Analytical methodology to measure periodontal bone morphometry following orthodontic tooth movement in mice.

INTRODUCTION: Basic research in orthodontics is commonly conducted in rodents. However, experimental studies on orthodontic tooth movement (OTM) lack a standard method to examine OTM and periodontal changes. This study describes a unifying protocol for the analysis of OTM and associated bone microarchitectural changes in mice using microcomputed tomography (µCT). METHODS: Mice (10 animals/group) were divided into control and OTM groups. OTM was generated by anchoring a nickel-titanium closed-coil spring to the upper incisors to pull the upper left first molar. A third group of TNFα -/- mice was added since these are known to have slower OTM. Using µCT, we implemented and tested a number of methods to measure OTM distance and examine 3D bone morphometric parameters associated with OTM in mice. RESULTS: In total, we tested five methods to measure the OTM distance in mice. The results indicated that measuring the intermolar diastema, and assessing tooth movement relative to the anterior root of the zygomatic arch, displayed the lowest standard deviation and enabled optimal detection of intergroup differences. We also developed two protocols for µCT analysis of the periradicular bone that yielded no false-positive results. Our results revealed that including the width of the periodontal ligament rather than excluding it from the region of interest in mice detected more statistically significant differences in the morphometric parameters between the OTM and control sides and between WT and TNFα -/- mice despite more subtle differences. CONCLUSIONS: We, therefore, propose new guidelines for a standardized µCT-based method to analyse OTM and the extent of the periradicular bone structural changes in mice.

Ablation of the mammalian lectin galectin-8 induces bone defects in mice.

Mice overexpressing galectin-8 [gal-8 transgenic (Tg)], a secreted mammalian lectin, exhibit enhanced bone turnover and reduced bone mass, similar to cases of postmenopausal osteoporosis. Here, we show that gal-8 knockout (KO) mice have increased bone mass accrual at a young age but exhibit accelerated bone loss during adulthood. These phenotypes can be attributed to a gal-8-mediated increase in receptor activator of NF-κB ligand (RANKL) expression that promotes osteoclastogenesis, combined with direct inhibition of osteoblast differentiation, evident by reduced bone morphogenetic protein (BMP) signaling, reduced phosphorylation of receptor regulated mothers against decapentaplegic homolog (R-SMAD) and reduced expression of osteoblast differentiation markers osterix, osteocalcin, runt-related transcription factor 2 (RUNX2), dentin matrix acidic phosphoprotein-1 (DMP1), and alkaline phosphatase. At the same time, gal-8 promotes expression of estrogen receptor α (ESR1). Accordingly, the rate of bone loss is accelerated in ovariectomized, estrogen-deficient gal-8 Tg mice, whereas gal-8 KO mice, having low levels of ESR1, are refractory to ovariectomy. Finally, gal-8 mRNA positively correlates with the mRNA levels of osteoclastogenic markers RANKL, tartrate-resistant acid phosphatase, and cathepsin K in human femurs. Collectively, these findings identify gal-8 as a new physiologic player in the regulation of bone mass.-Vinik, Y., Shatz-Azoulay, H., Hiram-Bab, S., Kandel, L., Gabet, Y., Rivkin, G., Zick, Y. Ablation of the mammalian lectin galectin-8 induces bone defects in mice.

A new device for improving dental implants anchorage: a histological and micro-computed tomography study in the rabbit.

OBJECTIVE: In the present study, a new healing cap that could generate a pulsed electromagnetic field (PEMF) around titanium implants to stimulate peri-implant osteogenesis was tested in the rabbit model. MATERIALS AND METHODS: A total of 22 implants were inserted in the proximal tibial metaphysis of 22 rabbits. A healing cap containing the active device was inserted in half of the implants (11 test implants); an "empty" healing cap was inserted in the other ones (11 control implants). The animals were euthanized after 2 and 4 weeks, and the samples were processed for micro-computed tomography and histology. The peri-implant volume was divided into coronal (where the PEMF was the strongest) and apical regions. RESULTS: Most of the effects of the tested device were confined to the coronal region. Two weeks post-implantation, test implants showed a significant 56% higher trabecular bone fraction (BV/TV), associated with enhanced trabecular number (Tb.N, +37%) and connectivity density (Conn.D, +73%) as compared to the control group; at 4 weeks, the PEMF induced a 69% increase in BV/TV and 34% increase of Tb.N. There was no difference in the trabecular thickness (Tb.Th) at either time point. Furthermore, we observed a 48% higher bone-to-implant contact (BIC) in the test implants vs. controls after 2 weeks; this increase tended to remain stable until the fourth week. Mature trabecular and woven bone were observed in direct contact with the implant surface with no gaps or connective tissue at the bone-implant interface. CONCLUSIONS: These results indicate that the PEMF device stimulated early bone formation around dental implants resulting in higher peri-implant BIC and bone mass already after 2 weeks which suggests an acceleration of the osseointegration process by more than three times.

Orthodontic force and extracorporeal shock wave therapy: Assessment of orthodontic tooth movement and bone morphometry in a rat model.

OBJECTIVE: The objective was to investigate the effect of extracorporeal shock wave therapy (ESWT) on the magnitude of orthodontic tooth movement, in a rat model, based on a previously established treatment protocol. DESIGN: In conjunction with orthodontic force commencement, rats underwent ESWT. The amount of tooth movement along with different microarchitectural parameters were measured after three weeks by means of microcomputed tomography. In addition, the percentage of cells expressing vascular endothelial growth factor, the number of tartrate-resistant acid phosphatase (TRAP) positive cells/area and blood vessel density were evaluated both for the pressure and tension sides. RESULTS: The addition of ESWT to the orthodontic force after three weeks more than doubled the average tooth movement. The addition of ESWT on the pressure side induced a significant decrease in volumetric bone mineral density. Blood vessel density and the number of TRAP positive cells were higher after the application of ESWT. CONCLUSION: The induction of ESWT during orthodontic tooth movement in a rat model increases the rate of tooth movement by accelerating bone resorption on the pressure side and possibly enhances bone formation on the tension side.

Microcomputed Tomography-Based Analysis of Neovascularization within Bioengineered Vascularized Tissues.

In the field of tissue engineering, evaluating newly formed vascular networks is considered a fundamental step in deciphering the processes underlying tissue development. Several common modalities exist to study vessel network formation and function. However, a proper methodology that allows through three-dimensional visualization of neovessels in a reproducible manner is required. Here, we describe in-depth exploration, visualization, and analysis of vessels within newly formed tissues by utilizing a contrast agent perfusion protocol and high-resolution microcomputed tomography. Bioengineered constructs consisting of porous, biocompatible, and biodegradable scaffolds are loaded with cocultures of adipose-derived microvascular endothelial cells (HAMECs) and dental pulp stem cells (DPSCs) and implanted in a rat femoral bundle model. After 14 days of in vivo maturation, we performed the optimized perfusion protocol to allow host penetrating vascular visualization and assessment within neotissues. Following high-resolution microCT scanning of DPSC:HAMEC explants, we performed the volumetric and spatial analysis of neovasculature. Eventually, the process was repeated with a previously published coculture system for prevascularization based on adipose-derived mesenchymal stromal cells (MSCs) and HAMECs. Overall, our approach allows a comprehensive understanding of vessel organization during engraftment and development of neotissues.

The Balance between Orthodontic Force and Radiation in the Jawbone: Microstructural, Histological, and Molecular Study in a Rat Model.

Irradiation of facial bones is associated with a lifelong risk of osteonecrosis. In a rat model, maxillae were exposed to a single 5 Gy dose of external beam radiation and orthodontic force was applied for 2 weeks on the first maxillary molar; control rats were treated identically without radiation. Tooth movement in irradiated jaws was 30% less than in controls, representing radiation-related damage. Micro-CT, histological, and molecular outcomes of orthodontic tooth movement were studied. Microstructurally, bone parameters (trabecular thickness, bone volume fraction, bone mineral density) were significantly affected by orthodontic force but not by radiation. Histological parameters were influenced only by orthodontic force, especially by an increase in osteoclasts. A molecular study revealed a differential distribution of cells expressing pre-osteoclast markers (RANK+-majority, CD11b+, CD14+-minority), with changes being influenced by orthodontic force (increased CD11b+ and CD14+ cells) and also by radiation (decreased RANK+ cells). The activation status of osteoclasts (TRAP staining) showed an orthodontic-force-related increase, which probably could not fully compensate for the radiation-associated impairment. The overall balance showed that orthodontic force had elicited a substantial microstructural, histological, and functional normalization process in irradiated maxillae but a radiation-induced impact was still conspicuous. Additional studies are needed to validate these findings.

Therapeutic Potential of Vasoactive Intestinal Peptide and its Derivative Stearyl-Norleucine-VIP in Inflammation-Induced Osteolysis.

The common use of dental and orthopedic implants calls for special attention to the immune response leading to peri-prosthetic bone loss and implant failure. In addition to the well-established microbial etiology for oral implant failure, wear debris and in particular titanium (Ti) particles (TiP) in the implant vicinity are an important trigger of inflammation and activation of bone resorption around oral and orthopedic implants, presenting an unmet medical need. Here, we employed bacterial-derived lipopolysaccharides (LPS) to model infection and TiP to model aseptic inflammation and osteolysis. We assessed inflammation in vitro by measuring IL1ß, IL6 and TNFα mRNA expression in primary macrophages, osteoclastogenesis in RANKL-induced bone marrow derived pre-osteoclasts and osteolysis in vivo in a mouse calvarial model. We also assessed the trans-epithelial penetrability and safety of the tested compound in rats. Our results show that a lipophilic super-active derivative of vasoactive intestinal peptide (VIP), namely stearyl-norleucine-VIP (SNV) presented superior anti-inflammatory and anti-osteoclastogenic effects compared to VIP in vitro. In the bacterial infection model (LPS), SNV significantly reduced IL1ß expression, while VIP increased IL6 expression. In the aseptic models of osteolysis, SNV showed greater suppression of in vitro osteoclastogenesis than VIP, and significantly inhibited inflammation-induced osteolysis in vivo. We also observed that expression levels of the VIP receptor VPAC-2, but not that of VPAC-1, dramatically decreased during osteoclast differentiation. Importantly, SNV previously shown to have an increased stability compared to VIP, showed here significant trans-epithelial penetration and a clean toxicological profile, presenting a novel drug candidate that could be applied topically to counter both aseptic and infection-related bone destruction.

Effects of Extracorporeal Shock Wave Therapy on Distraction Osteogenesis in Rat Mandible.

BACKGROUND: Distraction osteogenesis has widespread clinical use in the treatment of congenital and acquired craniofacial deformities. Nonetheless, during the prolonged consolidation period, the newly regenerated bone carries the risk of complications. A known method for enhancing bone healing is extracorporeal shock wave therapy, which has been shown to induce neovascularization and promote tissue regeneration. The authors investigated whether extracorporeal shock wave therapy can accelerate bony consolidation and regeneration in distraction osteogenesis of the rat mandible and at which stage of distraction osteogenesis it should be applied. METHODS: Twenty-four male Sprague-Dawley rats were subjected to distraction osteogenesis of the right mandible (latency period, 3 days; distraction period, 10 days; 0.5 mm/day). Experimental groups consisted of the following: group I (control), no extracorporeal shock wave therapy; group II, extracorporeal shock wave therapy (0.18 mJ/mm(2)) at the latency period; and group III, extracorporeal shock wave therapy (0.18 mJ/mm(2)) at the consolidation period. Explants were removed for evaluation after 4 weeks of consolidation. RESULTS: Histologic evaluation showed well-developed cortical cortex and a higher degree of bone formation and mature bone in group III; micro-computed tomography showed significantly increased bone mineral density, bone volume fraction, and trabecular thickness; immunohistochemistry demonstrated significantly increased expression of bone morphogenetic protein-2, vascular endothelial growth factor, and proliferating cell nuclear antigen. CONCLUSION: Extracorporeal shock wave therapy application at the consolidation period during distraction osteogenesis in the rat mandible enhances bone formation and osteogenic and angiogenic growth factors, improves bone mechanical properties, and accelerates bone mineralization.

Mechanism and Prevention of Titanium Particle-Induced Inflammation and Osteolysis.

The worldwide number of dental implants and orthopedic prostheses is steadily increasing. Orthopedic implant loosening, in the absence of infection, is mostly attributable to the generation of wear debris. Dental peri-implantitis is characterized by a multifactorial etiology and is the main cause of implant failure. It consists of a peri-implant inflammatory lesion that often results in loss of supporting bone. Disease management includes cleaning the surrounding flora by hand instruments, ultrasonic tips, lasers, or chemical agents. We recently published a paper indicating that US scaling of titanium (Ti) implants releases particles that provoke an inflammatory response and osteolysis. Here we show that a strong inflammatory response occurs; however, very few of the titanium particles are phagocytosed by the macrophages. We then measured a dramatic Ti particle-induced stimulation of IL1ß, IL6, and TNFα secretion by these macrophages using multiplex immunoassay. The particle-induced expression profile, examined by FACS, also indicated an M1 macrophage polarization. To assess how the secreted cytokines contributed to the paracrine exacerbation of the inflammatory response and to osteoclastogenesis, we treated macrophage/preosteoclast cultures with neutralizing antibodies against IL1ß, IL6, or TNFα. We found that anti-TNFα antibodies attenuated the overall expression of both the inflammatory cytokines and osteoclastogenesis. On the other hand, anti-IL1ß antibodies affected osteoclastogenesis but not the paracrine expression of inflammatory cytokines, whereas anti-IL6 antibodies did the opposite. We then tested these neutralizing antibodies in vivo using our mouse calvarial model of Ti particle-induced osteolysis and microCT analysis. Here, all neutralizing antibodies, administered by intraperitoneal injection, completely abrogated the particle-induced osteolysis. This suggests that blockage of paracrine inflammatory stimulation and osteoclastogenesis are similarly effective in preventing bone resorption induced by Ti particles. Blocking both the inflammation and osteoclastogenesis by anti-TNFα antibodies, incorporated locally into a slow-release membrane, also significantly prevented osteolysis. The osteolytic inflammatory response, fueled by ultrasonic scaling of Ti implants, results from an inflammatory positive feedback loop and osteoclastogenic stimulation. Our findings suggest that blocking IL1ß, IL6, and/or TNFα systemically or locally around titanium implants is a promising therapeutic approach for the clinical management of peri-implant bone loss.

Scaling of titanium implants entrains inflammation-induced osteolysis.

With millions of new dental and orthopedic implants inserted annually, periprosthetic osteolysis becomes a major concern. In dentistry, peri-implantitis management includes cleaning using ultrasonic scaling. We examined whether ultrasonic scaling releases titanium particles and induces inflammation and osteolysis. Titanium discs with machined, sandblasted/acid-etched and sandblasted surfaces were subjected to ultrasonic scaling and we physically and chemically characterized the released particles. These particles induced a severe inflammatory response in macrophages and stimulated osteoclastogenesis. The number of released particles and their chemical composition and nanotopography had a significant effect on the inflammatory response. Sandblasted surfaces released the highest number of particles with the greatest nanoroughness properties. Particles from sandblasted/acid-etched discs induced a milder inflammatory response than those from sandblasted discs but a stronger inflammatory response than those from machined discs. Titanium particles were then embedded in fibrin membranes placed on mouse calvariae for 5 weeks. Using micro-CT, we observed that particles from sandblasted discs induced more osteolysis than those from sandblasted/acid-etched discs. In summary, ultrasonic scaling of titanium implants releases particles in a surface type-dependent manner and may aggravate peri-implantitis. Future studies should assess whether surface roughening affects the extent of released wear particles and aseptic loosening of orthopedic implants.

Parathyroid hormone 1-34 enhances titanium implant anchorage in low-density trabecular bone: a correlative micro-computed tomographic and biomechanical analysis.

The use of endosseous titanium implants is the standard of care in dentistry and orthopaedic surgery. Nevertheless, implantation in low-density bone has a poor prognosis and experimental studies show delayed implant anchorage following gonadectomy-induced bone loss. Intermittently administered human parathyroid hormone 1-34 [iahPTH(1-34)] is the leading bone anabolic therapy. Hence, this study assessed whether iahPTH(1-34) enhances titanium implant integration in low-density bone. Threaded titanium implants, 0.9 mm in diameter, were inserted horizontally into the proximal tibial metaphysis of 5-month-old rats, 7 weeks postorchiectomy (ORX). Subcutaneous administration of iahPTH(1-34), at 5, 25 and 75 microg/kg/day commenced immediately thereafter and lasted for 8 weeks. Quantitative micro-computed tomography (muCT) at the implantation site was carried out at 15 microm resolution using high energy and long integration time to minimize artifacts resulting from the high implant radiopacity. Osseointegration (OI) was calculated as percent implant surface in contact with bone (%OI) quantified as the ratio of "bone"-to-total voxels in contact with the implant. Additionally, the trabecular bone volume density (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and connectivity density (Conn.D) were measured in the peri-implant bone. All microCT parameters were stimulated by iahPTH(1-34) dose-dependently; the percent maximal enhancement was %OI = 143, BV/TV = 257, Tb.Th = 150, Tb.N = 140 and Conn.D = 193. The maximal values of %OI, BV/TV and Tb.Th in iahPTH(1-34)-treated ORX rats exceeded significantly those measured in the implantation site of untreated sham-ORX controls. The same specimens were then subjected to pullout biomechanical testing. The biomechanical parameters were also enhanced by iahPTH(1-34) dose-dependently, exceeding the values recorded in the sham-ORX controls. The percent iahPTH(1-34)-induced maximal enhancement was: ultimate force = 315, stiffness = 270 and toughness = 395. Except for the BV/TV and Tb.Th, there was no significant difference between the effect of the 25 and 75 microg/kg/day doses. There was a highly significant correlation between the morphometric and biomechanical parameters suggesting the use of quantitative CT as predictive of the implant mechanical properties. These findings demonstrate that iahPTH(1-34) effectively stimulates implant anchorage in low-density trabecular bone and thus the feasibility of administering iahPTH(1-34) to improve the clinical prognosis in low-density trabecular bone sites.

Three-dimensional quantification of alveolar bone loss in Porphyromonas gingivalis-infected mice using micro-computed tomography.

BACKGROUND: Animal models are routinely used for the study of the pathogenesis of periodontal disease. However, some of the methods used for evaluating alveolar bone loss are limited to linear or two dimension measurements, while other methods, such as histology, are labor consuming. The present study was designed to evaluate a novel method for assessing the volume of alveolar bone loss in mice and to compare it to the traditional morphometric (linear) technique. METHODS: Seven- to 8-week-old BALB/c mice were divided into three equal groups of six each; two groups were infected orally with Porphyromonas gingivalis (P. gingivalis) 381 or 53,977, while the third group was used as non-infected control. Forty-two days following infection, serum samples were obtained and maxillae were harvested. Bone loss was evaluated by micro-computed tomography (micro-CT) and by the morphometric technique. RESULTS: Significant decrease in residual supportive bone volumes (RSBV) was observed in both P. gingivalis-infected groups compared to the control group (P<0.0001). The P. gingivalis 53,977-infected group showed less residual supportive bone volume compared to the P. gingivalis 381-infected group, but there were no significant differences between these two groups. Using the morphometric technique, the differences between the three groups were not found to be statistically significant (P>0.05). CONCLUSIONS: The present study describes a novel micro-CT technique for volumetric evaluation of alveolar bone loss in mice. This technique is relatively simple and accurate, and due to its high sensitivity, enables the investigator to reduce the number of animals needed in each experimental group.

Quantification of in vitro wear of a synthetic meniscus implant using gravimetric and micro-CT measurements.

A synthetic meniscus implant was recently developed for the treatment of patients with mild to moderate osteoarthritis with knee pain associated with medial joint overload. The implant is distinctively different from most orthopedic implants in its pliable construction, and non-anchored design, which enables implantation through a mini-arthrotomy without disruption to the bone, cartilage, and ligaments. Due to these features, it is important to show that the material and design can withstand knee joint conditions. This study evaluated the long-term performance of this device by simulating loading for a total of 5 million gait cycles (Mc), corresponding to approximately five years of service in-vivo. All five implants remained in good condition and did not dislodge from the joint space during the simulation. Mild abrasion was detected by electron microscopy, but µ-CT scans of the implants confirmed that the damage was confined to the superficial surfaces. The average gravimetric wear rate was 14.5 mg/Mc, whereas volumetric changes in reconstructed µ-CT scans point to an average wear rate of 15.76 mm(3)/Mc (18.8 mg/Mc). Particles isolated from the lubricant had average diameter of 15 µm. The wear performance of this polycarbonate-urethane meniscus implant concept under ISO-14243 loading conditions is encouraging.

New approach to quantifying developmental variation in the dentition using serial microtomographic imaging.

Dental morphogenesis and cellular differentiation are expressed in the fully formed tooth by the topography of the dentin-enamel junction and outer enamel surface. These boundaries can be differentiated using a variety of imaging systems. In this study, we used serial microCT imaging to provide accurate 3D reconstructions of developing lower human second deciduous molars. These were used to quantify the volume of enamel and dentin of individual cusps in relation to basal crown height. As growth and differentiation proceed apically, the spatial orientation of cusp tips and their bases were used to estimate their order of initiation and coalescence. We found that the order of coalescence differed from the order of initiation. We also found that dentin cusp height and volume as well as rate and quantity of enamel apposition varied along mesio-distal and bucco-lingual axes, and were independent of order of initiation and duration of growth. These results demonstrate that the potential for variation in crown size and form is maintained throughout development. We propose that the microCT model developed in this study constitutes a new approach for the investigation of developmental variation and its contribution to phylogenetic variation expressed in crown form and size.

Endosseous implant anchorage is critically dependent on mechanostructural determinants of peri-implant bone trabeculae.

Low bone mass is highly prevalent among patients receiving endosseous implants. In turn, the implantation prognosis in low-density skeletal sites is poor. However, little is known about the mechanostructural determinants of implant anchorage. Using metabolic manipulations that lead to low bone density and to its rescue, we show here that anchorage is critically dependent on the peri-implant bone (PIB). Titanium implants were inserted horizontally into the proximal tibial metaphysis of adult rats 6 weeks after orchiectomy (ORX) or sham ORX. Systemic intermittent administration of human parathyroid hormone (1-34) [iahPTH(1-34)] or vehicle commenced immediately thereafter for 6 weeks. The bone-implant apparatus was then subjected to image-guided failure assessment, which assesses biomechanical properties and microstructural deformation concomitantly. Anchorage failure occurred mainly in PIB trabeculae, 0.5 to 1.0 mm away from the implant. Mechanically, the anchorage performed poorly in ORX-induced low-density bone, attributable mainly to decreased trabecular number. iahPTH(1-34) rescued the PIB density and implant mechanical function by augmenting trabecular thickness (Tb.Th). However, implant biomechanical properties in low-density bone were relatively insensitive to implant surface treatment that affected only the osseointegration (%bone-implant contact). These results support a model wherein anchorage failure involves buckling of the weakest trabecular struts followed by sequential failure of the stronger trabeculae. Treatment with iahPTH(1-34) induced thicker struts, which were able to delay and even prevent failure of individual elements, thus implicating trabecular thickness as a prime target for enhancing implant anchorage by systemic bone anabolic therapy.