Resolving differences between animal models for expedited orthodontic tooth movement.

OBJECTIVE: To highlight differences in commonly used animal models of orthodontic tooth movement (OTM). METHODS: Narrative review. RESULTS: A critical review of the literature on animal models has become increasingly important in the last decade as methods to increase the rate of tooth movement have been intensely sought. We provide a review focusing on the rodent and canine models for expedited OTM and describe the challenges in extrapolation of the results to the clinical practice of orthodontics. We review and contrast the two (rodent, canine) most commonly used animal models for OTM. We then describe animal models to study bone adaptation, remodelling and modelling, which are keys events in describing and quantifying OTM. CONCLUSIONS: Understanding the differences between animal models and their adaptation biology as it relates to OTM is key to make valid conclusions. The rodent model can be used to understand the initial phases of tooth movement. Unlike the dog model, the rodent model does not lend itself to understand prolonged adaptation in response to bodily tooth movement. Extrapolation of rate of tooth movement to humans is more challenging from rodent model data.

Preface to COAST 2016 innovators' workshop on personalized and precision orthodontic therapy.

OBJECTIVE: A second focused workshop explored how to transfer novel findings into clinical orthodontic practice. SETTING AND SAMPLE POPULATION: Participants met in West Palm Beach (Florida, USA), on 9-11 September 2016 for the Consortium for Orthodontic Advances in Science and Technology 2016 Innovators' Workshop (COAST). Approximately 65 registered attendees considered and discussed information from 27 to 34 speakers, 8 to 15 poster presenters and four lunch-hour focus group leaders. MATERIAL AND METHODS: The innovators' workshops were organized according to five themed sessions. The aims of the discussion sessions were to identify the following: i) the strength and impact of the evidenced-based discoveries, ii) required steps to enable further development and iii) required steps to translate these new discoveries into orthodontic practice. RESULTS: The role of gene-environment interactions that underlie complex craniofacial traits was the focus of several sessions. It was agreed that diverse approaches are called for, such as (i) large-scale collaborative efforts for future genetic studies of complex traits; (ii) deep genome sequencing to address the issues of isolated mutations; (iii) quantifying epigenetic-environmental variables in diverse areas myofascial pain, alveolar remodelling and mandibular growth. Common needs identified from the themed sessions were multiscale/multispecies modelling and experimentation using controlled and quantified mechanics and translation of the findings in bone biology between species. Panel discussions led to the consensus that a consortium approach to establish standards for intra-oral scanning and 3D imaging should be initiated. CONCLUSIONS: Current and emerging technologies still require supported research to translate new findings from the laboratory to orthodontic practice.

Oncologic doses of zoledronic acid induce site specific suppression of bone modelling in rice rats.

OBJECTIVES: To examine the effect of zoledronic acid (ZOL) on cortical bone modelling and healing of extraction sockets in the jaw bones of a rodent model. We hypothesized ZOL suppresses both the bone formation in the modelling mode in the jaw bones and alters the extraction site healing. MATERIAL & METHODS: Rice rats were administered saline solution and two dose regimens of ZOL: 0.1 mg/kg, twice a week, for 4 weeks (n=17, saline=8 & ZOL=9) and a higher dose of 0.4 mg/kg, weekly, for 9 weeks (n=30, saline=15 & ZOL=15). Two pairs of fluorochrome bone labels were administered. Extraction of maxillary teeth was performed in maxilla. Mineral apposition rate, mineralizing surface and bone formation rate (BFR) were quantified on periodontal (PDL), alveolar and basal bone surfaces, and in the trabecular bone of proximal tibia. Bone volume (BV) was evaluated at extraction sockets. Multivariate Gaussian models were used to account for repeated measurements, and analyzes were conducted in SAS V9.3. RESULTS: ZOL suppressed bone modelling (BFR/BS) at the PDL surfaces in the mandible (P<.05), but its effect was not significant at the periosteal surfaces of both jaws. BV for the healing sockets of ZOL treated animals was not significantly different (P=.07) compared to the saline group. ZOL suppressive effect was higher in the tibia compared to the jaws. CONCLUSION: ZOL severely suppresses coupled remodelling in the tibia, and the suppression of bone formation in the modelling mode in the jaws demonstrates the site specific effects of ZOL in rice rats.

In vitro evaluation of osteoblast responses to carbon nanotube-coated titanium surfaces.

BACKGROUND: The effects of surface roughness and carboxyl functionalization of multi-walled carbon nanotubes (MWCNTs) mixed with collagen coated onto titanium (Ti) substrates on MC3T3-E1 osteoblasts were evaluated. METHODS: The proliferation, differentiation, and matrix mineralization were investigated using (1) smooth-surfaced Ti discs, (2) Ti discs coated with collagen and MWCNT (Ti-MWCNT), and (3) Ti discs coated with collagen and MWCNT-COOH (Ti-MWCNT-COOH) for applications in orthodontic mini screw implants (MSIs). The coatings were uniform when analyzed using scanning electron microscopy (SEM), and surface roughness was evaluated by surface profilometry that demonstrated similar surface roughness (R a , mean ± SD) in the MWCNT (0.83 ± 0.02 µm) and MWCNT-COOH (0.84 ± 0.01 µm) groups. MTT (3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide) assay was performed after days 1, 3, and 7 to assess proliferation. Alkaline phosphatase (ALP)-specific activity was assessed after day 7 to quantify differentiation. Alizarin red staining was measured after day 28 to quantify matrix mineralization. All data were analyzed with JMP Pro11 software (SAS, USA) with a statistical significance of p < 0.05. RESULTS: Surface profilometry demonstrated similar surface roughness (R a , mean ± SD) in the MWCNT (0.83 ± 0.02 µm) and MWCNT-COOH (0.84 ± 0.01 µm) groups. On day 7, ALP assay showed that MWCNT-COOH (mean ± SD 0.98 ± 0.26 U/µg of protein) enhanced cell differentiation when compared to the uncoated group (p = 0.05). Alizarin red staining after 28 days of cell culture revealed that MWCNT-COOH (mean ± SD 1.5 ± 0.2 OD405) increased (p = 0.03) matrix mineralization when compared to the uncoated group (0.9 ± 0.09 OD405). CONCLUSIONS: This study showed that coatings containing MWCNT-COOH (increased hydrophilic surface chemistry) influence osteoblast proliferation, differentiation, and matrix mineralization and should be further studied for applications in orthodontic MSIs.

Personalized and precision orthodontic therapy.

OBJECTIVE: To bring together orthodontic stakeholders from academics, industry, and private practice for a series of thematically focused workshops to explore and develop the transfer of novel approaches into clinical orthodontic practice. SETTING AND SAMPLE POPULATION: Twenty-seven invited speakers, eight poster presenters, and participants of the Consortium for Orthodontic Advances in Science and Technology (COAST) 2014 Innovators' Workshop at the Eaglewood Resort and Spa, Itasca, Illinois, September 11-14, 2014. MATERIAL AND METHODS: Five themed sessions involving between 4-7 presentations followed by panel discussions were organized. The aims of the discussion sessions were to highlight important findings and consider the strength of evidence for these, indicate next steps and needed research or technological developments to move forward, and to weigh the expected benefits from these findings and steps to implement in clinical practice. RESULTS: Among important areas for attention identified were need for multiscale and multispecies modeling and experimentation for interspecies translation of results; large-scale collaborative efforts within the profession to address the need for adequate sample sizes for future genetic studies of complex traits such as malocclusion; a consortium approach to improve new technologies such as intra-oral scanning and 3D imaging by establishing standards; and harnessing the growing body of knowledge about bone biology for application in orthodontics. CONCLUSIONS: With increased awareness of the potential of current and emerging technologies, translation of personalized and precision approaches in the field of orthodontics holds ever-increasing promise.

Bone anchors--can you hitch up your wagon?

The purpose of this review was to address and understand the current status of mini-screw implants (MSI) that are used in orthodontics. Understanding the biologic adaptation of MSI to its adjacent bone is one of the critical factors to their success. The review explores factors that are associated with failure of MSI, with special focus on an understanding of osseointegration as it relates to MSI. The rationale and importance of measuring bone contact and dynamic bone remodeling in animal studies are outlined. The utility of microcomputed tomography (µCT) as a substitute for conventional histomorphometry is debated. Finally, alveolar physiology and rigidity of implants are explored to understand potential reasons for the high failure rate of MSI when compared to endosseous implants.

Alveolar bone preservation subsequent to miniscrew implant placement in a canine model.

OBJECTIVE: To assess the effects of transcortical screws on alveolar (bone) ridge preservation following extraction. DESIGN: Four adult beagle dogs had mandibular premolars extracted bilaterally. After 6 weeks, using a split-mouth design, two transcortical screws were inserted unilaterally below the alveolar crest on the experimental side in the region of the extraction. The dogs were killed after 12 weeks. The bone at the extraction sites was analyzed using µCT and 3D analysis. A cylindrical core was placed around the actual and a virtual screw placed in the identical location on the control side. The bone volume within the cylinders was quantified. An insertion of a dental implant was simulated bilaterally at the insertion site. The height of the clinical crown and the alveolar crest were determined on both sides. The bone turnover was assessed histomorphometrically on un-decalcified bucco-lingual sections stained with basic fuchsine and toluidine blue. RESULTS: Comparison of the two sides revealed a significant difference both with regard to the bone volume and morphology. The transcortical screw caused an increase in bone density and less ridge atrophy. When simulating a dental implant placement on both sides, the bone preservation on the experimental side led to a need for a shorter clinical crown compared to the control side. A higher activity level of the bone in the experimental side was demonstrated histologically. CONCLUSION: In this dog model the insertion of a mini-implant across the healing alveolar process results in increased density not only adjacent to the screws, but also in the region where a potential dental implant would be inserted. In humans, the insertion of transcortical screws may maintain bone when for various reasons insertion of a permanent dental implant has to be postponed.

Quasi-static and harmonic indentation of osteonal bone.

UNLABELLED: The purpose of the study was to compare Quasi-Static (QS) and harmonic (CSM) methods of indentation testing. Bone sections were obtained from mid-femoral diaphyses of dogs which received a pair of calcein labels. Labeled (n = 35) and unlabeled (n = 112) osteons were identified. Indentation modulus (IM) and hardness (H) for the CSM method were collected during the entire loading cycle to peak depth, while IM and H for QS method were calculated at a peak depth of 500 nm. RESULTS: The mean (SD) of the IM and H for labeled osteons were as follows: QS IM = 15.3 GPa (3.85) versus CSM IM = 14.7 GPa (3.58); P = .52 and QS H = .39 GPa (.171) versus CSM H = .42 GPa (.146); P = .32. The mean (SD) of the IM and H for unlabeled osteons were as follows: QS IM = 21.5 GPa (2.80) versus CSM IM = 20.6 GPa (2.53); P = .054 and QS H = .64 GPa (.117) versus CSM H = .70 GPa (.120); P = .017. There was no difference in IM and H for the two methods, except for H of the unlabeled osteons. In addition, for the CSM method, IM at 100 nm, 200 nm, 300 nm, 400 nm and 500 nm were not statistically significant different (P = .06). Bone is viscoelastic at an organ level. However, this component of its behavior was not detected at the length scale examined.

Zoledronic acid decreases bone formation without causing osteocyte death in mice.

Bisphosphonates have been associated with osteonecrosis of the jaw. The purpose of this study was to examine the effect of a potent bisphosphonate, zoledronic acid (ZA) on osteocyte viability and bone formation. Ten experimental C57BL/6 mice were administered ZA (0.1 mg/kg-i.p.) weekly for 9 weeks while four control mice did not receive the drug. A pair of calcein (30 mg/kg) labels was administered 10 and 3 days prior to sacrifice of the 34-week-old mice. Fresh mandibular and femoral sections were obtained to evaluate osteocyte viability using a lactate dehydrogenase (LDH) assay. In addition, sections from the femur, mandible and maxilla were prepared for standard histomorphometry. The operator was blinded for data collection to eliminate bias. Data on necrotic area/total bone area from the LDH sections were collected. In addition, standard histomorphometric variables including bone formation rate were calculated. Mixed models were used to analyse data. The osteocytes were overwhelmingly viable and no necrotic areas were detected in the mandible and femur of both groups. ZA was not directly cytotoxic to the mouse osteocytes. There was suppression in indices of bone formation at all skeletal sites of the ZA group compared to the control group. While ZA administration in mice does not produce necrotic osteocytes, it severely suppresses bone formation. Such reductions can have a profound effect on bone healing.

Comparison of hand-traced and computer-based cephalometric superimpositions.

OBJECTIVE: To determine the ability to produce comparable superimpositions using hand tracing and digital methods (Dolphin v10). In addition, if the two methods were comparable, we wanted to determine if a difference existed between the best-fit cranial base superimposition and S-N superimpositions using the digital method. METHODS AND MATERIALS: Sixty-four initial (T(1)) and final (T(2)) cephalometric film radiographs were obtained. Cranial base and regional superimpositions were completed independently for each pair of radiographs by either hand tracing and digital methods. To quantitatively evaluate the differences between the two methods, the hand and digital superimpositions were digitized to obtain x-y coordinates of routine cephalometric landmarks at T(2). Linear distance between multiple corresponding (hand and digital) T(2) cephalometric landmark locations (e.g., A point) were measured and defined as the T(2) landmark distance (T(2) LD). Additionally, 61 patient records were used to compare the digital method for best-fit cranial base superimpositions versus S-N superimpositions. A Friedman test was applied to examine for differences. RESULTS: The upper 95% confidence limit for the mean of the T(2) LD for hand and digital superimposition methods was <1 mm for all landmarks except maxillary incisor tip and apex. The upper 95% confidence interval for best-fit vs S-N was >1 mm for most landmarks. CONCLUSION: This study validates the use of superimpositions produced by Dolphin Imaging version 10 and is a necessary step forward toward widespread acceptance of digital superimpositions.

Tissue level mechanical properties of cortical bone in skeletally immature and mature dogs.

OBJECTIVES: The purpose of this study was to quantify the tissue level mechanical properties of cortical bone of skeletally immature (~five-month-old) Beagle dogs and compare them to data from mature dogs measured in a previous study. METHODS: Eight femoral cross sectional specimens (two bone sections / dog) were obtained from four skeletally immature dogs. A pair of calcein bone labels were administered intravenously to the dogs to mark sites of active mineralization prior to euthanasia. Prepared bone specimens were placed in a nano- indenter specimen holder and the previously identified calcein labelled osteons were located. Labelled (n = 128) and neighbouring unlabelled (n = 127) osteons in skeletally immature femurs were examined by instrumented indentation testing. Indents were made to a depth of 500 nm at a loading rate of 10 nm/s. Indentation modulus (IM) and hardness (H) were obtained. RESULTS: The overall IM of the cortical bone in the skeletally mature groups was significantly greater than in the immature group (p = 0.0011), however overall H was not significantly different. The differences between the groups in IM were significant for the unlabelled osteons (p = 0.001), but not for the labelled osteons (p = 0.56). CONCLUSION: There are differences in the IM of unlabelled osteons in skeletally immature and mature groups of Beagle dogs. In contrast to whole bone mechanical tests, where there are obvious differences between growing and mature bones, there are only small differences in the micro-mechanical properties.

Indentation modulus of the alveolar process in dogs.

One mechanism of bone adaptation is alteration in tissue level material properties. We hypothesized that alteration in the indentation modulus of the alveolar process is an adaptive response to the localized mechanical environment. Forty-eight specimens representing anterior and posterior regions of the maxilla and mandible were obtained from 6 mature male beagle dogs. The indentation properties of the alveolar bone proper and more distant osteonal cortical bone were estimated. The bone types were further divided into 3 regions (coronal, middle, and apical), with 27 indents being made in each region of tooth-supporting bone. There was a significant difference (p < 0.001) in the indentation moduli of the jaws (maxilla/mandible), location (anterior/posterior), and bone type (alveolar bone proper vs. cortical bone). However, statistical interactions exist which preclude the simple interpretation of results. The distribution of relative stiffness provides a better understanding of bone adaptations in the alveolar process.

Indentation properties of young and old osteons.

The purpose of this study was to quantify differences in indentation modulus and microhardness between labeled osteons identified by epifluorescent microscopy and neighboring unlabeled osteons. In microradiographs and backscattered images, newly formed osteons appear more radiolucent (darker) than older osteons. This is ascribed to incomplete mineralization of the osteon. However, the mechanical properties of these young osteons are unknown. Nine femoral cross-sectional specimens were obtained from five skeletally mature dogs. Prior to death, the dogs received a pair of calcein bone labels. Labeled osteons were identified under an epiflourescent microscope. Bone specimens were transferred to a nanoindenter specimen holder, and the previous identified labeled osteons were located. Labeled (n = 102) and unlabeled (n = 101) osteons were examined by instrumented indentation testing. Indents were made to a depth of 500 nm at a loading rate of 10 nm/second. There were significant differences in the indentation modulus (P < 0.001) of labeled (10.02 +/- 3.61 gigapascal (GPa), mean +/- standard deviation) and unlabeled (15.11 +/- 3.72 GPa) osteons. Similar differences existed in microhardness measurements. Newly formed osteons had lower modulus (34%) and hardness (41%) than older osteons found in femoral cross sections. These data provide information on the indentation moduli of osteons during an early phase of mineralization compared to osteons that have completed mineralization.

Biomechanical and histomorphometric analyses of monocortical screws at placement and 6 weeks postinsertion.

Maxillofacial screws are increasingly being used in orthodontics to provide anchorage for tooth movement. The objective of this study was to determine the biomechanical stability as well as the bone tissue response of screws at 6 weeks postinsertion in a canine model. Seven skeletally mature male dogs received 102 screws (2 x 6 mm or 2 x 8 mm) at predetermined sites. Twenty screws became loose or were lost during the 6-week undisturbed healing period. Forty-eight screws were randomized for mechanical testing and 34 for histology. Peak pullout strength was recorded and approximately 80-microm sections were examined for histomorphometric parameters. Statistical analyses were conducted by analysis of variance and Tukey-Kramer method. Mean +/- SE peak pullout strengths for the various sites ranged from 153.5 +/- 37.6 N to 389.3 +/- 32.5 N with no significant (P < .05) differences at immediate placement and 6 weeks postinsertion. Bone contact ranged from 79% to 95%. Histomorphometric analyses indicated higher bone formation rate in the mandible than in the maxilla and a gradient of decreasing turnover with increasing distance from the screw interface. These results provide the clinical orthodontist with an estimate of the holding power of these screws and an understanding of early biological healing response associated with self-drilling screws.

Development of a fluorescent light technique for evaluating microdamage in bone subjected to fatigue loading.

A new method using fluorescent light microscopy has been developed to visualize and evaluate bone microdamage. We report the findings of two different experiments with a common aim of comparing the fluorescent light technique to the brightfield method for quantifying microdamage in bone. In Experiment 1, 36 canine femurs were tested in four-point cyclic bending until they had lost between 5 and 43% of their stiffness. The loaded portion of the bone was stained en bloc with basic fuchsin for the presence of damage. Standard point counting techniques were used to calculate fractional damaged area (Dm.Ar = Cr.Ar/B.Ar, mm2/mm2) under brightfield and fluorescent microscopy. In Experiment 2, bone microdamage adjacent to endosseous implants, subjected to fatigue loading (150,000 cycles, 2 Hz and 37 degrees C) ex vivo was examined. The bone around the implant was either allowed to heal (adapted specimen) for 12 weeks after placement in dog mid-femoral diaphyses prior to testing or was loaded immediately to simulate non-healed bone surrounding endosseous implants (non-adapted). Crack numerical density (Cr.Dn = Cr.N/B.Ar, #/mm2), crack surface density (Cr.S.Dn = Tt.Cr.Le/B.Ar, mm/mm2) and fractional damaged area were calculated separately by both techniques in the adapted and non-adapted specimens. In both Experiments 1 and 2, significantly more microdamage was detected by the fluorescent technique than by the brightfield method. Also, there was a trend towards higher intraobserver repeatability when using the fluorescent method. These results suggest that the brightfield technique underestimates microdamage accumulation and that the fluorescent technique better represents the actual amounts of microdamage present. The results demonstrate that the fluorescent method provides an accurate and precise approach for bone microdamage evaluation, and that it improves the prediction of stiffness loss from damage accumulation.

Microdamage adjacent to endosseous implants.

Intense remodeling occurs in lamellar bone adjacent to osseointegrated endosseous implants. The purpose of this study was to compare microdamage accumulation subsequent to ex vivo fatigue loading of bone that surrounds an endosseous implant, (a) immediately after placement (nonadapted bone) and (b) following a 12 week healing period after placement (adapted bone). We hypothesize that there is less microdamage in the more compliant adapted bone than in the older nonadapted bone. Nonthreaded titanium plasma sprayed (TPS)-coated endosseous implants were placed into dog mid-femoral diaphyses and allowed to heal for 12 weeks. Block sections of bone, each containing one implant, were cut anteroposteriorly, resulting in an implant containing lateral cortex, and a medial cortex that was used for testing the nonadapted specimens. Control specimens (n = 14 each for adapted and nonadapted) were loaded at 0 N. Experimental specimens (n = 13, adapted; n = 14, nonadapted) were loaded at 100 N in cantilever bending for 150,000 cycles at 2 Hz, at 37 degrees C on a Bionix 858 testing machine. Specimens were bulk stained with basic fuchsin and 120-140 microm sections were obtained. Crack numerical density (Cr.Dn = Cr.N/ B.Ar, #/mm2), crack surface density (Cr.S.Dn = Tt.Cr.Le/ B.Ar, mm/mm2), and percent damage area (Dm.Ar = Cr.Ar x 100/B.Ar, mm2/mm2) were measured at x 250. Statistically significant differences (p < 0.0001) were seen for Cr.Dn, Cr.S.Dn, and Dm.Ar on the compressed cortices suggesting that adapted bone near the implant accumulated significantly less microdamage than nonadapted bone. Also, the adapted nonloaded control specimens had approximately 20-fold less damage than the respective nonadapted specimens. This study suggests that the compliant adapted bone adjacent to endosseous implants is relatively resistant to fatigue loads. The high success rates of endosseous implants may be due to the presence of a rapidly remodeling region that maintains tissue compliance and limits microdamage initiation.

Effects of callus and bonding on strains in bone surrounding an implant under bending.

Descriptions of the healing and adaptation of endosseous implants have been provided; however, their effects on mechanical parameters such as maximum and minimum principal strains, strain energy density, and maximum shear strain have not been addressed. Three linear, elastic, and partially anisotropic finite element models were generated to simulate the immediate postoperative period, time of provisional loading, and long-term adaptation of bone surrounding implants. In each model, unbonded and bonded interface conditions were imposed. Bone geometry was estimated from dental implants placed in femurs of hounds. A lateral load was applied and the mechanical parameters were calculated. Interface bonding decreased the peak minimum principal strain 2.6 to 6.4 fold, while the presence of a callus reduced it 3 to 7 fold. These data document the critical stabilizing roles of callus and bond formation.

Microhardness and anisotropy of the vital osseous interface and endosseous implant supporting bone.

Limited information is available on the mechanical properties of the rapidly remodeling bone that surrounds endosseous implants. Fifteen implant-bone blocks were obtained from the mid-femoral diaphyses of three mature male hounds 12 weeks after placement of the implants. To evaluate the microhardness and cortical anisotropy of bone, the implants were sectioned along their long axes. In this process, the femurs were sectioned transversely. Knoop microhardness measurements (HK) were made with a 50 g force on cortical bone and a 25 g force on periosteal callus, endocortical callus, and circumferential lamellar bone. The long diagonal of the indenter was placed parallel to the implant (in the radial bone direction). Measurements were made in cortical bone at 200, 400, 600, 800, 1,000, 1,500, 2,000, and 2,500 microm from both sides of the implant. To detect cortical anisotropy in the radial compared with the tangential direction, a second set of indentations was made perpendicular to the first. Microhardness of periosteal callus and endocortical callus and anisotropy of circumferential lamellar bone near the endocortical surfaces of the femur were also evaluated. Repeated measures analysis of variance showed significantly (p < 0.05) lower microhardness values (30.6 +/- 0.8 HK [mean +/- SEM]) for cortical bone at 200 microm than at any other location (range: 40.3-46.6 HK). Microhardness anisotropy was not detected in cortical bone. Furthermore, within 200 microm of the implant surface, the Knoop microhardness values were significantly lower for periosteal and endocortical calluses than for cortical bone. These data provide information about the mechanical properties of bone adjacent to endosseous implants at a microstructural level. The results are consistent with the high rate of remodeling seen adjacent to endosseous implants at 12 weeks after implantation.