Heritability of periodontal bone loss in mice.

BACKGROUND: Periodontitis is an inflammatory disease of the periodontal tissues that compromises tooth support and can lead to tooth loss. Although bacterial biofilm is central in disease pathogenesis, the host response plays an important role in the progression and severity of periodontitis. Indeed, clinical genetic studies indicate that periodontitis is 50% heritable. In this study, we hypothesized that lipopolysaccharide (LPS) injections lead to a strain-dependent periodontal bone loss pattern. MATERIAL AND METHODS: We utilized five inbred mouse strains that derive the recombinant strains of the hybrid mouse diversity panel. Mice received Porphyromonas gingivalis-LPS injections for 6 wk. RESULTS AND CONCLUSION: Micro-computed tomography analysis demonstrated a statistically significant strain-dependent bone loss. The most susceptible strain, C57BL/6J, had a fivefold higher LPS-induced bone loss compared to the most resistant strain, A/J. More importantly, periodontal bone loss revealed 49% heritability, which closely mimics periodontitis heritability for patients. To evaluate further the functional differences that underlie periodontal bone loss, osteoclast numbers of C57BL/6J and A/J mice were measured in vivo and in vitro. In vitro analysis of osteoclastogenic potential showed a higher number of osteoclasts in C57BL/6J compared to A/J mice. In vivo LPS injections statistically significantly increased osteoclast numbers in both groups. Importantly, the number of osteoclasts was higher in C57BL/6J vs. A/J mice. These data support a significant role of the genetic framework in LPS-induced periodontal bone loss and the feasibility of utilizing the hybrid mouse diversity panel to determine the genetic factors that affect periodontal bone loss. Expanding these studies will contribute in predicting patients genetically predisposed to periodontitis and in identifying the biological basis of disease susceptibility.

Ligature-induced peri-implantitis in mice.

BACKGROUND AND OBJECTIVE: Peri-implantitis has a prevalence of 11-47%, involves destruction of peri-implant bone and may lead to implant loss. A detailed understanding of the pathogenesis of peri-implantitis is lacking. The objective of this study was to develop a murine model of experimental peri-implantitis. MATERIAL AND METHODS: Machined, smooth-surface, screw-shaped titanium implants were placed in the healed alveolar bone of the left maxillary molars of C57BL/6J male mice, 8 wk after tooth extraction. Peri-implantitis was induced by securing silk ligatures around the head of the implant fixtures. Implant survival and peri-implant bone levels were analyzed by micro-computed tomography (micro-CT) scans and histology, 12 wk after ligature placement. RESULTS: Implant survival was 60% (six of 10) for implants with ligatures and 100% (eight of eight) for controls. Micro-CT revealed significantly greater bone loss around the implants that received ligatures and that survived, compared with controls. The radiographic findings were confirmed via histology and toluidine blue staining. CONCLUSION: This study describes a murine model of experimental peri-implantitis around screw-shaped titanium implants placed in the edentulous alveolar bone. This model should be a useful tool to dissect pathogenic mechanisms of peri-implantitis and evaluate potential treatment interventions.

MicroCT morphometry analysis of mouse cancellous bone: intra- and inter-system reproducibility.

The agreement between measurements and the relative performance reproducibility among different microcomputed tomography (microCT) systems, especially at voxel sizes close to the limit of the instruments, is not known. To compare this reproducibility 3D morphometric analyses of mouse cancellous bone from distal femoral epiphyses were performed using three different ex vivo microCT systems: GE eXplore Locus SP, Scanco µCT35 and Skyscan 1172. Scans were completed in triplicate at 12 µm and 8 µm voxel sizes and morphometry measurements, from which relative values and dependence on voxel size were examined. Global and individual visually assessed thresholds were compared. Variability from repeated scans at 12 µm voxel size was also examined. Bone volume fraction and trabecular separation values were similar, while values for relative bone surface, trabecular thickness and number varied significantly across the three systems. The greatest differences were measured in trabecular thickness (up to 236%) and number (up to 218%). The relative dependence of measurements on voxel size was highly variable for the trabecular number (from 0% to 20% relative difference between measurements from 12 µm and 8 µm voxel size scans, depending on the system). The intra-system reproducibility of all trabecular measurements was also highly variable across the systems and improved for BV/TV in all the systems when a smaller voxel size was used. It improved using a smaller voxel size in all the other parameters examined for the Scanco system, but not consistently so for the GE or the Skyscan system. Our results indicate trabecular morphometry measurements should not be directly compared across microCT systems. In addition, the conditions, including voxel size, for trabecular morphometry studies in mouse bone should be chosen based on the specific microCT system and the measurements of main interest.

Osteogenic potential of mandibular vs. long-bone marrow stromal cells.

Although fundamentally similar to other bones, the jaws demonstrate discrete responses to developmental, mechanical, and homeostatic regulatory signals. Here, we hypothesized that rat mandible vs. long-bone marrow-derived cells possess different osteogenic potential. We established a protocol for rat mandible and long-bone marrow stromal cell (BMSC) isolation and culture. Mandible BMSC cultures formed more colonies, suggesting an increased CFU-F population. Both mandible and long-bone BMSCs differentiated into osteoblasts. However, mandible BMSCs demonstrated augmented alkaline phosphatase activity, mineralization, and osteoblast gene expression. Importantly, upon implantation into nude mice, mandible BMSCs formed 70% larger bone nodules containing three-fold more mineralized bone compared with long-bone BMSCs. Analysis of these data demonstrates an increased osteogenic potential and augmented capacity of mandible BMSCs to induce bone formation in vitro and in vivo. Our findings support differences in the mechanisms underlying mandible homeostasis and the pathophysiology of diseases unique to the jaws.

Overexpression of IGF-binding protein 5 alters mineral and matrix properties in mouse femora: an infrared imaging study.

The anabolic effects of insulin-like growth factors (IGFs) are modulated by a family of IGF-binding proteins (IGFBPs). Among the six known IGFBPs, IGFBP-5 is considered to play a role in bone formation. To investigate the effects of IGFBP-5 on bone mineral and matrix properties, femurs from transgenic mice overexpressing IGFBP-5 under the control of the osteocalcin promoter were evaluated by Fourier Transform Infrared Imaging (FTIRI). Analyses were done at the time of maximal osteocalcin expression (5 weeks). The spectroscopic parameters monitored were mineral-to-matrix ratio (indicative of the relative amount of mineral present), mineral crystallinity (index of the mineral crystal size and perfection) and collagen maturity (reflecting the ratio of non-reducible and reducible collagen cross-links). Multiple fields were selected for each femur, ranging from epiphysis to diaphysis. Previously, we showed that these transgenic mice display decreased osteoblastic function and osteopenia. In the present work, FTIRI showed that transgenic mice as compared to wild types have a different pattern of bone mineralization and matrix maturation. Specifically, cortical bone, primary spongiosa, and secondary ossification centers had lower values for mineral-to-matrix ratio and collagen maturity. Differences were not statistically significant in all cases although the trends were consistent. The mineral crystallinity did not vary significantly between the two groups, implying that the crystal maturation of mineral was not affected by IGFBP-5 overexpression. This study demonstrates that femurs from transgenic mice over expressing IGFBP-5 under the control of the osteocalcin promoter have modest alterations in mineral and matrix distribution, consistent with a role of IGF in osteoblast maturation.

Distribution of collagen cross-links in normal human trabecular bone.

UNLABELLED: Infrared imaging analysis of normal human iliac crest biopsy specimens shows a characteristic spatial variation in the nonreducible:reducible collagen cross-links at trabecular surfaces, depending on the surfaces' metabolic status. INTRODUCTION: Bone is a composite material consisting of mineral, collagen, non-collagenous proteins, and lipids. Bone collagen, mainly type I, provides the scaffold on which mineral is deposited and imparts specific mechanical properties, determined in part by the amount of collagen present, its orientation and fibril diameter, and the distribution of its cross-links. MATERIALS AND METHODS: In this study, the technique of Fourier transform infrared imaging (FTIRI) was used to determine the ratio of nonreducible:reducible cross-links, in 2- to 4-microm-thick sections from human iliac crest biopsy specimens (N = 14) at trabecular surfaces as a function of surface activity (forming versus resorbing), with an approximately 6.3-mm spatial resolution. The biopsy specimens were obtained from patients devoid of any metabolic bone disease based on histomorphometric and bone densitometric parameters. RESULTS AND CONCLUSIONS: Distributions of collagen cross-links within the first 50 mm at forming trabecular surfaces demonstrated a progressive increase in the nonreducible:reducible collagen cross-link ratio, unlike in the case of resorbing surfaces, in which the collagen cross-links ratio (as defined for the purposes of the present report) was relatively constant.

Spectroscopic imaging of mineral maturation in bovine dentin.

Dentin is a useful model for the study of mineral maturation. Using Fourier Transform Infrared Imaging (FTIRI), we characterized distinct regions in developing dentin at 7- micro m spatial resolution. Mineral-to-matrix ratio and crystallinity in bovine dentin from cervical and incisal parts of 3rd-trimester fetal compared with one-year-old incisor crowns showed that virtually all maturation stages in dentin could be spectroscopically isolated and analyzed. In the fetal incisors, mantle and circumpulpal dentin presented distinct patterns of mineral maturation. Gradients in both mineral properties examined were observed at the mineralization front and at the dentino-enamel junction.

Effects of transforming growth factor-beta deficiency on bone development: a Fourier transform-infrared imaging analysis.

Transforming growth factor-beta 1 (TGF-beta1) is a cytokine member of the TGF-beta superfamily involved in the control of proliferation and differentiation of various cell types. TGF-beta1 plays an important role in bone formation and resorption. To determine the effect of TGF-beta1 deficiency on bone mineral and matrix, tibias from mice in which TGF-beta1 expression had been ablated (TGF-beta1 null) were analyzed and compared with background- and age-matched wild-type (WT) control animals by Fourier transform-infrared imaging (FTIRI) and histochemistry. FTIRI allows the characterization of nondemineralized thin tissue sections at the ultrastructural level with a spatial resolution of approximately 7 microm. The spectroscopic parameters calculated were: mineral-to-matrix ratio (previously shown to correspond to ash weight); mineral crystallinity (related to the crystallographically determined crystallite size and perfection in the apatite c-axis direction); and collagen maturity (related to the ratio of pyridinoline:deH-DHLNL collagen cross-links). Several fields were selected to represent different stages of bone development within the same specimen from the secondary ossification center to the distal diaphysis. Anatomically equivalent areas were compared as a function of age and genotype. The spectroscopic results were expressed both as color-coded images and as pixel population distributions for each of the three parameters monitored. Based on comparisons of histochemistry and FTIRI, there were distinctive age and genotype variations. At all ages examined, in the TGF-beta1 null mice growth plates, alkaline phosphatase (ALP) activity and collagen maturity were reduced, but no effect on mineral content or crystallinity was noted. In the TGF-beta1 null mice metaphyses, there was a persistence of trabeculae, but no significant alterations in mineral content or crystallinity. In contrast, mineral content, mineral crystallinity, and collagen maturity were reduced in the secondary ossification center and cortical bone of the TGF-beta1 null mice. These results, consistent with a mechanism of impaired bone maturation in the TGF-beta1 null mice, may be directly related to TGF-beta1 deficiency and indirectly to increased expression of inflammatory cytokines in the TGFbeta1 null mice.

Variceal pressure is a strong predictor of variceal haemorrhage in patients with cirrhosis as well as in patients with non-cirrhotic portal hypertension.

BACKGROUND: Variceal pressure is a strong predictor for a first variceal bleed in patients with cirrhosis. AIMS: To evaluate whether variceal pressure is also a determinant of the risk of a first variceal bleed in patients with non-cirrhotic portal hypertension. METHODS: Variceal pressure was measured non-invasively in 25 patients with non-cirrhotic portal hypertension and large varices while receiving a stable therapeutic regimen. Factors predictive of bleeding were compared with those observed in 87 cirrhotics. RESULTS: The one year incidence of variceal bleeding was 32% (n=28) for the cirrhotic and 20% (n=5) for the non-cirrhotic patients. There was no difference in factors predicting the risk of bleeding between the groups, except for variceal pressure. For the same level of variceal pressure, the risk of variceal bleeding was lower in patients with non-cirrhotic portal hypertension. Multiple logistic regression analysis revealed the following variables as having a significant predictive power: variceal pressure (p=0.0001), red spots (p=0.004), and the time interval between the first observation of the varices and the moment of variceal pressure measurement (p=0. 0046). For the non-cirrhotics the risk of bleeding increased with higher Child-Pugh score (p=0.0024); this was not the case for the cirrhotic patients (p=0.9521). CONCLUSION: Variceal pressure is a major predictor of variceal bleeding in patients with cirrhosis as well as in patients with non-cirrhotic portal hypertension. The risk of bleeding in non-cirrhotics is less than in cirrhotics for the same level of variceal pressure. In patients with non-cirrhotic portal hypertension the risk of variceal bleeding increases more with advancing disease.

Inhibition of basal and corticotropin-releasing hormone-stimulated adenylate cyclase activity and cytosolic Ca2+ levels by somatostatin in human corticotropin-secreting pituitary adenomas.

The effects of CRH and somatostatin (SRIH) on adenylate cyclase (AC) activity, intracellular free calcium concentrations [( Ca2+]i) and in vitro ACTH release were investigated in six human ACTH-secreting pituitary adenomas. In all tumors, CRH induced a marked stimulation (from 69-210% at 10 nM), whereas SRIH caused a definite inhibition (from 29-50% at 100 nM) of membrane AC. When added together, CRH and SRIH caused a purely additive effect on AC. In adenomatous corticotrophs CRH (10 nM) caused [Ca2+]i to rise from 160 +/- 30 nM (mean +/- SD) to 410 +/- 95 nM. CRH-induced transients were biphasic, with an initial peak predominantly due to redistribution from intracellular Ca2+ stores and a secondary phase due to Ca2+ influx. The effects of CRH on [Ca2+]i were totally independent of the stimulation of AC. In fact, cAMP-elevating agents other than CRH did not modify [Ca2+]i. SRIH (100 nM) decreased resting [Ca2+]i (approximately 20-40%) as well as [Ca2+]i rises induced by CRH, arginine vasopressin, or high K+. The effect of SRIH on [Ca2+]i was maintained in presence of high cAMP levels, while was totally abolished after pertussis toxin pretreatment. CRH (10 nM) stimulated ACTH release (from 22.5 +/- 3.5 to 45.0 +/- 8.5 pmol/L) by an extent similar to that elicited by calcium ionophore and forskolin. By contrast, SRIH (0.1 microM) inhibited both basal and CRH-stimulated ACTH release. In conclusion, in human adenomatous corticotrophs SRIH exerts an inhibitory action by reducing both AC activity and, independently, [Ca2+]i. In this way, SRIH can efficiently counteract the stimulatory action of CRH that in these cells involves activation of both cAMP and Ca2+ pathways.