The purpose of this study is to test predicted form-function relationships between cranial suture complexity and masticatory muscle mass and biomechanics in a mouse model. Specifically, to test the hypothesis that increased masticatory muscle mass increases sagittal suture complexity, we measured the fractal dimension (FD), temporalis mass, and temporalis bite force in myostatin-deficient (GDF8(-/-)) mice and wild-type CD-1 mice (all male, 6 months old). Myostatin is a negative regulator of muscle mass, and myostatin-deficient mice show a marked increase in muscle mass compared to normal mice. We predicted that increased sagittal suture complexity would decrease suture stiffness. The data presented here demonstrate that increased suture complexity (measured as FD) was observed in a hypermuscular mouse model (GDF8(-/-)) with significantly increased temporalis muscle mass and bite forces. Hypermuscular mice were also found to possess suture connective tissue that was less stiff (i.e., underwent more displacement before failure occurred) when loaded in tension. By decreasing stiffness, suture complexity apparently helps to dissipate mechanical loads within the cranium that are related to chewing. These results suggest that cranial suture connective tissue locally adapts to functional demands of the biomechanical suture environment. As such, cranial sutures provide a novel model for studies in connective tissue mechanotransduction.

Age-dependent bone loss has been well documented in both human and animal models. Although the underlying causal mechanisms are probably multifactorial, it has been hypothesized that alterations in progenitor cell number or function are important. Little is known regarding the properties of bone marrow stromal cells (BMSCs) or bone progenitor cells during the aging process, so the question of whether aging alters BMSC/progenitor osteogenic differentiation remains unanswered. In this study, we examined agedependent changes in bone marrow progenitor cell number and differentiation potential between mature (3 and 6 mo old), middle-aged (12 and 18 mo old), and aged (24 mo old) C57BL/6 mice. BMSCs or progenitors were isolated from five age groups of C57BL/6 mice using negative immunodepletion and positive immunoselection approaches. The osteogenic differentiation potential of multipotent BMSCs was determined using standard osteogenic differentiation procedures. Our results show that both BMSC/progenitor number and differentiation potential increase between the ages of 3 and 18 mo and decrease rapidly thereafter with advancing age. These results are consistent with the changes of the mRNA levels of osteoblast lineageassociated genes. Our data suggest that the decline in BMSC number and osteogenic differentiation capacity are important factors contributing to age-related bone loss.

In an effort to extend our studies on Ca2+ pumps to animal models, we developed a new monoclonal antibody (5F10) prepared against the human erythrocyte Ca2+-Mg2+-adenosinetriphosphatase (ATPase) that recognizes a protein of approximately 140 kDa in rat kidney homogenates. Enzyme-linked immunosorbent assays show that monoclonal antibody 5F10 binds purified Ca2+-Mg2+-ATPase and rat kidney membrane extracts in a concentration-dependent manner. In paraffin-embedded tissue sections, antibody 5F10 binds to an epitope in the basolateral membranes of rat kidney distal convoluted tubule principal cells. The antibody does not bind to intercalated cells. The latter cells were characterized by the presence of large amounts of carbonic anhydrase C. Polyclonal antibodies directed against chick intestinal 28-kDa vitamin D-dependent calcium binding protein (28-kDa CaBP) also bind epitopes in distal convoluted tubule cells, connecting tubules, and portions of collecting duct but not intercalated cells. Western blot and 45Ca blot analysis of renal cytosolic proteins showed that the polyclonal 28-kDa CaBP-directed antibody detects a protein which also binds calcium. Western blot analysis with monoclonal antibody 5F10 shows binding to both the authentic purified erythrocyte Ca2+ pump (approximately 138 kDa) and to tryptic fragments of this pump. Antibody JA3, previously used for staining of human kidney tubules, reacts with a different set of tryptic fragments, showing that the two antibodies are directed against different regions or conformational determinants on the pump molecule. We show that Ca2+-Mg2+-ATPase and 28-kDa CaBP are present in the principal cells of the distal convoluted tubule of the rat and are absent in intercalated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Bisphosphonates such as alendronate (ALD), although controversial, are worthy of investigation for the enhancement of implant osseointegration in patients with low bone mass who are already taking bisphosphonates for osteoporosis. These patients may receive additional benefits and be acceptable candidates for dental implants without needing to change their medication regimen and possibly as a result of their medication regimen. The purpose of this study was to compare implant osseointegration in maxillary bone of normal rats with a rat model of postmenopausal estrogen deficiency (ovariectomized [OVX]), with and without ALD. An experimental group of 32 rats was divided in 4 groups: ALD-OVX (n=8 OVX with ALD), OVX (n=8 OVX without ALD), ALD (n=8 normal rats with ALD), and control (n=8 normal rats). All rats received one titanium microscrew implant in the left edentulous region of the maxillary arch. The ALD-OVX and ALD groups received subcutaneous injections of ALD 3 times a week. On the fourth week after ALD administration, an implant was placed in all 32 rats. The maxilla of each rat was radiographed 4 times: at 0, 7, 14, and 28 days. On day 28 after implant placement, all rats were killed, and the peri-implant tissue was embedded in plastic or paraffin for histological examination. The X rays were used for a chronologic calculation of the contact ratio between implant and bone surfaces. Radiographic bone density was determined at 3 points: mesial, apical, and distal. The results show that osseointegration of the implants was impaired in the estrogen-deficient OVX rats compared with the ALD-OVX rats. Fifty percent of the implants were lost at 2 weeks in the OVX group. Radiographic evidence suggested that none of the implants in the OVX group osseointegrated. In the histologic examination more bone was observed around implants from the ALD-OVX and ALD groups than around implants from the OVX group. The OVX group presented a dramatic reduction in implant bone contact at 2 weeks and a significant 13% reduction at 4 weeks vs day of implant (P = .006). The ALD-OVX group presented 50% more bone density than the OVX group (P = .0003). Both ALD groups (ALD and ALD-OVX) had significantly higher radiographic bone density than the other groups (P < .01 for each comparison). In conclusion, osseointegration of implants was enhanced by ALD. Radiographic bone density and contact ratio improved with ALD administration. Implant osseointegration was impaired by estrogen deficiency in the OVX group.

Introduction Endogenous dentin matrix metalloproteinases (MMPs) contribute to extracellular collagen matrix degradation in hybrid layers following adhesive dentin bonding procedures. Endodontic irrigants, including chlorhexidine (CHX) and ethylenediaminetetraacetic acid (EDTA) may help protect the hybrid layer from this process. The objective of the present study was to determine the exposure time necessary for EDTA to inactivate endogenous MMP activity in human dentin. Methods Dentin beams (2×1×3 mm) were prepared from mid-coronal dentin of extracted third molars. The beams were demineralized in 10 wt% phosphoric acid which also activated endogenous MMPs, and were divided into four experimental groups based on exposure time to 17% EDTA (0, 1, 2 or 5 min). A generic colorimetric MMP assay measured MMP activity via absorbance at 412 nm. Data were evaluated by Kruskal Wallis ANOVA, followed by Dunn’s pair-wise comparisons at α = 0.05. Results All exposure times resulted in significant inhibition (P<0.001) compared to unexposed controls. Specifically, percent inhibition for 1-, 2-, and 5-minute exposure times were 55.1±21.5%, 72.8±11.7%, and 74.7±19.7%, respectively. Conclusions 17% EDTA significantly inhibits endogenous MMP activity of human dentin within 1–2 min. This may minimize hybrid layer degradation following resin bonding procedures in the root canal space.

Bone remodeling in response to force requires the coordinated action of osteoblasts, osteoclasts, osteocytes, and periodontal ligament cells. Coordination among these cells may be mediated, in part, by cell-to-cell communication via gap junctions. This study tests the hypothesis that the regulation of expression of connexin 43, a gap junction protein, is part of the transduction mechanism between force as applied to bone during orthodontic tooth movement and bone remodeling. To test this hypothesis, we examined connexin 433 expression in a rat model system of experimental tooth movement. To establish the model, we extracted maxillary first molars to initiate supra-eruption of opposing mandibular molars. The rats were killed at 0, 6, 12, 24, and 48 hrs post-extraction. The mandibles were removed, demineralized, and embedded in paraffin. To localize connexin 43 protein and mRNA, we used a specific antibody for immunohistochemistry and a specific cDNA probe for in situ hybridization. Western and Northern blot analyses were used to assess the specificity of the connexin 43 antibody and cDNA probe, respectively. We found connexin 43 protein expressed by osteoclasts (++ ++) and periodontal ligament cells (++ +) in compression zones, and by osteoblasts (++ ++) and osteocytes (++ ++) in tension zones of the periodontal ligament. In addition, connexin 43 mRNA was found in some bone and periodontal ligament cells. Connexin 43 protein was found, by densitometric analysis, to be higher in the periodontal ligament after exposure to force compared with controls (P < 0.001). The number of osteocytes expressing connexin 43 48 hrs after molar extraction was also significantly greater in bone subjected to tension when compared with controls (P < 0.001). The results of this study support the hypothesis that connexin 43 plays a role in the coordination of events during experimentally induced alveolar bone remodeling.

This study aims to develop a reproducible rat model for post-traumatic bisphosphonate-related osteonecrosis of the jaw (BRONJ). In our previous studies using dental extraction as an inducing factor, only 30% - 60% of zoledronate-treated animals fulfilled the definition of clinical BRONJ. We modified the zoledronate regimen and introduced repeated surgical extraction to illicit quantifiable BRONJ in all animals. Eighty retired-breeder female Sprague-Dawley rats were divided between the treatment (IV zoledronate; 80 μg/kg/week for 13 weeks) and control (saline) groups. On week 13, the left mandibular first molar was surgically extracted, followed by the second molar a week later. Animals were euthanized at 1-week, 2-weeks, and 8-weeks following extraction. The occurrence and severity of BRONJ were scored in each animal based on gross and MicroCT analysis. Parameters of bone formation and osteoclast functions at the extraction site were compared between groups. All zoledronate-treated animals developed a severe case of BRONJ that fulfilled the clinical definition of the condition in humans. Osteoclast attachment continued to be defective eight weeks after stopping the treatment. There were no signs of kidney or liver toxicity. Our data confirmed that repeated surgical extraction (major trauma) by itself consistently precipitated massive bone necrosis in ZA-treated animals, eliminating the need to induce pre-existing infection or comorbidity. These results will be the basis for further studies examining the in-vivo pathogenesis and prevention of BRONJ.

Systemic administration of PTH significantly stimulates local bone formation. Bone formation was significantly limited by the beta-TCP biomaterial.