Dynamic loading stimulates mandibular condyle remodeling.

BACKGROUND: We and others have reported that low-magnitude high-frequency dynamic loading has an osteogenic effect on alveolar bone. Since chondrocytes and osteoblasts originate from the same progenitor cells, we reasoned that dynamic loading may stimulate a similar response in chondrocytes. A stimulating effect could be beneficial for patients with damaged condylar cartilage or mandibular deficiency. METHODS: Studies were conducted on growing Sprague-Dawley rats divided into three groups: control, static load, and dynamic load. The dynamic load group received a dynamic load on the lower right molars 5 minutes per day with a 0.3 g acceleration and peak strain of 30 µÎµ registered by accelerometer and strain gauge. The static load group received an equivalent magnitude of static force (30 µÎµ). The control group did not receive any treatment. Samples were collected at days 0, 28, and 56 for reverse transcriptase polymerase chain reaction analysis, microcomputed tomography, and histology and fluorescent microscopy analysis. RESULTS: Our experiments showed that dynamic loading had a striking effect on condylar cartilage, increasing the proliferation and differentiation of mesenchymal cells into chondrocytes, and promoting chondrocyte maturation. This effect was accompanied by increased endochondral bone formation resulting in lengthening of the condylar process. CONCLUSIONS: Low-magnitude, high-frequency dynamic loading can have a positive effect on condylar cartilage and endochondral bone formation in vivo. This effect has the potential to be used as a treatment for regenerating condylar cartilage and to enhance the effect of orthopedic appliances on mandibular growth.

Therapeutic effect of localized vibration on alveolar bone of osteoporotic rats.

OBJECTIVES: Vibration, in the form of high frequency acceleration (HFA), stimulates alveolar bone formation under physiologic conditions and during healing after dental extractions. It is not known if HFA has an anabolic effect on osteoporotic alveolar bone. Our objective is to determine if HFA has a regenerative effect on osteoporotic alveolar bone. METHODS AND MATERIALS: Adult female Sprague-Dawley rats were divided into five groups: 1) Ovariectomized Group (OVX), 2) Sham-OVX Group that received surgery without ovariectomy, 3) OVX-HFA Group that was ovariectomized and treated daily with HFA, 4) OVX+Static Force Group that was ovariectomized and received the same force as HFA, but without vibration, and 5) Control Group that did not receive any treatment. All animals were fed a low mineral diet for 3 months. Osteoporosis was confirmed by micro-CT of the fifth lumbar vertebra and femoral head. HFA was applied to the maxillary first molar for 5 minutes/day for 28 and 56 days. Maxillae were collected for micro-CT, histology, fluorescent microscopy, protein and RNA analysis, and three-point bending mechanical testing. RESULTS: Micro-CT analysis revealed significant alveolar bone osteoporosis in the OVX group. Vibration restored the quality and quantity of alveolar bone to levels similar to the Sham-OVX group. Animals exposed to HFA demonstrated higher osteoblast activity and lower osteoclast activity. Osteogenic transcription factors (RUNX2, Foxo1, Osterix and Wnt signaling factors) were upregulated following vibration, while RANKL/RANK and Sclerostin were downregulated. HFA did not affect serum TRAcP-5b or CTx-1 levels. The osteogenic effect was highest at the point of HFA application and extended along the hemimaxillae this effect did not cross to the contra-lateral side. CONCLUSIONS: Local application of vibration generated gradients of increased anabolic metabolism and decreased catabolic metabolism in alveolar bone of osteoporotic rats. Our findings suggest that HFA could be a predictable treatment for diminished alveolar bone levels in osteoporosis patients.

Different duration of parathyroid hormone exposure distinctively regulates primary response genes Nurr1 and RANKL in osteoblasts.

Parathyroid hormone (PTH) exerts dual effects, anabolic or catabolic, on bone when administrated intermittently or continuously, via mechanisms that remain largely unknown. PTH binding to cells induces PTH-responsive genes including primary response genes (PRGs). PRGs are rapidly induced without the need for de novo protein synthesis, thereby playing pivotal roles in directing subsequent molecular responses. In this study, to understand the role of PRGs in mediating osteoblastic cellular responses to PTH, we investigated whether various durations of PTH differentially induce PRGs in primary osteoblasts and MC3T3-E1. Nurr1 and RANKL, PRGs known for their anabolic and catabolic roles in bone metabolism respectively, presented distinctive transient vs. sustained induction kinetics. Corroborating their roles, maximum induction of Nurr1 was sufficiently achieved by brief PTH in as little as 30 minutes and continued beyond that, while maximum induction of RANKL was achieved only by prolonged PTH over 4 hours. Our data suggested distinctive regulatory mechanisms for Nurr1 and RANKL: PKA-mediated chromatin rearrangement for transcriptional regulation of both PRGs and ERK-mediated transcriptional regulation for RANKL but not Nurr1. Lastly, we classified PRGs into two groups based on the induction kinetics: The group that required brief PTH for maximum induction included Nur77, cox-2, and Nurr1, all of which are reported to play roles in bone formation. The other group that required prolonged PTH for maximum induction included IL-6 and RANKL, which play roles in bone resorption. Together, our data suggested the crucial role of PRG groups in mediating differential osteoblastic cellular responses to intermittent vs. continuous PTH. Continued research into the regulatory mechanisms of PKA and ERK for PRGs will help us better understand the molecular mechanisms underlying the dual effects of PTH, thereby optimizing the current therapeutic use of PTH for osteoporosis.

Deletion of Orai1 leads to bone loss aggravated with aging and impairs function of osteoblast lineage cells.

Osteoblast lineage cells, a group of cells including mesenchymal progenitors, osteoblasts, and osteocytes, are tightly controlled for differentiation, proliferation and stage-specific functions in processes of skeletal development, growth and maintenance. Recently, the plasma membrane calcium channel Orai1 was highlighted for its role in skeletal development and osteoblast differentiation. Yet the roles of Orai1 in osteoblast lineage cells at various stages of maturation have not been investigated. Herein we report the severe bone loss that occurred in Orai1-/- mice, aggravated by aging, as shown by the microcomputed tomography (mCT) and bone histomorphometry analysis of 8-week and 12-week old Orai1-/- mice and sex-matched WT littermates. We also report that Orai1 deficiency affected the differentiation, proliferation, and type I collagen secretion of primary calvarial osteoblasts, mesenchymal progenitors, and osteocytes in Orai1-/- mice; specifically, our study revealed a significant decrease in the expression of osteocytic genes Fgf23, DMP1 and Phex in the cortical long bone of Orai1-/- mice; a defective cellular and nuclear morphology of Orai1-/- osteocytes; and defective osteogenic differentiation of Orai1-/- primary calvarial osteoblasts (pOBs), including a decrease in extracellular-secretion of type I collagen. An increase in the mesenchymal progenitor population of Orai1-/- bone marrow cells was indicated by a colony forming unit-fibroblasts (CFU-F) assay, and the increased proliferation of Orai1-/- pOBs was indicated by an MTT assay. Notably, Orai1 deficiency reduced the nuclear localization and transcription activity of the Nuclear Factor of Activated T-cell c1 (NFATc1), a calcium-regulated transcription factor, in pOBs. Altogether, our study demonstrated the crucial role of Orai1 in bone development and maintenance, via its diverse effects on osteoblast lineage cells from mesenchymal progenitors to osteocytes.

Vibration paradox in orthodontics: Anabolic and catabolic effects.

Vibration in the form of High Frequency Acceleration (HFA) is anabolic on the craniofacial skeleton in the absence of inflammation. Orthodontic forces trigger an inflammation-dependent catabolic cascade that is crucial for tooth movement. It is unknown what effect HFA has on alveolar bone if applied during orthodontic treatment. The objectives of this study are to examine the effect of HFA on the rate of tooth movement and alveolar bone, and determine the mechanism by which HFA affects tooth movement. Adult Sprague Dawley rats were divided to control, orthodontic force alone (OTM), and different experimental groups that received the same orthodontic forces and different HFA regimens. Orthodontic tooth movement was assessed when HFA parameters, frequency, acceleration, duration of exposure, and direct or indirect application were varied. We found that HFA treatment significantly enhanced the inflammation-dependent catabolic cascade during orthodontic tooth movement. HFA treatment increased inflammatory mediators and osteoclastogenesis, and decreased alveolar bone density during orthodontic tooth movement. Each of the HFA variables produced significant changes in the rate of tooth movement and the effect was PDL-dependent. This is the first report that HFA enhances inflammation-dependent catabolic cascades in bone. The clinical implications of our study are highly significant, as HFA can be utilized to enhance the rate of orthodontic tooth movement during the catabolic phase of treatment and subsequently be utilized to enhance retention during the anabolic remodeling phase after orthodontic forces are removed.

Age-dependent biologic response to orthodontic forces.

INTRODUCTION: Orthodontic tooth movement results from increased inflammation and osteoclast activation. Since patients of all ages now routinely seek orthodontics treatment, we investigated whether age-dependent biologic responses to orthodontic force correlate with the rate of tooth movement. METHODS: We studied 18 healthy subjects, adolescents (11-14 years) and adults (21-45 years), with Class II Division 1 malocclusion requiring 4 first premolar extractions. Canines were retracted with a constant force of 50 cN. Gingival crevicular fluid was collected before orthodontic treatment and at days 1, 7, 14, and 28 after the canine retraction. Cytokine (IL-1ß, CCL2, TNF-α) and osteoclast markers (RANKL and MMP-9) were measured using antibody-based protein assays. Pain and discomfort were monitored with a numeric rating scale. The canine retraction rate was measured from study models taken at days 28 and 56. RESULTS: Although the cytokine and osteoclast markers increased significantly in both age groups at days 1, 7, and 14, the increases were greater in adults than in adolescents. Interestingly, the rate of tooth movement in adults was significantly slower than in adolescents over the 56-day study period. Adults also reported significantly more discomfort and pain. CONCLUSIONS: Age is a significant variable contributing to the biologic response to orthodontic tooth movement. Adults exhibited a significantly higher level of cytokine and osteoclasts activity but, counterintuitively, had a significantly slower rate of tooth movement.

Skeletal and soft-tissue incidental findings on cone-beam computed tomography images.

Cone-beam computed tomography provides orthodontists with 3-dimensional images of the craniofacial region and valuable information for diagnosis and treatment planning of craniofacial or dental anomalies. However, a narrow focus on the skeletal and dental contributions to malocclusion can cause failure to identify skeletal or soft-tissue pathologies of the craniofacial structures unrelated to the orthodontic concerns. Two cases are presented that demonstrate skeletal and soft-tissue anomalies identified as incidental findings on cone-beam computed tomography scans of asymptomatic orthodontics patients. One patient was diagnosed with craniofacial fibrous dysplasia; the other had an intrahemispheric lipoma. Their cone-beam computed tomography images are presented, along with a literature review on their pathologies.

Regulation of Nur77 gene expression by prostanoids in cementoblastic cells.

OBJECTIVE: The inflammatory cytokine interleukin-1 (IL-1) decreases mineralisation by immortalized mouse-derived cementoblastic cells (OC-CM cells), whilst various prostanoids, including fluprostenol (flup) increase it. Subtraction hybridisation conducted on flup minus IL-1-treated OC-CM cells revealed that one of the primary response genes preferentially induced by flup is the transcription factor Nur77. The objective of this study was to examine the signal transduction cascades regulating prostanoid induction of Nur77 gene expression in OC-CM cells. METHODS: Confluent OC-CM cells were treated with prostaglandin E(2) (PGE(2)), prostaglandin F(2alpha) (PGF(2alpha)), specific activators of the various EP prostanoid receptors and of the FP prostanoid receptor, and direct activators/inhibitors of the cyclic AMP-protein kinase A (PKA), protein kinase C (PKC) and intracellular calcium pathways. Nur77 gene expression was examined by mRNA extraction and Northern blot analysis. RESULTS: PGE(2) and PGF(2alpha) treatment of OC-CM cells significantly increased Nur77 mRNA expression in a time- and dose-dependent fashion. Both the EP1 prostanoid receptor-specific activator 16,16-dimethyl-PGE(2) and the FP prostanoid receptor-specific activator flup significantly increased Nur77 gene expression by OC-CM cells as compared to vehicle-treated controls. Increase in Nur77 gene expression was also observed when direct activators of the PKA, PKC and intracellular calcium pathways were used to treat OC-CM cells. Direct inhibition of the PKA, PKC and intracellular calcium pathways abrogated Nur77 gene expression induced by OC-CM cell treatment with PGE(2) and PGF(2alpha). CONCLUSION: Nur77 is a primary gene expressed by OC-CM cells and its induction appears to be mediated by the PKA, PKC and intracellular calcium pathways. Nur77 may affect expression of downstream target genes in OC-CM cells and partially regulate cementoblast cell function.

Nuclear receptor profile in calvarial bone cells undergoing osteogenic versus adipogenic differentiation.

Nuclear receptors (NRs) are key regulators of cell function and differentiation. We examined NR expression during osteogenic versus adipogenic differentiation of primary mouse calvarial osteoblasts (MOBs). MOBs were cultured for 21 days in osteogenic or adipogenic differentiation media. von Kossa and Oil Red O staining, and qRT-PCR of marker genes and 49 NRs were performed. PCR amplicons were subcloned to establish correct sequences and absolute standard curves. Forty-three NRs were detected at days 0-21. Uncentered average linkage hierarchical clustering identified four expression clusters: NRs (1) upregulated during osteogenic, but not adipogenic, differentiation, (2) upregulated in both conditions, with greater upregulation during adipogenic differentiation, (3) upregulated equally in both conditions, (4) downregulated during adipogenic, but not osteogenic, differentiation. One-way ANOVA with contrast revealed 20 NRs upregulated during osteogenic differentiation and 12 NRs upregulated during adipogenic differentiation. Two-way ANOVA demonstrated that 18 NRs were higher in osteogenic media, while 9 NRs were higher in adipogenic media. The time effect revealed 16 upregulated NRs. The interaction of condition with time revealed 6 NRs with higher expression rate during adipogenic differentiation and 3 NRs with higher expression rate during osteogenic differentiation. Relative NR abundance at days 0 and 21 were ranked. Basal ranking changed at least 5 positions for 13 NRs in osteogenic media and 9 NRs in adipogenic media. Osteogenic and adipogenic differentiation significantly altered NR expression in MOBs. These differences offer a fingerprint of cellular commitment and may provide clues to the underlying mechanisms of osteogenic versus adipogenic differentiation.

Prostanoid- and interleukin-1-induced primary genes in cementoblastic cells.

BACKGROUND: Cementum is a key component of a functional periodontal organ. However, regenerating lost cementum is difficult and often incomplete. Identifying molecular mediators of cementoblast differentiation and function should lead to better targeted treatment for periodontitis. Prostaglandins increase mineralization of murine cementoblastic OCCM cells and alveolar bone formation, whereas the cytokine interleukin-1 (IL-1) inhibits alveolar bone formation. We hypothesized that differentially induced primary genes in OCCM cells may mediate anabolic and catabolic responses. Our objective was to identify primary genes differentially induced by the synthetic prostanoid fluprostenol and IL-1 in cementoblastic cells. METHODS: Confluent OCCM cells were pretreated with the protein synthesis inhibitor cycloheximide followed by fluprostenol or IL-1 for 1.5 hours. cDNA generated from each group was used for cDNA subtraction hybridization to identify differentially induced genes. Preferential gene induction was verified by Northern blot analysis. RESULTS: Thirteen fluprostenol- and seven IL-1-regulated genes were identified. Among the fluprostenol-induced genes was mitogen-activated protein (MAP) kinase phosphatase 1 (MKP1), a negative regulator of MAP kinase signaling. To verify the cDNA subtraction hybridization results, OCCM cells were treated with fluprostenol or prostaglandin F2 (PGF2), and MKP1 mRNA levels were determined. The 0.001 to 1 microM fluprostenol and 0.01 to 1 microM PGF2 significantly induced MKP1 mRNA levels, which peaked at 1 hour of treatment and returned to baseline at 2 hours. CONCLUSIONS: Fluprostenol enhanced, whereas IL-1 inhibited, OCCM mineralization. Using cDNA subtraction hybridization, we identified primary genes that correlate with the observed anabolic and catabolic responses. These findings further our understanding of cementoblast function and suggest that differentially induced genes may mediate cementum formation and resorption.

PGC-1alpha is induced by parathyroid hormone and coactivates Nurr1-mediated promoter activity in osteoblasts.

Parathyroid hormone (PTH) potently activates cAMP-protein kinase A (PKA)-driven molecular cascades in osteoblasts. The NR4A/NGFI-B orphan nuclear receptor (NR) Nurr1 is a PTH-induced, cAMP-responsive primary response gene (PRG) that transactivates osteocalcin (Ocn) expression through a putative NGFI-B response element (NBRE) in the proximal promoter. As a true orphan NR, Nurr1's expression level and coactivator recruitment regulate its transactivation capacity. We postulated that Nurr1's induction through cAMP-PKA signaling might favor a coactivator that is likewise cAMP-dependent. A possible candidate is the cAMP-inducible coactivator PPARgamma coactivator-1alpha (PGC-1alpha). We hypothesize that PGC-1alpha is a PTH-induced PRG that synergizes with Nurr1 to induce target gene transcription in osteoblasts. We show that 10 nM PTH for 2 h maximally induced PGC-1alpha mRNA in primary mouse osteoblasts (MOBs) and calvariae. Selective signaling agonists and antagonists demonstrated that PTH induced PGC-1alpha mRNA primarily through the cAMP-PKA pathway. Protein synthesis inhibition sustained PTH-induced PGC-1alpha expression. PGC-1alpha enhanced Nurr1-induced transactivation of a consensus 3xNBRE-luciferase construct and the rat (-1050)Ocn promoter-luciferase construct from 3.7- to 9.6- and 10.1-fold, respectively. This synergy required Nurr1-DNA binding, since a mutation of the Ocn promoter NBRE abolished both Nurr1- and Nurr1-PGC-1alpha-induced transactivation. Using GST pull-down assays, PGC-1alpha directly interacted with in vitro-generated and nuclear Nurr1. We conclude that PGC-1alpha is a PTH-induced, cAMP-dependent PRG that directly synergizes with Nurr1 to transactivate target genes in osteoblasts. Taken together with published data, our findings suggest that Nurr1 and PGC-1alpha may be pivotal mediators of cAMP-induced osteoblast gene expression and osteoblast function.

Parathyroid hormone induces the NR4A family of nuclear orphan receptors in vivo.

Parathyroid hormone (PTH) has both anabolic and catabolic effects on bone metabolism, although the molecular mechanisms mediating these effects are largely unknown. Among the transcription factors induced by PTH in osteoblasts are the nerve growth factor-inducible factor B (NR4A; NGFI-B) family of orphan nuclear receptors: Nurr1, Nur77, and NOR-1. PTH induces NR4A members through the cAMP-protein kinase A (PKA) pathway in vitro. We report here that PTH rapidly and transiently induced expression of all three NR4A genes in PTH-target tissues in vivo. In calvaria, long bones, and kidneys, NR4A induction was maximal 0.5-1 h after a single intraperitoneal (i.p.) injection of 80 microg/kg PTH. Nur77 demonstrated the highest expression, followed, in order, by Nurr1 and NOR-1. In calvaria and long bone, PTH-induced expression of each NR4A gene was detectable at 10 microg/kg i.p. with maximum induction at 40-80 microg/kg. PTH (3-34) did not induce NR4A mRNA levels in calvaria, long bone, and kidney in vivo, confirming our in vitro results that NR4A genes are induced primarily through the cAMP-PKA pathway. The magnitude of PTH-induced NR4A expression was comparable in vivo and in vitro. However, NR4A mRNA levels peaked and returned to baseline faster in vivo. Both in vivo and in vitro, PTH induced NR4A pre-mRNA levels suggesting that induction of these genes is, at least in part, through activation of mRNA synthesis. The in vivo induction of the NR4A family members by PTH suggests their involvement in, at least some, PTH-induced changes in bone metabolism.

Nuclear orphan receptor Nurr1 directly transactivates the osteocalcin gene in osteoblasts.

Nurr1, an NGFI-B nuclear orphan receptor, which transactivates promoters through an NGFI-B response element (NBRE), is strongly induced by parathyroid hormone through the cAMP-protein kinase A signaling pathway in osteoblasts. Here, we demonstrate that multiple agents activating diverse signaling pathways in osteoblasts induce Nurr1. The strongest Nurr1 inducers were activators of cAMP-protein kinase A-coupled signaling, followed by protein kinase C- and calcium-coupled signaling activators. Receptor tyrosine kinase activators had minimal effect, whereas serine/threonine kinase activators had no effect on basal Nurr1 mRNA levels. Computer analysis of osteoblastic promoters indicated two potential NBREs in the rat osteocalcin (Ocn) promoter. Intriguingly, the proximal site maps to the cAMP-responsive cis-element. We tested whether Nurr1 induces Ocn expression through the NBRE-like site. Recombinant and endogenous Nurr1 protein from primary mouse osteoblasts bound to a consensus NBRE in EMSAs. Nurr1 induced a consensus 3 x NBRE-luciferase reporter construct in mouse osteoblasts. Recombinant and endogenous Nurr1 protein bound to the proximal NBRE-like site in the Ocn promoter in EMSAs. Endogenous Nurr1 protein bound to this site as a monomer, because neither retinoid X receptor alpha nor retinoid X receptor beta antibody supershifted the protein-DNA complex. Ocn promoter-luciferase constructs lacking or containing a mutated proximal NBRE-like site had markedly blunted responses to Nurr1 overexpression. Finally, adenovirally expressed Nurr1 protein bound to the proximal NBRE-like site in chromatin immunoprecipitation assays and induced Ocn mRNA in primary rat osteoblasts. We conclude that Ocn is a Nurr1 target gene, which positions Nurr1 in the core of transcriptional factors regulating osteoblastic gene expression.

Parathyroid hormone induces the nuclear orphan receptor NOR-1 in osteoblasts.

Parathyroid hormone (PTH) significantly affects osteoblast function by altering gene expression. We have identified neuron-derived orphan receptor-1 (NOR-1) as a PTH-induced primary gene in osteoblastic cells. NOR-1, Nurr1, and Nur77 comprise the NGFI-B nuclear orphan receptor family and Nurr1 and Nur77 are PTH-induced primary osteoblastic genes. Ten nM PTH maximally induced NOR-1 mRNA at 2h in primary mouse osteoblasts and at 1h in mouse calvariae. Cycloheximide pretreatment did not inhibit PTH-induced NOR-1 mRNA. PTH activates cAMP-protein kinase A (PKA), protein kinase C (PKC), and calcium signaling. Forskolin (PKA activator) and PMA (PKC activator) mimicked PTH-induced NOR-1 mRNA. Ionomycin (calcium ionophore) and PTH(3-34), which do not activate PKA, failed to induce NOR-1 mRNA. PKA inhibition with H89 blocked PTH- and FSK-induced NOR-1 mRNA. PMA pretreatment to deplete PKC inhibited PMA-induced, but not PTH-induced, NOR-1 mRNA. We conclude that NOR-1 is a PTH-regulated primary osteoblastic gene that is induced mainly through cAMP-PKA signaling.