Hematopoietic Stem Cells as a Novel Source of Dental Tissue Cells.

While earlier studies have suggested that cells positive for hematopoietic markers can be found in dental tissues, it has yet to be confirmed. To conclusively demonstrate this, we utilized a unique transgenic model in which all hematopoietic cells are green fluorescent protein+ (GFP+). Pulp, periodontal ligament (PDL) and alveolar bone (AvB) cell culture analysis demonstrated numerous GFP+ cells, which were also CD45+ (indicating hematopoietic origin) and co-expressed markers of cellular populations in pulp (dentin matrix protein-1, dentin sialophosphoprotein, alpha smooth muscle actin [ASMA], osteocalcin), in PDL (periostin, ASMA, vimentin, osteocalcin) and in AvB (Runx-2, bone sialoprotein, alkaline phosphatase, osteocalcin). Transplantation of clonal population derived from a single GFP+ hematopoietic stem cell (HSC), into lethally irradiated recipient mice, demonstrated numerous GFP+ cells within dental tissues of recipient mice, which also stained for markers of cell populations in pulp, PDL and AvB (used above), indicating that transplanted HSCs can differentiate into cells in dental tissues. These hematopoietic-derived cells deposited collagen and can differentiate in osteogenic media, indicating that they are functional. Thus, our studies demonstrate, for the first time, that cells in pulp, PDL and AvB can have a hematopoietic origin, thereby opening new avenues of therapy for dental diseases and injuries.

Mechanical Stretching of Mouse Calvarial Osteoblasts In Vitro Models Changes in MMP-2 and MMP-9 Expression at the Bone-Implant Interface.

Bone to mechanical loading elicits a biological response that has clinical significance for several areas in dental medicine, including orthodontic tooth movement, tempromandibular joint disease, and endosseous dental implant osseointegration. Human orthopedic studies of failed hip implant sites have identified increased mRNA expression of several collagen-degrading matrix metalloproteinases (MMPs), while in vitro experiments have shown increases in MMP secretion after exposure to inflammatory mediators. This investigation evaluates the effects of mechanical deformation on in vitro osteoblasts by assessing changes in MMP gene expression and enzyme activity. We seeded mouse neonatal calvarial osteoblasts onto flexible 6-well plates and subjected to continuous cyclic mechanical stretching. The expression and activity of mRNA for several MMPs (2, 3, 9, and 10) was assessed. When subjected to mechanical stress in culture, only mRNA specific for MMP-9 was significantly increased compared to nonstretched controls (P < .005). Measurement of MMP activity by gelatin zymography demonstrated that none of the MMPs showed increased activity with stretching; however, MMP-2 activity decreased. Our results suggest that in response to stretch, MMP-2 responds rapidly by inhibiting conversion of a MMP-2 to the active form, while a slower up-regulation of MMP-9 may play a role in the long-term remodeling of extracellular matrix in response to continuous mechanical loading. This study suggests that the regulation of metalloproteinases at both the mRNA and protein level are important in the response of bone to mechanical stress.

Removal of pamidronate from bone in rats using systemic and local chelation.

OBJECTIVES: Bisphosphonates become adsorbed on hydroxyapatite crystals in the bone matrix. In case of side-effects, stopping the treatment would not affect the bisphosphonates already deposited in bone. This study tests the feasibility of in-vivo targeted removal of bisphosphonates from bone using chelating agents. DESIGN: 32 Sprague Dawley rats were given an injection of fluorescent pamidronate (OsteoSense EX; 0.16nmol/g). They were treated with either systemic (cadmium) or local [ethylenediaminetetraacetic (EDTA) or citric acid (CA)] chelating agents to induce the removal of the bisphosphonate from bone. We evaluated the decrease in fluorescence in the alveolar bone, femur, tibia, and vertebrae. We also analyzed the systemic effects of treatment. RESULTS: Systemic chelation reduced the pamidronate signal universally. However, the maximum reduction was observed in the alveolar bone and femur (22% and 21%, p values 0.008 and 0.028, respectively). Systemic chelation did not impair calcium homeostasis. The chelation effect was not due to a systemic toxic effect on the liver or kidney. On the other hand local chelation at the extraction site significantly (p=0.011) decreased the pamidronate signal at bony surfaces of the socket. CONCLUSIONS: Systemic and local chelating agents can remove bisphosphonate from bone. This study establishes a new concept for the prevention of side effects of bisphosphonates during high-risk situations.

A Model for Osteonecrosis of the Jaw with Zoledronate Treatment following Repeated Major Trauma.

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 (i.v. 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.

Correlation of changes in the mandible and retina/choroid vasculature of a rat model of BRONJ.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) causes bones of the mandible and maxilla to become necrotic and protrude into the oral cavity. Compromised blood supply to bone is also a feature of BRONJ. The design of this study was first to use our established technique of molar extraction and IV bisphosphonate injection to produce features of BRONJ in rats that mimic the human disease; second to confirm vascular changes in the mandible and eye using micro-CT of vascular casts, and image analysis of retina/choroid images; and third to show parallel bisphosphonate-induced changes in the structure and markers of the vasculature of the bone and eye. The results of this study show structural changes in the eye and mandible as well as biochemical changes including the up-regulation of VEGF in response to the bisphosphonate-associated ischemia. These changes are not associated with angiogenesis in either the eye or mandible as determined by reduced vascular complexity. These results suggest that observations of direct changes to the vasculature in the retina/choroid structures of the eye in patients taking bisphosphonates could serve as a window to the progression of debilitating changes occurring as a result of bisphosphonate therapy.

In vitro study of the adverse effect of nicotine and physical strain on human gingival fibroblasts as a model of the healing of wounds commonly found in the military.

BACKGROUND: Significant adverse effects on fibroblast growth and metabolism are observed with nicotine. We investigated the synergistic effects of nicotine and cyclical mechanical strain (CMS) on human gingival fibroblasts (HGFs) in a wound-healing model. METHODS: HGFs were isolated and grown in Dulbecco's modified Eagle's medium. Three-millimeter wounds were created on a confluent cell monolayer grown in a media containing 0, 1, 2, or 4 mM nicotine, with or without CMS. The applied deformation regimen remains constant for 6 days. On days 1, 2, 4, and 6, the cells were stained with hematoxylin and eosin Y for the evaluation of wound repopulation. RESULTS: The application of CMS alone demonstrates a biphasic response, with an initial stimulatory effect on wound repopulation (days 1-2) and less repopulation during the later phase (days 4-6). The addition of nicotine clearly demonstrated a time and inverse dose-dependent relationship on wound repopulation, with no effect during the early phase and reduced wound repopulation during the later phase. CONCLUSIONS: Initial treatment of HGF wounds with CMS resulted in faster wound repopulation regardless of nicotine presence. By day 6, wound healing of HGF exposed to both nicotine and CMS is delayed. These findings suggest that CMS and nicotine may affect fibroblasts and delay wound healing at other sites in the body as well.

Histologic Evaluation of Osseous Regeneration Following Combination Therapy With Platelet-Rich Plasma and Bio-Oss in a Rat Calvarial Critical-Size Defect Model.

Platelet-rich plasma (PRP) is an autogenous source of growth factors shown to facilitate human bone growth. Bio-Oss, an osteoconductive xenograft, is used clinically to regenerate periodontal defects, restore dental alveolar ridges, and facilitate sinus-lift procedures. The purpose of this study was to analyze whether a combination of PRP and Bio-Oss would enhance bone regeneration better than either material alone. PRP and/or Bio-Oss were administered in an 8-mm critical-size defect (CSD) rat calvarial model of bone defect between 2 polytetrafluoroethylene membranes to prevent soft tissue incursion. Eight weeks after the induction of the CSD, histologic sections were stained with hematoxylin and eosin stain and analyzed via light microscopy. Qualitative analyses revealed new bone regeneration in all 4 groups. The Bio-Oss and PRP plus Bio-Oss groups demonstrated greater areas of closure in the defects than the control or PRP-only groups because of the space-maintaining ability of Bio-Oss. The groups grafted with Bio-Oss showed close contact with new bone growth throughout the defects, suggesting a stronger graft. The use of PRP alone or in combination with Bio-Oss, however, did not appear to enhance osseous regeneration at 8 weeks. Areas grafted with Bio-Oss demonstrated greater space-maintaining capacity than controls, and PRP was an effective vehicle for placement of the Bio-Oss. However, at 8 weeks this study was unable to demonstrate a significant advantage of using PRP plus Bio-Oss over using Bio-Oss alone.

Vascular alterations in the sprague-dawley rat mandible during intravenous bisphosphonate therapy.

Long-term use of intravenous bisphosphonates, such as zoledronic acid (zoledronate), has been linked to bisphosphonate-related osteonecrosis of the jaw (BRONJ). Invasive dental surgery seems to trigger the bone necrosis in most cases. To determine the effects of zoledronic acid on the vascular structure of the rat mandible. Extracted of the mandibular first molar in rats that received 2 IV injections of zoledronate (20 µg/kg), 4 weeks apart. Zoledronate-treated rats (n = 18) were then compared to a control group of untreated rats (n = 18). At the fourth, eighth, and 12th week after molar extraction, 8 rat mandibles from each group were perfused with 35% radiopaque triphenylbismuth in methyl methacrylate via carotid artery perfusion. Mandibles were harvested and examined by micro-CT to assess the spatial and dimensional changes of the vasculature as a result of zoledronate treatment. The micro-CT analysis showed that zoledronic acid-treated rats had blood vessels that were thicker, less connected, and less ordered than control rats that were not exposed to zoledronic acid. This study demonstrated that treatment with zoledronic acid in rats is associated with vascular changes in alveolar bone. Further studies are underway to explore whether these vascular changes contribute to the pathogenesis of BRONJ.

Effect of metformin on periimplant wound healing in a rat model of type 2 diabetes.

PURPOSE: To investigate the effects of hyperglycemia and metformin (a popular biguanide antidiabetic) on periimplant healing. METHODS: Thirty-six male rats were assigned to 3 groups: (1) nondiabetic Wistar-Kyoto rats (controls), (2) Goto-Kakizaki (GK) spontaneously diabetic rats (GK group), and (3) GK rats were fed metformin (100 mg/kg body weight per day) in their water for 4 weeks (GK + Met group). The right maxillary first molars were extracted and sites were allowed 1 month to heal. Titanium implants (1 × 3 mm) were placed in healed extraction sites. Six rats from each group were analyzed at weeks 1 and 4 by micro computed tomography for bone/implant contact ratio, percent bone volume, trabecular number, and bone mineral density. Blood was also analyzed for glucose, HbA1c, and pyridinoline (PYD). RESULTS: At week 1, glucose levels in the GK-Met rats were high, and all bone parameters were similar to GK rats (lower bone parameters and higher PYD than controls). At week 4, glucose levels in the GK-Met rats and all parameters were similar to controls. CONCLUSIONS: Hyperglycemic GK type 2 diabetic rats showed improved blood glucose and wound healing around oral implants after metformin administration.

Development of a rat model of bisphosphonate-related osteonecrosis of the jaw (BRONJ).

The purpose of this study was to develop a rat model predictive of bisphosphonate-related osteonecrosis of the jaw (BRONJ) after exodontias. Thirty female rats were randomized into 2 groups, control and experimental. The experimental group received 2 intravenous injections of zoledronate (20 µg/kg). The mesial root of the right mandibular first molar was extracted. Rats were euthanized at 0, 4, and 8 weeks. Bone mineral density (BMD), collagen breakdown (pyridinium [PYD]), vascular regeneration (VEGF), and histology were examined. A trend toward higher PYD values was suggested in control vs experimental groups after wounding. Serum VEGF increased significantly after wounding for both control and experimental groups. After 8 weeks, VEGF continued to rise for the experimental group only. In the extraction socket area, BMD was significantly lower after wounding in control vs. zoledronate-treated rats. Histology sections from experimental groups showed bacteria and bone necrosis. Consistent findings of BRONJ features similar to those in humans were observed after zoledronate treatment.

Inhibition of endogenous dentin matrix metalloproteinases by ethylenediaminetetraacetic acid.

INTRODUCTION: Endogenous dentin matrix metalloproteinases (MMPs) contribute to extracellular collagen matrix degradation in hybrid layers after adhesive dentin bonding procedures. Endodontic irrigants, including chlorhexidine and ethylenediaminetetraacetic acid (EDTA), might 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 4 experimental groups on the basis of exposure time to 17% EDTA (0, 1, 2, or 5 minutes). A generic colorimetric MMP assay measured MMP activity via absorbance at 412 nm. Data were evaluated by Kruskal-Wallis analysis of variance, followed by Dunn pair-wise comparisons at α = 0.05. RESULTS: All exposure times resulted in significant inhibition (P < .001) compared with unexposed controls. Specifically, percent inhibition for 1-, 2-, and 5-minute exposure times was 55.1% ± 21.5%, 72.8% ± 11.7%, and 74.7% ± 19.7%, respectively. CONCLUSIONS: Seventeen percent EDTA significantly inhibits endogenous MMP activity of human dentin within 1-2 minutes. This might minimize hybrid layer degradation after resin bonding procedures in the root canal space.

Preparation, physical-chemical characterization, and cytocompatibility of polymeric calcium phosphate cements.

Aim. Physicochemical mechanical and in vitro biological properties of novel formulations of polymeric calcium phosphate cements (CPCs) were investigated. Methods. Monocalcium phosphate, calcium oxide, and synthetic hydroxyapatite were combined with either modified polyacrylic acid, light activated polyalkenoic acid, or polymethyl vinyl ether maleic acid to obtain Types I, II, and III CPCs. Setting time, compressive and diametral strength of CPCs was compared with zinc polycarboxylate cement (control). Specimens were characterized using X-ray diffraction, scanning electron microscopy, and infrared spectroscopy. In vitro cytotoxicity of CPCs and control was assessed. Results. X-ray diffraction analysis showed hydroxyapatite, monetite, and brushite. Acid-base reaction was confirmed by the appearance of stretching peaks in IR spectra of set cements. SEM revealed rod-like crystals and platy crystals. Setting time of cements was 5-12 min. Type III showed significantly higher strength values compared to control. Type III yielded high biocompatibility. Conclusions. Type III CPCs show promise for dental applications.

Polymeric-calcium phosphate cement composites-material properties: in vitro and in vivo investigations.

New polymeric calcium phosphate cement composites (CPCs) were developed. Cement powder consisting of 60 wt% tetracalcium phosphate, 30 wt% dicalcium phosphate dihydrate, and 10 wt% tricalcium phosphate was combined with either 35% w/w poly methyl vinyl ether maleic acid or polyacrylic acid to obtain CPC-1 and CPC-2. The setting time and compressive and diametral tensile strength of the CPCs were evaluated and compared with that of a commercial hydroxyapatite cement. In vitro cytotoxicity and in vivo biocompatibility of the two CPCs and hydroxyapatite cement were assessed. The setting time of the cements was 5-15 min. CPC-1 and CPC-2 showed significantly higher compressive and diametral strength values compared to hydroxyapatite cement. CPC-1 and CPC-2 were equivalent to Teflon controls after 1 week. CPC-1, CPC-2, and hydroxyapatite cement elicited a moderate to intense inflammatory reaction at 7 days which decreased over time. CPC-1 and CPC-2 show promise for orthopedic applications.

Advanced glycation endproducts and rat dental implant osseointegration.

Advanced glycation endproducts (AGEs) are a diverse group of molecular adducts formed in environments high in reducing sugars that accumulate with aging and in diabetes. This study tests the hypothesis that AGEs inhibit the stabile osseointegration of dental implants through tissue interactions that interfere with bone turnover and compromise the biomechanical properties at the bone-implant interface. Maxillary first molars were extracted from 32 rats and allowed to heal for 4 weeks. Titanium implants (1 mm x 3 mm) were placed in the healed sockets of 2 groups of 16 rats consisting of 8 rats injected 3 times/wk for 1 month with AGE (prepared from glucose and lysine) and 8 rats injected with vehicle as a control. AGE injections continued for an additional 14 or 28 days before sacrifice. X-ray images, blood, and tissues were collected to examine bone/implant contact ratio, serum pyridinoline ([PYD] a collagen breakdown marker), osteocalcin ([OSC] a bone formation marker), and for immunohistochemistry with antibodies to AGE and the bone turnover-marker protein matrix metalloproteinase1. Compared with the AGE-treated groups, the controls showed significantly higher bone/implant contact at both 14- and 28-day time points. PYD (P < .05) and OSC (trend) levels from controls showed decreases at 28 days when compared with AGE-treated groups. Immunohistochemistry with AGE-specific and bone turnover marker antibodies showed stronger staining associated with the implant/tissue interface in AGE-treated rats. Our studies indicate an association between AGE and inhibition of bone turnover, suggesting that the formation of AGE in high glycemic conditions, such as diabetes, may contribute to a slower rate of osseointegration that negatively affects implant stability.

Cytotoxicity, calcium release, and pH changes generated by novel calcium phosphate cement formulations.

Few published studies describe the biological properties of calcium phosphate cements (CPCs) for dental applications. We measured several biologically relevant properties of 3 CPCs over an extended (8 wk) interval. Monocalcium phosphate, calcium oxide, and synthetic hydroxyapatite were combined with either modified polyacrylic acid, light-activated modified polyalkenoic acid, or 35% w/w polymethyl vinyl ether maleic acid to obtain Types I, II, and III CPCs, respectively. Set cements were placed in direct contact with L929 fibroblasts for up to 8 weeks. Media Ca(+2) and pH were determined by atomic absorption spectroscopy and pH electrode respectively. Cell mitochondrial function was measured by MTT assay. Type I cements suppressed mitochondrial activity > 90% (vs. Teflon controls), but significantly (p < 0.05) improved to control levels over 8 weeks. Type II cements suppressed mitochondrial activity > 90% at all times. Type III cements elevated mitochondrial activity significantly after 7 wks. The pH profiles approached neutrality by 24 h, and all cements released calcium into the storage medium at all periods (24 h - 8 wk). We concluded that several types of cements had long-term biological profiles that show promise for dental applications.

Effect of systemic parathyroid hormone (1-34) and a beta-tricalcium phosphate biomaterial on local bone formation in a critical-size rat calvarial defect model.

OBJECTIVE: The objective of this study was to evaluate local bone formation following systemic administration of parathyroid hormone (1-34) (PTH), a surgically implanted synthetic beta-tricalcium phosphate (beta-TCP) bone biomaterial serving as a matrix to support new bone formation. MATERIALS AND METHODS: Critical-size, 8 mm, calvarial through-and-through osteotomy defects were surgically created in 100 adult male Sprague-Dawley rats. The animals were randomized into five groups of 20 animals each to receive one of the following treatments: PTH (15 microg PTH/kg/day; subcutaneously), PTH/beta-TCP, beta-TCP, or particulate human demineralized freeze-dried bone (DFDB), and sham-surgery controls. Ten animals/group were euthanized at 4 and 8 weeks post-surgery for radiographic and histometric analysis. RESULTS: The histometric analysis showed that systemic PTH significantly enhanced local bone formation, bone fill averaging (+/-SE) 32.2+/-4.0% compared with PTH/beta-TCP (15.7+/-2.4%), beta-TCP (12.5+/-2.3%), DFDB (14.5+/-2.3%), and sham-surgery control (10.0+/-1.5%) at 4 weeks (p<0.014). Systemic PTH showed significantly enhanced bone formation (41.5+/-4.0%) compared with PTH/beta-TCP (22.4+/-3.0%), beta-TCP (21.3+/-4.4%), and with the sham-surgery control (23.8+/-4.2%) at 8 weeks (p<0.025). The DFDB group showed significantly increased bone formation from 4 (14.5+/-2.3%) to 8 weeks (32.0+/-3.2%) (p<0.006). The PTH/beta-TCP and beta-TCP groups both showed limited biomaterials resorption. The radiographic analysis was not diagnostic to distinguish local bone formation from the radiopaque beta-TCP biomaterial. CONCLUSIONS: Systemic administration of PTH significantly stimulates local bone formation. Bone formation was significantly limited by the beta-TCP biomaterial.

Rat model for studying tissue changes induced by the mechanical environment surrounding loaded titanium implants.

PURPOSE: This study presents a new rat oral implant model for assessing histologic changes in the mechanical environment surrounding loaded and unloaded dental implants. MATERIALS AND METHODS: The maxillary left first molar from retired breeder rats was extracted, and the site was allowed to heal for 1 month. A titanium miniscrew implant was then placed into the site and allowed to heal for 21 days. The mandibular left first molars in one group of rats were extracted to create an unloaded condition; in a second group of rats the mandibular left first molars were left in occlusion with the opposing screw head to simulate loading. Radiographs were taken on the day of placement and again at 7 days, 14 days, and 21 days after placement and were used to estimate the bone-implant contact ratio. The rats were sacrificed after 21 days. Peri-implant tissue samples from day 21 were processed for histology and immunohistochemistry with antibodies to osteocalcin and matrix metalloproteinase 13 (MMP-13). Two-dimensional finite element models were created from images of the histologic sections and immunohistochemical samples to observe tissue changes. RESULTS: Areas of high shear stress adjacent to the helical threads of loaded implants were associated with osteocalcin localization and bone formation but only minimal localization of MMP-13. Bone adjacent to unloaded implants showed fibrous tissue and extensive MMP-13 localization surrounding the apical two-thirds of each implant. These results agree with estimated bone-implant contact ratios, which showed a steady decrease in contact ratio for the unloaded implant group but a significantly higher contact ratio in the loaded group between 14 and 21 days. CONCLUSION: The rat oral implant model is useful for studies of the mechanical and physiologic environment affecting osseointegration in loaded and unloaded implants.

Tensile strain-induced Ets-2 phosphorylation by CaMKII and the homeostasis of cranial sutures.

BACKGROUND: Mechanotransduction underpins the homeostasis of musculoskeletal tissues, including cranial sutures. Intracellular calcium, [Ca 2+]ic, and protein phosphorylation are two intermediate variables in signal relay during mechanotransduction. This project establishes a chain of cause and effect, linking cellular strain to substrate phosphorylation, and identifies the agent and target sites of phosphorylation. METHODS: Cyclic tensile force (0.5 N at 1 Hz) was applied to 1-day-old rat sagittal sutures. [Ca 2+]ic was measured by FURA-2. Ets-2 phosphorylation by CaMKII was tested using Western blot autoradiography. Peptide array was constructed to determine the precise sites of phosphorylation. The results were confirmed with mass spectroscopy and Western blots using phospho-specific antibodies. RESULTS: [Ca 2+]ic increased rapidly in response to tensile stress. In the presence of Ca2+, CaMKII caused Ets-2 phosphorylation. Of the three possible sites for phosphorylation of Ets-2 by CaMKII, RVPS, FESF, RLSS, Serine 246, 310, and 313 were the targets. Furthermore, the contiguous sequence modified this effect. Mass spectroscopy showed 80 Da (molecular weight of phosphate group, -PO3) right shifts consistent with phosphorylation. There was cytosolic translocation of Ets-2 on tensile deformation of suture cells. CaMKII binding of Ets-2 occurred within 30 minutes after the onset of tensile strain. CONCLUSIONS: Cranial suture cells can respond to tensile forces by increasing [Ca 2+]ic, which causes CaMKII to phosphorylate Ets-2, thus altering Ets-2 binding to its downstream promoters. Of note, Ets-2 is at the intersection of three key pathways important in craniosynostosis: fibroblast growth factor-2, transforming growth factor-beta, and mechanotransduction.

Effect of ovariectomy and alendronate on implant osseointegration in rat maxillary bone.

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.

Age-related changes in the osteogenic differentiation potential of mouse bone marrow stromal cells.

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 age-dependent 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 lineage-associated genes. Our data suggest that the decline in BMSC number and osteogenic differentiation capacity are important factors contributing to age-related bone loss.