Wnt/ß-catenin Signaling Controls Maxillofacial Hyperostosis.

The roles of Wnt/ß-catenin signaling in regulating the morphology and microstructure of craniomaxillofacial (CMF) bones was explored using mice carrying a constitutively active form of ß-catenin in activating Dmp1-expressing cells (e.g., daßcatOt mice). By postnatal day 24, daßcatOt mice exhibited midfacial truncations coupled with maxillary and mandibular hyperostosis that progressively worsened with age. Mechanistic insights into the basis for the hyperostotic facial phenotype were gained through molecular and cellular analyses, which revealed that constitutively activated ß-catenin in Dmp1-expressing cells resulted in an increase in osteoblast number and an increased rate of mineral apposition. An increase in osteoblasts was accompanied by an increase in osteocytes, but they failed to mature. The resulting CMF bone matrix also had an abundance of osteoid, and in locations where compact lamellar bone typically forms, it was replaced by porous, woven bone. The hyperostotic facial phenotype was progressive. These findings identify for the first time a ligand-independent positive feedback loop whereby unrestrained Wnt/ß-catenin signaling results in a CMF phenotype of progressive hyperostosis combined with architecturally abnormal, poorly mineralized matrix that is reminiscent of craniotubular disorders in humans.

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.

Mechanoadaptive Responses in the Periodontium Are Coordinated by Wnt.

Despite an extensive literature documenting the adaptive changes of bones and ligaments to mechanical forces, our understanding of how tissues actually mount a coordinated response to physical loading is astonishingly inadequate. Here, using finite element (FE) modeling and an in vivo murine model, we demonstrate the stress distributions within the periodontal ligament (PDL) caused by occlusal hyperloading. In direct response, a spatially restricted pattern of apoptosis is triggered in the stressed PDL, the temporal peak of which is coordinated with a spatially restricted burst in PDL cell proliferation. This culminates in increased collagen deposition and a thicker, stiffer PDL that is adapted to its new hyperloading status. Meanwhile, in the adjacent alveolar bone, hyperloading activates bone resorption, the peak of which is followed by a bone formation phase, leading ultimately to an accelerated rate of mineral apposition and an increase in alveolar bone density. All of these adaptive responses are orchestrated by a population of Wnt-responsive stem/progenitor cells residing in the PDL and bone, whose death and revival are ultimately responsible for directly giving rise to new PDL fibers and new bone.

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.

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.

Relationships among Bone Quality, Implant Osseointegration, and Wnt Signaling.

A variety of clinical classification schemes have been proposed as a means to identify sites in the oral cavity where implant osseointegration is likely to be successful. Most schemes are based on structural characteristics of the bone, for example, the relative proportion of densely compact, homogenous (type I) bone versus more trabeculated, cancellous (type III) bone. None of these schemes, however, consider potential biological characteristics of the bone. Here, we employed multiscale analyses to identify and characterize type I and type III bones in murine jaws. We then combined these analytical tools with in vivo models of osteotomy healing and implant osseointegration to determine if one type of bone healed faster and supported osseointegration better than another. Collectively, these studies revealed a strong positive correlation between bone remodeling rates, mitotic activity, and osteotomy site healing in type III bone and high endogenous Wnt signaling. This positive correlation was strengthened by observations showing that the osteoid matrix that is responsible for implant osseointegration originates from Wnt-responsive cells and their progeny. The potential application of this knowledge to clinical practice is discussed, along with a theory unifying the role that biology and mechanics play in implant osseointegration.

Effects of Condensation on Peri-implant Bone Density and Remodeling.

Bone condensation is thought to densify interfacial bone and thus improve implant primary stability, but scant data substantiate either claim. We developed a murine oral implant model to test these hypotheses. Osteotomies were created in healed maxillary extraction sites 1) by drilling or 2) by drilling followed by stepwise condensation with tapered osteotomes. Condensation increased interfacial bone density, as measured by a significant change in bone volume/total volume and trabecular spacing, but it simultaneously damaged the bone. On postimplant day 1, the condensed bone interface exhibited microfractures and osteoclast activity. Finite element modeling, mechanical testing, and immunohistochemical analyses at multiple time points throughout the osseointegration period demonstrated that condensation caused very high interfacial strains, marginal bone resorption, and no improvement in implant stability. Collectively, these multiscale analyses demonstrate that condensation does not positively contribute to implant stability.

Axin2-expressing cells execute regeneration after skeletal injury.

The mammalian skeleton performs a diverse range of vital functions, requiring mechanisms of regeneration that restore functional skeletal cell populations after injury. We hypothesized that the Wnt pathway specifies distinct functional subsets of skeletal cell types, and that lineage tracing of Wnt-responding cells (WRCs) using the Axin2 gene in mice identifies a population of long-lived skeletal cells on the periosteum of long bone. Ablation of these WRCs disrupts healing after injury, and three-dimensional finite element modeling of the regenerate delineates their essential role in functional bone regeneration. These progenitor cells in the periosteum are activated upon injury and give rise to both cartilage and bone. Indeed, our findings suggest that WRCs may serve as a therapeutic target in the setting of impaired skeletal regeneration.

Mechanoresponsive Properties of the Periodontal Ligament.

The periodontal ligament (PDL) functions as an enthesis, a connective tissue attachment that dissipates strains created by mechanical loading. Entheses are mechanoresponsive structures that rapidly adapt to changes in their mechanical loading; here we asked which features of the PDL are sensitive to such in vivo loading. We evaluated the PDL in 4 physiologically relevant mechanical environments, focusing on mitotic activity, cell density, collagen content, osteogenic protein expression, and organization of the tissue. In addition to examining PDLs that supported teeth under masticatory loading and eruptive forces, 2 additional mechanical conditions were created and analyzed: hypoloading and experimental tooth movement. Collectively, these data revealed that the adult PDL is a remarkably quiescent tissue and that only when it is subjected to increased loads--such as those associated with mastication, eruption, and orthodontic tooth movement-does the tissue increase its rate of cell proliferation and collagen production. These data have relevance in clinical scenarios where PDL acclimatization can be exploited to optimize tooth movement.

Multiscale analyses of the bone-implant interface.

Implants placed with high insertion torque (IT) typically exhibit primary stability, which enables early loading. Whether high IT has a negative impact on peri-implant bone health, however, remains to be determined. The purpose of this study was to ascertain how peri-implant bone responds to strains and stresses created when implants are placed with low and high IT. Titanium micro-implants were inserted into murine femurs with low and high IT using torque values that were scaled to approximate those used to place clinically sized implants. Torque created in peri-implant tissues a distribution and magnitude of strains, which were calculated through finite element modeling. Stiffness tests quantified primary and secondary implant stability. At multiple time points, molecular, cellular, and histomorphometric analyses were performed to quantitatively determine the effect of high and low strains on apoptosis, mineralization, resorption, and collagen matrix deposition in peri-implant bone. Preparation of an osteotomy results in a narrow zone of dead and dying osteocytes in peri-implant bone that is not significantly enlarged in response to implants placed with low IT. Placing implants with high IT more than doubles this zone of dead and dying osteocytes. As a result, peri-implant bone develops micro-fractures, bone resorption is increased, and bone formation is decreased. Using high IT to place an implant creates high interfacial stress and strain that are associated with damage to peri-implant bone and therefore should be avoided to best preserve the viability of this tissue.

A pre-clinical murine model of oral implant osseointegration.

Many of our assumptions concerning oral implant osseointegration are extrapolated from experimental models studying skeletal tissue repair in long bones. This disconnect between clinical practice and experimental research hampers our understanding of bone formation around oral implants and how this process can be improved. We postulated that oral implant osseointegration would be fundamentally equivalent to implant osseointegration elsewhere in the body. Mice underwent implant placement in the edentulous ridge anterior to the first molar and peri-implant tissues were evaluated at various timepoints after surgery. Our hypothesis was disproven; oral implant osseointegration is substantially different from osseointegration in long bones. For example, in the maxilla peri-implant pre-osteoblasts are derived from cranial neural crest whereas in the tibia peri-implant osteoblasts are derived from mesoderm. In the maxilla, new osteoid arises from periostea of the maxillary bone but in the tibia the new osteoid arises from the marrow space. Cellular and molecular analyses indicate that osteoblast activity and mineralization proceeds from the surfaces of the native bone and osteoclastic activity is responsible for extensive remodeling of the new peri-implant bone. In addition to histologic features of implant osseointegration, molecular and cellular assays conducted in a murine model provide new insights into the sequelae of implant placement and the process by which bone is generated around implants.

Primary cilia act as mechanosensors during bone healing around an implant.

The primary cilium is an organelle that senses cues in a cell's local environment. Some of these cues constitute molecular signals; here, we investigate the extent to which primary cilia can also sense mechanical stimuli. We used a conditional approach to delete Kif3a in pre-osteoblasts and then employed a motion device that generated a spatial distribution of strain around an intra-osseous implant positioned in the mouse tibia. We correlated interfacial strain fields with cell behaviors ranging from proliferation through all stages of osteogenic differentiation. We found that peri-implant cells in the Col1Cre;Kif3a(fl/fl) mice were unable to proliferate in response to a mechanical stimulus, failed to deposit and then orient collagen fibers to the strain fields caused by implant displacement, and failed to differentiate into bone-forming osteoblasts. Collectively, these data demonstrate that the lack of a functioning primary cilium blunts the normal response of a cell to a defined mechanical stimulus. The ability to manipulate the genetic background of peri-implant cells within the context of a whole, living tissue provides a rare opportunity to explore mechanotransduction from a multi-scale perspective.

Molecular analysis of healing at a bone-implant interface.

While bone healing occurs around implants, the extent to which this differs from healing at sites without implants remains unknown. We tested the hypothesis that an implant surface may affect the early stages of healing. In a new mouse model, we made cellular and molecular evaluations of healing at bone-implant interfaces vs. empty cortical defects. We assessed healing around Ti-6Al-4V, poly(L-lactide-co-D,L,-lactide), and 303 stainless steel implants with surface characteristics comparable with those of commercial implants. Our qualitative cellular and molecular evaluations showed that osteoblast differentiation and new bone deposition began sooner around the implants, suggesting that the implant surface and microenvironment around implants favored osteogenesis. The general stages of healing in this mouse model resembled those in larger animal models, and supported the use of this new model as a test bed for studying cellular and molecular responses to biomaterial and biomechanical conditions.

Microstrain fields for cortical bone in uniaxial tension: optical analysis method.

This study employed an optical strain measurement method, called microdisplacements by machine vision photogrammetry (DISMAP), to measure both the global and local strain fields in microtensile specimens of cortical bone subjected to controlled uniaxial tension. The variation of local maximum principal strains was measured within the gauge region of samples as a function of applied tensile stress during testing. High gradients of local strain appeared around microstructural features in stressed bone even while the global strain for the entire gauge region showed a strong linear correlation with increasing tensile stress (r2 = 0.98, p < 0.0001). The highest local strain around microstructural features in bone was 11.5-79.5 times higher than the global strain.

Real-time in vivo loading in the lumbar spine: part 1. Interbody implant: load cell design and preliminary results.

STUDY DESIGN: Instrumented interbody implants were placed into the disc space of a motion segment in two baboons. During the animal's activities, implants directly measured in vivo loads in the lumbar spine by telemetry transmitter. OBJECTIVES: Develop and test an interbody implant-load cell and use the implant to measure directly loads imposed on the lumbar spine of the baboon, a semiupright animal. SUMMARY OF BACKGROUND DATA: In vivo forces in the lumbar spine have been estimated using body weight calculations, moment arm models, dynamic chain models, electromyogram measurements, and intervertebral disc pressure measurements. METHODS: An analytical model was used to determine the force-strain relation in a customized interbody implant. After validation by finite element modeling, strain gauges were mounted onto the implant and connected to a telemetry transmitter. Implants were placed surgically into the L4-L5 disc space of skeletally mature baboons and the transmitter in the flank. After surgery, load data were collected from the animals during activities. Radiographs were taken monthly to assess fusion. RESULTS: The implant-load cell is sufficiently sensitive to monitor dynamic changes in strain and load. During extreme activity, highest measurable strain values were indicative of loads in excess of 2.8 times body weight. CONCLUSIONS: The study technique and technology are efficacious for measuring real-time in vivo loads in the spine. Measuring load on an intradiscal implant over the course of healing provides key information about the mechanics of this process. Loads may be used to indicate performance demands on the intervertebral disc and interbody implants for subsequent implant design.

Biomaterials and biomechanics of oral and maxillofacial implants: current status and future developments.

Research in biomaterials and biomechanics has fueled a large part of the significant revolution associated with osseointegrated implants. Additional key areas that may become even more important--such as guided tissue regeneration, growth factors, and tissue engineering--could not be included in this review because of space limitations. All of this work will no doubt continue unabated; indeed, it is probably even accelerating as more clinical applications are found for implant technology and related therapies. An excellent overall summary of oral biology and dental implants recently appeared in a dedicated issue of Advances in Dental Research. Many advances have been made in the understanding of events at the interface between bone and implants and in developing methods for controlling these events. However, several important questions still remain. What is the relationship between tissue structure, matrix composition, and biomechanical properties of the interface? Do surface modifications alter the interfacial tissue structure and composition and the rate at which it forms? If surface modifications change the initial interface structure and composition, are these changes retained? Do surface modifications enhance biomechanical properties of the interface? As current understanding of the bone-implant interface progresses, so will development of proactive implants that can help promote desired outcomes. However, in the midst of the excitement born out of this activity, it is necessary to remember that the needs of the patient must remain paramount. It is also worth noting another as-yet unsatisfied need. With all of the new developments, continuing education of clinicians in the expert use of all of these research advances is needed. For example, in the area of biomechanical treatment planning, there are still no well-accepted biomaterials/biomechanics "building codes" that can be passed on to clinicians. Also, there are no readily available treatment-planning tools that clinicians can use to explore "what-if" scenarios and other design calculations of the sort done in modern engineering. No doubt such approaches could be developed based on materials already in the literature, but unfortunately much of what is done now by clinicians remains empirical. A worthwhile task for the future is to find ways to more effectively deliver products of research into the hands of clinicians.

In vivo bone response to biomechanical loading at the bone/dental-implant interface.

Since dental implants must withstand relatively large forces and moments in function, a better understanding of in vivo bone response to loading would aid implant design. The following topics are essential in this problem. (1) Theoretical models and experimental data are available for understanding implant loading as an aid to case planning. (2) At least for several months after surgery, bone healing in gaps between implant and bone as well as in pre-existing damaged bone will determine interface structure and properties. The ongoing healing creates a complicated environment. (3) Recent studies reveal that an interfacial cement line exists between the implant surface and bone for titanium and hydroxyapatite (HA). Since cement lines in normal bone have been identified as weak interfaces, a cement line at a bone-biomaterial interface may also be a weak point. Indeed, data on interfacial shear and tensile "bond" strengths are consistent with this idea. (4) Excessive interfacial micromotion early after implantation interferes with local bone healing and predisposes to a fibrous tissue interface instead of osseointegration. (5) Large strains can damage bone. For implants that have healed in situ for several months before being loaded, data support the hypothesis that interfacial overload occurs if the strains are excessive in interfacial bone. While bone "adaptation" to loading is a long-standing concept in bone physiology, researchers may sometimes be too willing to accept this paradigm as an exclusive explanation of in vivo tissue responses during experiments, while overlooking confounding variables, alternative (non-mechanical) explanations, and the possibility that different types of bone (e.g., woven bone, Haversian bone, plexiform bone) may have different sensitivities to loading under healing vs. quiescent conditions.

Mechanical and morphologic investigation of the tensile strength of a bone-hydroxyapatite interface.

For load-bearing calcium-phosphate biomaterials, it is important to understand the relative contributions of direct physical-chemical bonding vs. mechanical interlocking to interfacial strength. In the limit of a perfectly smooth hydroxyapatite (HA) surface, a tensile test of the bone-HA interface affords an opportunity to isolate the bonding contribution related to HA surface chemistry alone. This study measured the bone-HA interfacial tensile strength for highly polished (approximately 0.05 micron alumina) dense HA disks (5.25 mm in diameter, 1.3 in mm thickness) in rabbit tibiae. Each of five rabbits received four HA disks, two per proximal tibia. Pull-off loads ranged from 3.14 +/- 2.38N at 55 days after implantation to 18.35 +/- 11.9N at 88 days; nominal interfacial tensile strengths were 0.15 +/- 0.11 MPa and 0.85 +/- 0.55 MPa, respectively. SEM of failed interfaces revealed failures between HA and bone, within the HA itself and within adjacent bone. Tissue remnants on HA were identified as mineralized bone with either a lamellar or trabecular structure. Oriented collagen fibers in the bone intricately interdigitated with the HA surface, which frequently showed breakdown at material grain boundaries and a rougher surface than originally implanted. Mechanical interlocking could not be eliminated as a mode of tissue attachment and contribution to bone-HA bonding, even after implanting an extremely smooth HA surface.

A dynamic modal testing technique for noninvasive assessment of bone-dental implant interfaces.

A dynamic modal testing technique has been developed to noninvasively assess the interface surrounding an endosseous dental implant with a lateral tap from an impedance head hammer. The technique assesses the rotational stiffness of the interface based on the shape of the power spectrum of the force-time curve produced on impact. In vitro experiments were performed to determine the sensitivity of the technique for detecting clinically relevant structural differences between interfaces. The modal test data were able to distinguish interfaces based on the type of bone at the interface and the degree of fixation between the implant and the interface.