Histomorphometric Measurements Research Articles

Abstract P351: Statins Prevent Expansive Remodeling in Arterialized Vein Grafts

Vein grafts (VG) account for more than 70% of conduits used in aortocoronary bypass surgery, but have high failure rates by 10 years. We have previously shown in an experimental model that VG undergo marked expansion by 4 weeks, which was followed by increased total LDL retention at 12 weeks. The objective of this study was to determine whether statin therapy would prevent these expansive remodelling changes. Methods and Results Reversed jugular vein-to-common carotid artery interposition graft were constructed in 24 cholesterol-fed (0.5%) rabbits. Rabbits were randomized to either control or atorvastatin (5 mg/kg/day) groups, starting two weeks prior to vein graft implantation and continuing until sacrifice at 1 or 12 weeks post-surgery. Ultrasound measurements of arterial luminal cross-sectional area (LCSA) were done at day 3 and at 4, 8 and 12 weeks post-surgery. Histomorphometric measurements were performed following sacrifice at 12 weeks. Atorvastatin treatment significantly decreased total plasma cholesterol levels at 12 weeks (6.70±4.19mmol/L vs placebo 38.65±10.55mmol/L, p<0.001). Atorvastatin significantly reduced expansive remodelling at all time points, including at 12 weeks (44.6±6.62mm 2 vs placebo 77.6±10.68mm 2 , p<0.001). Intimal CSA by histomorphometry was significantly reduced by atorvastatin at 12 weeks (5.59±2.19mm 2 vs placebo 9.57±2.43mm 2 , p<0.05). At 1 week, macrophage infiltration was significantly reduced (Ram11 positive intimal staining 0.018±0.019 vs 0.189±0.049, p<0.05). Atorvastatin treated samples at 1 week showed reduction in both MMP 2 activity (gelatin zymography) and metalloelastase activity (casein zymography). Conclusion: Atorvastatin prevents expansive remodelling in arterialized VG, likely through inhibition of macrophage infiltration and reduction of tissue proteolytic activity. This may be an important mechanism preventing VG atherosclerosis and late VG failure.

Dynamic Ultrasound Assessment of the Effects of Knee and Ankle Position on Achilles Tendon Apposition Following Acute Rupture

Previous reports have suggested that knee flexion improves tendon edge apposition following acute rupture of the Achilles tendon. The aim of the present study was to determine, with use of ultrasonography, the effects of knee and ankle position on the Achilles tendon gap distance in patients with an acute rupture. Twenty-six patients with a unilateral acute complete Achilles tendon rupture that had been confirmed with ultrasonography were recruited within a week after the injury. The mean age at the time of presentation was forty-one years. Ultrasound measurements included the location of the rupture and the gap distance between the superficial tendon edges with the ankle in neutral and the knee extended. The gap distance was sequentially measured with the foot in maximum equinus at the ankle and with 0°, 30°, 60°, and 90° of knee flexion. The mean distance of the rupture from the calcaneal enthesis was 52 mm (range, 40 to 76 mm). The mean gap distance with the ankle in neutral and the knee extended was 12 mm (95% confidence interval, 10 to 13 mm). This distance decreased to 5 mm (95% confidence interval, 4 to 7 mm) when the foot was placed in maximum ankle equinus with 0° of knee flexion and to 4 mm (95% confidence interval, 3 to 5 mm) with 30° of knee flexion, 3 mm (95% confidence interval, 2 to 4 mm) with 60° of knee flexion, and 2 mm (95% confidence interval, 1 to 2 mm) with 90° of knee flexion. Expressing the reduction in gap distance with each successive position as a percentage of the gap distance with the knee extended and the ankle in neutral revealed a mean reduction of 55.7%, 64.4%, 75.4%, and 84.8% with maximum ankle equinus and 0°, 30°, 60°, and 90° of knee flexion, respectively. The difference in gap distance between each of these positions was significant (p < 0.05). Maximum ankle equinus alone significantly reduces the gap distance after acute Achilles tendon rupture. Increasing knee flexion further reduces the gap distance by small increments that, although significant, may not be clinically important.

Osteoinductivity of Demineralized Bone Matrix Is Independent of Donor Bisphosphonate Use

Demineralized bone matrix is commonly used as a bone graft substitute, either alone or to supplement an osteoconductive material, because of its osteoinductive properties. The aging of the population has led to an increase in the number of prospective donors of demineralized bone matrix who have taken bisphosphonates to prevent osteoclast-mediated bone resorption. The aim of this study was to determine whether oral bisphosphonate usage affects the osteoinductivity of demineralized bone matrix from donors. Sex-matched and age-matched pairs of samples were provided by four tissue banks (three or four pairs per bank). Demineralized bone matrix donors without bisphosphonate treatment had a mean age (and standard deviation) of 69.1 ± 2.5 years, and donors with bisphosphonate treatment had a mean age of 68.9 ± 2.0 years. Each pair included one donor known to have taken bisphosphonates and one who had not taken bisphosphonates. Demineralized bone matrix previously confirmed as osteoinductive was the positive control, and heat-inactivated demineralized bone matrix was the negative control. Demineralized bone matrix incubated with 1 mL of phosphate-buffered saline solution containing 0, 0.002, 2.0, or 2000 ng/mL of alendronate was also tested. Gelatin capsules containing 15 mg of demineralized bone matrix were implanted bilaterally in the gastrocnemius muscle of male nude mice (eight implants per group). The mice were killed thirty-five days after implantation, and hind limbs were recovered and processed for histological analysis. Osteoinductivity was measured with use of a qualitative score and by histomorphometry. Nine of fifteen samples from donors who had had bisphosphonate treatment and ten of fifteen samples from patients who had not had bisphosphonate treatment were osteoinductive. Qualitative mean scores were comparable (1.7 ± 0.4 for those without bisphosphonates and 1.9 ± 0.7 for those with bisphosphonates). Osteoinductive demineralized bone matrix samples produced ossicles of comparable size, regardless of bisphosphonate usage. Histomorphometric measurements of the area of new bone formation and residual demineralized bone matrix were also comparable. The addition of alendronate to control demineralized bone matrix did not affect its osteoinductivity. Demineralized bone matrix samples from donors treated with bisphosphonates and donors not treated with bisphosphonates have the same ability to induce bone formation. However, it is not known if the quality of the new bone is affected, with subsequent consequences affecting bone remodeling.

Abstract 9819: Comparison of Optical Coherence Tomography and Histopathology Findings Following Paclitaxel-Coating Balloon in Porcine In-Stent Stenosis Model

Background: Paclitaxel-Coating balloon (PCB) has shown efficacy for treatment of coronary artery disease. Optical coherence tomography (OCT) is a promising endovascular imaging devise and has been reported to have a high correlation with histomorphometric measurement. However, the association of in-stent stenosis (ISR) by OCT and histopathology response is still not fully addressed. The current study was designed to compare the OCT and histopathological findings following PCB treatment in a porcine ISR model. Materials and Methods: Thirty-two coronary arteries (LAD: n=12, LCX: n=11, RCA: n=9) of 24 Yorkshire pigs were assigned to two (2) groups randomly. PCB (SeQuent, length 20mm, n=16) or non-coating balloon (NCB, length 20mm n=16) were inflated for 60 seconds with B:A 1.2:1 followed by BMS (length 12mm) implantation (S/A 1.5:1) within the target region. At 28 days, animals underwent OCT and histopathology evaluation. Results: There were no significant differences between groups among parameters measured by OCT. The thickness of neointimal hyperplasia on stent strut and the percent of ISR were 585 ± 277 μ m, 46.8 ± 19.0 % for PCB, and 661 ± 237 μ m, 52.4 ± 15.3 % for NCB (p=NS, respectively). However, inflammation (PEB: 3.40 ± 0.84, NCB: 2.27 ± 1.18, p=0.003) and fibrin deposition scores (PCB: 2.31 ± 0.93, NCB: 0.81 ± 0.86, p&lt;0.001) were significantly higher in PCB compared to NCB. The OCT ISR% was positively correlated with inflammation score (PCB: r=0.62, and NCB: r=0.85; p&lt;0.05 respectively). Interestingly, fibrin deposition score only in PCB was negatively correlated with the OCT ISR% (r=-0.68, p=0.005). OCT also showed an uncovered (33.3%) and insufficient non-uniformed neointima (13.3%) in PCB group (p=0.008 vs NCB). Conclusions: OCT can be used as a valuable imaging tool to evaluate vessel wall histopathological changes in vivo. The higher inflammation and fibrin deposition scores in PCB may indicate a delayed healing response in the current experimental setting.

Abstract 17237: The Underlying Necrotic Core is a Determinant of Occlusive Thrombosis

Background: It is well known that plaque rupture is not always associated acute coronary syndrome and in fact 10% of asymptomatic individuals dying of other causes have plaque rupture. To identify the underlying mechanisms of high risk plaque that leads to occlusive thrombosis, we examined coronary plaques from our registry of sudden coronary death victims. Methods and Results: From our sudden coronary death registry, 102 plaque ruptures (PR) were identified in proximal and middle segments of coronary trees; distal lesions were excluded. Histomorphometric area measurements included EEL, IEL, lumen, necrotic core (NC), calcification, and macrophage positivity (CD68) at the cross-section of rupture site. In addition, thinnest portion of fibrous cap adjacent to rupture site was measured. The lesions with PR were categorized into 2 groups: occlusive (&gt;75% luminal thrombus) or non-occlusive (&lt;75% luminal thrombus). PRs with occlusive thrombosis was associated with larger IEL area, larger NC, thicker fibrous cap as compared to those with non-occlusive thrombosis while EEL, lumen, calcification, and macrophage area were similar in both groups (Table). Multivariate regression analysis revealed that total NC volume is the best predictor of occlusive thrombus with an odds ratio of 1.15 (p=0.019), followed by cap thickness (odds: 1.04, p=0.024). ROC analysis of NC demonstrated the threshold of 2.89 mm 2 with sensitivity 0.61 and specificity 0.67 (AUC 0.67). Conclusion: These data suggest that strategies directed at detection of precise necrotic core area are necessary to assess high risk coronary plaques.

Prasugrel Aggravates the Inflammatory Response in a PG-PS-Induce Arthritis Model,

Abstract 3268Prasugrel is a very potent and effective P2Y12 receptor antagonist, used primarily to prevent thrombotic events in patients managed for percutaneous coronary intervention (PCI). It is not recommended as prophylaxis antithrombotic therapy because prasugrel increases patient’s risk of bleeding. Even though indication of prasugrel treatment has been limited to the PCI procedure, there are not reports of the effects of prasugrel in patients with associated inflammatory diseases that undergo for PCI. Thus, we evaluate the effects of prasugrel, for 21 days, in a peptidoglycan polysaccharide (PG-PS)-induced arthritis rat model. Lewis rats were randomly separated into four groups from which only half of the animals were induced with PG-PS. There was a significant increase in the inflammatory changes observed in the PG-PS-induced animals treated with prasugrel compared to the PG-PS-induced control including a) increase joint diameter associated with visible signs of inflammation (during days 4 to 6 [p<0.05] and days 13 to 21 [p<0.001]); b) microscopic studies of hind joints showed marked increase in histomorphometric measurements including leukocyte infiltration, synoviocyte hyperplasia, fibrosis and neovascularization (significant increase in vessel number and size); and c) significant increase in platelet count (1,047 ± 89.03 × 103 vs. 710.8 ± 52.65 × 103, p<0.05). Platelet factor 4 (PF4) plasma levels were increased in PG-PS induced control as well as in PG-PS treated with prasugrel animals, however no significant differences were observed between these two groups. Plasma levels of cytokines such as IL-1 beta, IL-6, MIP1 alpha, MCP1, and RANTES were increased in PG-PS-induced animals (both, control and prasugrel treated animals). Only MIP1 alpha levels were significantly increased with additional treatment of prasugrel (p<0.05). In contrast, IL-10 levels were lower in the PG-PS-induced treated with prasugrel (p<0.05). In summary, treatment with P2Y12 receptor antagonist drugs, such as prasugrel, increases the inflammatory changes in PG-PS-induced arthritis model. Further investigations are in need to elucidate the potential unwanted effect of the P2Y12 receptor antagonists in patients afflicted with inflammatory illnesses. Disclosures:No relevant conflicts of interest to declare.

Experimental evidence for interfacial biochemical bonding in osseointegrated titanium implants

(i) To identify and quantify an interfacial biochemical bond and the bonding strength of osseointegrated implants with bioactive titanium oxide chemistry, ATiO(x)B (A, metal cations; TiO(x) , titanium oxides/hydroxides; B, non-metal anions) and (ii) to provide quantitative evidence for the biochemical bond theory of osseointegration proposed by Sul et al. for description and explanation of why and how the implants with ATiO(x) B surface oxide chemistry may exhibit a significantly stronger bone response, in spite of the fact that the roughness values approached zero, or were equivalent to or significantly lower than those of the control implants. We applied a newly developed biochemical bond measurement (BBM) method to model implant surfaces that were "perfectly" smooth nanotopography near-zero roughness as the constant parameter, and used the bioactive surface chemistry of titanium oxide, ATiOx B chemistry as a variable parameter in rabbit tibiae for 10 weeks. In this manner, we determined an interfacial biochemical bond and quantified its bonding strength. The increase in biochemical bond strengths of the test implant relative to the control implant was determined to be 0.018 (±0.008) MPa (0.031 vs 0.021 MPa, n = 10) for tensile strength and 8.9 (±6.1) Ncm (33.0 vs 24.1 Ncm, n = 10) for removal torque. Tensile and removal torque show strong correlation in the Pearson test (r = 0.901, P ≤ 0.001). In addition, histomorphometric measurements including bone-to-metal-contact (BMC, P = 0.007), bone area and newly formed bone showed significant increases in the mean values for ATiO(x) B chemistry (P = 0.007, n = 10). Biochemical bond theory states that the surface oxide chemistry, ATiO(x) B must have more electrical and chemical molecular polarity that fractionally charges the surfaces denoted as δ(+) and δ(-) and leads to electrostatic and electrodynamic interactions with the bone healing cascade, eventually leading to the formation of biochemical bonding at the bone/implant interface. The present study has provided quantitative evidence for biochemical bond theory of osseointegration of implants with bioactive surface oxide chemistry, ATiO(x) B. The theory of biochemical bonds may provide a scientific rationale pertinent to recent emerging trends and technologies for surface chemistry modifications of implants.

Tissue integration of collagen‐based matrices: an experimental study in mice

To test whether or not tissue integration, biodegradation, and new blood vessel formation in two collagen-based matrices depend on the level of chemical cross-linking. Two collagen matrices with high (CM1) and low (CM2) levels of chemical cross-linking were randomly implanted in two pouches in 14 athymic nude mice. Three and 6 weeks later, the animals were euthanized. Histologic and histomorphometric measurements were performed on paraffin-embedded sections. Both collagen matrices integrated well into the surrounding soft tissues. The level of cross-linking and duration of implantation had an effect on the formation of new blood vessels. More blood vessels (n = in absolute numbers) were found in outer compartments compared to the central compartments of the matrices, reaching 5.6 (CM2) vs. 4.3 (CM1) at 3 weeks, and 5.3 (CM2) vs. 7.3 (CM1) at 6 weeks. Similarly, connective tissue formation increased for both matrices between 3 and 6 weeks, whereas the amount of remaining collagen network gradually decreased over time being more pronounced for CM1 (-50%) compared to CM2 (-15%). The degree of cross-linking was negatively correlated for all outcome measures resulting in improved tissue integration, superior matrix stability and enhanced angiogenic patterns for the less cross-linked collagen matrix (CM2) in this experimental study in mice.

Neuroimmunomodulative properties of dipeptidyl peptidase IV/CD26 in a TNBS‐induced model of colitis in mice

Causal connections between dipeptidyl peptidase IV, also known as CD26 molecule (DPP IV/CD26) and inflammatory bowel disease (IBD) have been shown, but mechanisms of these interactions are unclear. Our hypothesis was that DPP IV/CD26 could affect the neuroimmune response during inflammatory events. Therefore, we aimed to evaluate its possible role and the relevance of the gut-brain axis in a model of IBD in mice. Trinitrobenzenesulfonic acid-induced (TNBS) colitis was induced in CD26-deficient (CD26(-/-) ) and wild-type (C57BL/6) mice. Pathohistological and histomorphometrical measurements were done. Concentrations and protein expressions of DPP IV/CD26 substrates neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) were determined. Concentrations of IL-6 and IL-10 were evaluated. Investigations were conducted at systemic and local levels. Acute inflammation induced increased serum NPY concentrations in both mice strains, more enhanced in CD26(-/-) mice. Increased NPY concentrations were found in colon and brain of C57BL/6 mice, while in CD26(-/-) animals only in colon. VIP and IL-6 serum and tissue concentrations were increased in both mice strains in acute inflammation, more pronouncedly in CD26(-/-) mice. IL-10 concentrations, after a decrease in serum of both mice strains, increased promptly in CD26(-/-) mice. Decreased IL-10 concentration was found in brain of C57BL/6 mice, while it was increased in colon of CD26(-/-) mice in acute inflammation. DPP IV/CD26 deficiency affects the neuroimmune response at systemic and local levels during colitis development and resolution in mice. Inflammatory changes in the colon reflected on investigated parameters in the brain, suggesting an important role of the gut-brain axis in IBD pathogenesis.

Trabecular bone histomorphometry in humans with Type 1 Diabetes Mellitus.

Patients with Type 1 Diabetes Mellitus (DM) have markedly increased risk of fracture, but little is known about abnormalities in bone microarchitecture or remodeling properties that might give insight into the pathogenesis of skeletal fragility in these patients. We report here a case-control study comparing bone histomorphometric and micro-CT results from iliac biopsies in 18 otherwise healthy subjects with Type 1 Diabetes Mellitus with those from healthy age- and sex-matched non-diabetic control subjects. Five of the diabetics had histories of low-trauma fracture. Transilial bone biopsies were obtained after tetracycline labeling. The biopsy specimens were fixed, embedded, and scanned using a desktop μCT at 16 μm resolution. They were then sectioned and quantitative histomorphometry was performed as previously described by Recker et al. [1]. Two sections, >250 μm apart, were read from the central part of each biopsy. Overall there were no significant differences between diabetics and controls in histomorphometric or micro-CT measurements. However, fracturing diabetics had structural and dynamic trends different from nonfracturing diabetics by both methods of analysis. In conclusion, Type 1 Diabetes Mellitus does not result in abnormalities in bone histomorphometric or micro-CT variables in the absence of manifest complications from the diabetes. However, diabetics suffering fractures may have defects in their skeletal microarchitecture that may underlie the presence of excess skeletal fragility.

Tissue Integration of a New Titanium–Zirconium Dental Implant: A Comparative Histologic and Radiographic Study in the Canine

This study evaluates a newly developed titanium-zirconium implant (TiZr), comparing it to a commercially available pure titanium (Ti) implant subjected to the same surface treatment. In nine dogs, 12 implants (six TiZr and six Ti) were randomly placed in the mandible with the implant shoulder at the bone crest and subjected to submerged healing. Standardized radiographs were taken after implantation, and at the sacrifice of 2 weeks (three dogs), 4 weeks (three dogs), and 8 weeks (three dogs). Histologic and histomorphometric measurements were performed on non-decalcified histologic sections. The main outcome measures included the first bone-implant contact (fBIC) and BIC over time. For statistical analysis, Wilcoxon signed-rank test and mixed model regressions were applied. From baseline to 8 weeks, a mean bone loss of 0.09 ± 0.33 mm for TiZr and a gain of 0.02 ± 0.33 mm for Ti were calculated radiographically. The number of implants with the fBIC coronal to the reference point (implant shoulder) gradually increased over time, reaching 39% of all TiZr implants and 50% of all Ti implants at 8 weeks. The mean fBIC values for Ti and TiZr were 0.29 ± 0.42 mm and 0.26 ± 0.32 mm (2 weeks), -0.01 ± 0.20 mm and 0.10 ± 0.28 mm (4 weeks), and -0.06 ± 0.22 mm and 0.08 ± 0.30 mm (8 weeks), respectively. The mean BIC values peaked at 86.9% ± 6.8% (8 weeks) for TiZr and at 83.4% ± 5.9% (4 weeks) for Ti. No statistically significant differences were observed at any time point. TiZr and Ti bone level implants with chemically-modified, sandblasted, and acid-etched surfaces performed similarly in regards to osseointegration in this unloaded canine study.

Nanostructured implant surface effect on osteoblast gene expression and bone-to-implant contact in vivo

The aim of this study was to investigate the response of nanostructured implant surfaces at the level of osteoblast differentiation and its effects in bone-to-implant contact (BIC) and removal-torque values (RTV). CpTi grade IV implants (1.6 × 4.0 mm) were machined or machined and subsequently coated with an oxide solution. The surfaces were divided into: machined (M), titania-anatase (An), titania-rutile (Ru), and zirconia (Zr). Surfaces were examined by scanning electron microscopy, atomic force microscopy, and by X-ray microanalysis. Implants were inserted in rat tibia and harvested from 0 to 21 days for measurement of Alkaline Phosphatase, Bone Sialoprotein, Osteocalcin, Osteopontin, and RUNX-2 mRNA levels by real time PCR; from 0 to 56 days for RTV; and from 0 to 56 days for BIC. The roughness parameter (Sa) was compared by one-way ANOVA followed by Tukey Test. Comparison of Torque removal values and histomorphometric measurements on implants in vivo was performed by Kruskal–Wallis test and the significance level for all statistical analyses was set at p ≤ 0.05. mRNA levels on all nanostructured surfaces were increased compared to M. At 56 days, the mean RTV in Ncm was 11.6 ± 2.5, 11.3 ± 2.4, 11.1 ± 3.5, 9.7 ± 1.4 for An, Ru, Zr, and M, respectively. Higher BIC (%) was measured for all the nanostructured surfaces versus M at 21 and 56 days (p < 0.05). Nanostructured topographic features composed of TiO 2 or ZrO 2 applied to machined cpTi implant promoted greater mesenchymal stem cell commitment to the osteoblast phenotype and associated increased BIC and physical association with bone.

Soft tissue volume augmentation by the use of collagen‐based matrices in the dog mandible – a histological analysis

The aim was to test, whether or not soft tissue volume augmentation with a specifically designed collagen matrix (CM), leads to ridge width gain in chronic ridge defects similar to those obtained by an autogenous subepithelial connective tissue graft (SCTG). In six dogs, soft tissue volume augmentation was performed by randomly allocating three treatment modalities to chronic ridge defects [CM, SCTG and sham-operated control (Control)]. Dogs were sacrificed at 28 (n=3) and 84days (n=3). Descriptive histology and histomorphometric measurements were performed on non-decalcified sections. SCTG and CM demonstrated favourable tissue integration, and subsequent re-modelling over 84days. The overall mean amount of newly formed soft tissue (NMT) plus bone (NB) amounted to 3.8±1.2mm (Control), 6.4±0.9mm (CM) and 7.2±1.2mm (SCTG) at 28days. At 84days, the mean NMT plus NB reached 2.4±0.9mm (Control), 5.6±1.5mm (CM) and 6.0±2.1mm (SCTG). Statistically significant differences were observed between CM/SCTG and Control at both time-points (p<0.05). Within the limits of this animal model, the CM performed similar to the SCTG, based on histomorphometric outcomes combining NB and NMT.

Effect of vitamin C on bone formation in the expanded inter-premaxillary suture. Early bone changes

The aim of this experimental study was to histomorphometrically evaluate the effects of vitaminC administration on bone formation in response to expansion of the rat inter-premaxillary suture. A total of 30 50- to 60-day old Wistar male rats were divided into three groups of equal number. The inter-premaxillary suture in each animal was expanded with a 0.49N force applied to the upper incisors. At 24h after appliance placement, control animals received saline solution (groupI) and two groups were given a single dose of vitaminC using two different methods [locally into the suture (groupII) and systemically via intramuscular injection (groupIII)]. The area of new bone (µm(2)), the perimeter around the new bone (µm), Feret's diameter (µm), and the percentage of new bone to non-ossified tissue (%) were measured and compared. Kruskal-Wallis and Tukey tests were used for statistical evaluation at the p<0.05 level. We observed significant differences among the groups in all histomorphometric parameters. New bone area, bone perimeter, Feret's diameter, and percentage of new bone dimensions were significantly higher in groupIII than the others (p<0.001). Histomorphometric measurements of bone architecture revealed that this improved in the group administered with vitaminC systemically, while local injection revealed significantly less bone growth than the control group. The systemic administration of vitaminC during the early stages of inter-premaxillary suture expansion may stimulate bone growth. However, local injection of this antioxidant into an orthopedically-expanded suture area has negative effects on bone formation.

Accuracy of cone beam computed tomography in assessing peri‐implant bone defect regeneration: a histologically controlled study in dogs

To assess the accuracy of cone-beam computed tomography (CBCT) in terms of buccal bone-wall configuration and peri-implant bone defect regeneration after guided bone regeneration (GBR). Titanium implants were inserted into standardized box-shaped defects in the mandible of 12 foxhounds. Defects of one side were augmented following the principle of GBR, while the other side was left untreated. Radiological evaluation was performed using CBCT and compared with histomorphometrical measurements of the respective site serving as a validation method. Non-augmented control sites providing a horizontal bone width (BW) of<0.5 mm revealed a significantly lower accuracy between the radiological and the histological evaluation of the buccal defect depth (1.93 ± 1.59 mm) compared with the group providing a BW of >0.5 mm (0.7 ± 0.7 mm) (P<0.05, Mann-Whitney U-test). In GBR-treated defects, the subgroup <0.5 mm (1.49 ± 1.29 mm) revealed a significantly higher difference between CBCT and histology compared with >0.5 mm (0.82 ± 1.07) (P>0.05, Mann-Whitney U-test). However, a radiological discrimination between original bone, integrated and non-integrated bone substitute material was not reliable. Additionally, it was found that a minimum buccal BW of 0.5 mm was necessary for the detection of bone in radiology. The evaluation of peri-implant bone defect regeneration by means of CBCT is not accurate for sites providing a BW of <0.5 mm. Moreover, a safe assessment of the success of the GBR technique is not possible after the application of a radiopaque bone substitute material.

An experimental model of distraction osteogenesis in rat mandibular alveolar bone

Introduction: Osteogenesis distraction (DO) is a surgical technique that allows distraction of the fracture callus. It has been used extensively in endochondral long bones but not in the craniofacial skeleton of endomembranous origin. Mandibular alveolar bone of endomembranous origin contains teeth sockets; it is a functional bone that undergoes bone turnover at a higher rate than other bone tissues. The aim of our work was to present the development of an experimental model of DO in rat mandibular alveolar bone including all stages, i.e. animal procedures, laboratory determinations, histological procedures and histomorphometric measurements. Methods: 8 Wistar rats, 80 g bw, underwent extraction of all mandibular molars. At 400 g bw, right hemimandibular osteotomy was performed and the device was placed. After 6 days (latency) it was activated (0.175 mm/d once a day/ 6 days) and then left in place 28 days (consolidation phase). Rats were sacrificed and hemimandibles were resected. The technique was standardized to perform radiographs and hemimandible orientation was adjusted to allow obtaining comparable histologic sections. A method to record and compare these measurements was designed. H&E stained mesiodistally oriented hemimandible sections were obtained. Screw loosening and loss of the device during the consolidation phase were initial problems (SAIO, 2008). The original design was perfected to avoid mobility. Results: The new, improved device remained in place throughout the experiment. Comparable mesiodistally oriented sections were obtained. Conclusion: This experimental model of mandibular DO will allow studying the cellular and molecular events that take place during DO under different experimental conditions. This article is part of a Special Issue entitled AAOMM 2010 Abstracts.

Melatonin: A protective hormone of intestinal calcium absorption?

Introduction: Osteogenesis distraction (DO) is a surgical technique that allows distraction of the fracture callus. It has been used extensively in endochondral long bones but not in the craniofacial skeleton of endomembranous origin. Mandibular alveolar bone of endomembranous origin contains teeth sockets; it is a functional bone that undergoes bone turnover at a higher rate than other bone tissues. The aim of our work was to present the development of an experimental model of DO in rat mandibular alveolar bone including all stages, i.e. animal procedures, laboratory determinations, histological procedures and histomorphometric measurements. Methods: 8 Wistar rats, 80 g bw, underwent extraction of all mandibular molars. At 400 g bw, right hemimandibular osteotomy was performed and the device was placed. After 6 days (latency) it was activated (0.175 mm/d once a day/ 6 days) and then left in place 28 days (consolidation phase). Rats were sacrificed and hemimandibles were resected. The technique was standardized to perform radiographs and hemimandible orientation was adjusted to allow obtaining comparable histologic sections. A method to record and compare these measurements was designed. H&E stained mesiodistally oriented hemimandible sections were obtained. Screw loosening and loss of the device during the consolidation phase were initial problems (SAIO, 2008). The original design was perfected to avoid mobility. Results: The new, improved device remained in place throughout the experiment. Comparable mesiodistally oriented sections were obtained. Conclusion: This experimental model of mandibular DO will allow studying the cellular and molecular events that take place during DO under different experimental conditions. This article is part of a Special Issue entitled AAOMM 2010 Abstracts.

EVALUATION OF BONE-TO-IMPLANT CONTACT AND BONE DENSITY ADJACENT TO TITANIUM IMPLANTS USING A STEREOLOGICAL TECHNIQUE ON GROUND SECTIONS

When bone implants have to be examined in situ ground sections are required. Histomorphometric measurements are usually performed on two-dimensional sections, causing biased results when they are wrongly extrapolated to 3D without any knowledge of stereology. Unbiased results can only be obtained using stereological principles. The aim of the study was to describe an unbiased design for evaluating boneto-mplant contact (BIC) and peri-implant bone density (BD-i) in three-dimensions. The unbiased design was based on a fixed axis vertical random sampling technique. Three bone-implant blocks were collected from 3 rabbits. Four sections were obtained from each animal using a fixed axis vertical random sampling technique. The BIC was estimated by creating a stereological method based on a systematic test line set. The BD-i was estimated using a design based on a systematic point set. The efficiency of the systematic sampling was evaluated for each sampling level: Coefficient of error CE( i w ) for the systematic test line set, CESyst between the 4 rotated sections, CENoise for the reproducibility. These variances were compared to the biological variation (CV) between animals. The mean CE( i w ) was 5 to 6%; the CESyst was 7.8% for the BIC estimates and 5.7% for the BD-i estimates; the CENoise was 5.8% for the BIC and 7.7% for the BD-i. The CV was 19% for the BIC estimates and 24% for the BD-i estimates. These results demonstrated that the stereological technique used in the present study was a very efficient method to obtain unbiased estimates of BIC and BD-i in 3D on 2D implant-bone sections.

Small-Animal PET/CT Assessment of Bone Microdamage in Ovariectomized Rats

Microdamage in bone contributes to bone fragility in postmenopausal women. Therefore, it is important to find a noninvasive method to detect microdamage in living bone. PET with (18)F-fluoride has been used for skeletal imaging in clinical studies. However, few studies are undertaken to investigate bone microdamage associated with osteoporosis in vivo using noninvasive means. The aim of our study was to analyze the impact of osteoporosis due to estrogen deficiency on the occurrence of microdamage by observing the change in the uptake of (18)F-fluoride in the tibiae of ovariectomized rats after fatigue loading with small-animal PET/CT. We also explored the feasibility of noninvasive detection of bone microdamage in vivo using a small-animal PET/CT scanner specially designed for rodent study. Rats were randomized into 2 groups: ovariectomy and sham surgery. These rats were imaged using a dedicated small-animal PET scanner with (18)F-fluoride after the left tibiae were loaded cyclically under the axial compression. The fluoride uptake values were quantified in the tibial mid shafts, and the tibia was obtained for histomorphometric measurements of bone microdamage and osteocyte density. Bone mineral density at the fourth lumbar vertebra and right femur were measured using dual-energy x-ray absorptiometry. PET image intensity was significantly increased (P < 0.05) in the loaded tibia of the ovariectomy group, compared with that of the sham group. Histomorphometry showed that both crack density and crack length in the loaded tibia were significantly higher (P < 0.05) in ovariectomized rats than in sham rats. The PET image intensity in the loaded tibia was significantly positively correlated with crack length and crack density (which show in histomorphometric measurement) (P < 0.05). Both small-animal PET/CT and histomorphometric measurement provided evidence that bone microdamage is significantly increased after estrogen depletion. The strong correlation between these 2 measurements suggests that small-animal PET/CT is a useful noninvasive means to detect bone microdamage in vivo.

The effect of calcium ion concentration on the bone response to oxidized titanium implants

To investigate the effect of calcium concentration on the bone tissue response to Ca-incorporated titanium implants. Two titanium surfaces containing 4.2% and 6.6% calcium were prepared using the micro-arc oxidation process. The implants were inserted in the tibia of nine New Zealand White rabbits. After 6 weeks of healing, the bone response to the implants was quantitatively compared by biomechanical and histomorphometrical measurements. Ca 4.2% and Ca 6.6% containing implants revealed no distinctive differences in their qualitative surface chemistry; chemical bonding state of Ca in titanium oxide was mainly calcium titanates. No significant differences were observed between two implants in peak removal torque and shear strength comparisons (P>0.05). Histomorphometrical analyses presented no significant differences in bone-metal contact, bone area and newly formed bone measurements between two implants (P>0.05). From biomechanical and histomorphometrical measurements, the two calcium concentrations in this study did not differ significantly with respect to their influence on the bone tissue response. This similar bone response in rabbit tibiae may be explained by the similarity of the qualitative Ca chemistry in titanium surfaces.