, and microtubule flux.

TPX2 is a Ran-regulated spindle assembly factor that is required for kinetochore fiber formation and activation of the mitotic kinase Aurora A. TPX2 is enriched near spindle poles and is required near kinetochores, suggesting that it undergoes dynamic relocalization throughout mitosis. Using photoactivation, we measured the movement of PA-GFP-TPX2 in the mitotic spindle. TPX2 moves poleward in the half-spindle and is static in the interzone and near spindle poles. Poleward transport of TPX2 is sensitive to inhibition of dynein or Eg5 and to suppression of microtubule flux with nocodazole or antibodies to Kif2a. Poleward transport requires the C terminus of TPX2, a domain that interacts with Eg5. Overexpression of TPX2 lacking this domain induced excessive microtubule formation near kinetochores, defects in spindle assembly and blocked mitotic progression. Our data support a model in which poleward transport of TPX2 down-regulates its microtubule nucleating activity near kinetochores and links microtubules generated at kinetochores to dynein for incorporation into the spindle.

Peripheral, non-centrosome-associated microtubules contribute to spindle formation in centrosome-containing cells.

In centrosome-containing cells, microtubules utilized in spindle formation are thought to be nucleated at the centrosome. However, spindle formation can proceed following experimental destruction of centrosomes or in cells lacking centrosomes, suggesting that non-centrosome-associated microtubules may contribute to spindle formation, at least when centrosomes are absent. Direct observation of prometaphase cells expressing GFP-alpha-tubulin shows that peripheral, non-centrosome-associated microtubules are utilized in spindle formation, even in the presence of centrosomes. Clusters of peripheral microtubules moved into the centrosomal region, demonstrating that a centrosomal microtubule array can be composed of both centrosomally nucleated and peripheral microtubules. Peripheral bundles also moved laterally into the forming spindle between the spindle poles; 3D reconstructions of fixed cells reveal interactions between peripheral and centrosome-associated microtubules. The spindle pole component NuMA and gamma-tubulin were present at the foci of peripheral microtubule clusters, indicating that microtubules moved into the spindle with minus ends leading. Photobleach- and photoactivation-marking experiments of cells expressing GFP-tubulin or a photoactivatable variant of GFP-tubulin, respectively, demonstrate that microtubule motion into the forming spindle results from transport and sliding interactions, not treadmilling. Our results directly demonstrate that non-centrosome-associated microtubules contribute to spindle formation in centrosome-containing cells.