Connexin hemichannels drive lactation-induced osteocyte acidification and perilacunar-canalicular remodeling.

The maternal skeleton experiences significant bone loss during lactation, followed by rapid restoration post weaning. Parathyroid-related protein (PTHrP)-induced acidification of the perilacunar matrix by osteocytes is crucial in this process, yet its mechanism remains unclear. Here, we identify Cx43 hemichannels (HCs) as key mediators of osteocyte acidification and perilacunar-canalicular remodeling (PLR). Utilizing transgenic mouse models expressing dominant-negative Cx43 mutants, we show that mice with impaired Cx43 HCs exhibit attenuated lactation-induced responses compared to wild-type and only gap junction-impaired groups, including lacunar enlargement, upregulation of PLR genes, and bone loss with compromised mechanical properties. Furthermore, inhibition of HCs by a Cx43 antibody blunts PTHrP-induced calcium influx and protein kinase A activation, followed by impaired osteocyte acidification. Additionally, impeded HCs suppress bone recovery during the post-lactation period. Our findings highlight the pivotal role of Cx43 HCs in orchestrating dynamic bone changes during lactation and recovery by regulating acidification and remodeling enzyme expression.

Promotion of Joint Degeneration and Chondrocyte Metabolic Dysfunction by Excessive Growth Hormone in Mice.

OBJECTIVE: Many patients with acromegaly, a hormonal disorder with excessive growth hormone (GH) production, report pain in joints. We undertook this study to characterize the joint pathology of mice with overexpression of bovine GH (bGH) or a GH receptor antagonist (GHa) and to investigate the effect of GH on regulation of chondrocyte cellular metabolism. METHODS: Knee joints from mice overexpressing bGH or GHa and wild-type (WT) control mice were examined using histology and micro-computed tomography for osteoarthritic (OA) pathologies. Additionally, cartilage from bGH mice was used for metabolomics analysis. Mouse primary chondrocytes from bGH and WT mice, with or without pegvisomant treatment, were used for quantitative polymerase chain reaction and Seahorse respirometry analyses. RESULTS: Both male and female bGH mice at ~13 months of age had increased knee joint degeneration, which was characterized by loss of cartilage structure, expansion of hypertrophic chondrocytes, synovitis, and subchondral plate thinning. The joint pathologies were also demonstrated by significantly higher Osteoarthritis Research Society International and Mankin scores in bGH mice compared to WT control mice. Metabolomics analysis revealed changes in a wide range of metabolic pathways in bGH mice, including beta-alanine metabolism, tryptophan metabolism, lysine degradation, and ascorbate and aldarate metabolism. Also, bGH chondrocytes up-regulated fatty acid oxidation and increased expression of Col10a. Joints of GHa mice were remarkably protected from developing age-associated joint degeneration, with smooth articular joint surface. CONCLUSION: This study showed that an excessive amount of GH promotes joint degeneration in mice, which was associated with chondrocyte metabolic dysfunction and hypertrophic changes, whereas antagonizing GH action through a GHa protects mice from OA development.

Overexpression of ameloblastin in secretory ameloblasts results in demarcated, hypomineralized opacities in enamel.

Introduction: Developmental defects of the enamel manifest before tooth eruption and include amelogenesis imperfecta, a rare disease of underlying gene mutations, and molar-incisor hypomineralization (MIH), a prevalent disease in children originating from environmental and epigenetic factors. MIH enamel presents as the abnormal enamel marked by loss of translucency, demarcation between the healthy and affected enamel, and reduced mineral content. The pathophysiology of opaque, demarcated enamel lesions is not understood; however, the retention of enamel proteins in the matrix has been suggested. Ameloblastin (Ambn) is an enamel protein of the secreted calcium-binding phosphoproteins (SCPPs) critical for enamel formation. When the Ambn gene is mutated or deleted, teeth are affected by hypoplastic amelogenesis imperfecta. Methods: In this study, enamel formation in mice was analyzed when transgenic Ambn was overexpressed from the amelogenin promoter encoding full-length Ambn. Ambn was under- and overexpressed at six increasing concentrations in separate mouse lines. Results: Mice overexpressing Ambn displayed opaque enamel at low concentrations and demarcated lesions at high concentrations. The severity of enamel lesions increased starting from the inner enamel close to the dentino-enamel junction (DEJ) to span the entire width of the enamel layer in demarcated areas. Associated with the opaque enamel were 17-kDa Ambn cleavage products, a prolonged secretory stage, and a thin basement membrane in the maturation stage. Ambn accumulations found in the innermost enamel close to the DEJ and the mineralization front correlated with reduced mineral content. Demarcated enamel lesions were associated with Ambn species of 17 kDa and higher, prolonged secretory and transition stages, a thin basement membrane, and shortened maturation stages. Hypomineralized opacities were delineated against the surrounding mineralized enamel and adjacent to ameloblasts detached from the enamel surface. Inefficient Ambn cleavage, loss of contact between ameloblasts, and the altered basement membrane curtailed the endocytic activity; thus, enamel proteins remained unresorbed in the matrix. Ameloblasts have the ability to distinguish between Ambn concentration and Ambn cleavage products through finely tuned feedback mechanisms. The under- or overexpression of Ambn in murine secretory ameloblasts results in either hypoplastic amelogenesis imperfecta or hypomineralization with opaque or sharply demarcated boundaries of lesions, similar to MIH.

A New Method of Bone Stromal Cell Characterization by Flow Cytometry.

The bone microenvironment cellular composition plays an essential role in bone health and is disrupted in bone pathologies, such as osteoporosis, osteoarthritis, and cancer. Flow cytometry protocols for hematopoietic stem cell lineages are well defined and well established. Additionally, a consensus for mesenchymal stem cell flow markers has been developed. However, flow cytometry markers for bone-residing cells-osteoblasts, osteoclasts, and osteocytes-have not been proposed. Here, we describe a novel partial digestion method to separate these cells from the bone matrix and present new markers for enumerating these cells by flow cytometry. We optimized bone digestion and analyzed markers across murine, nonhuman primate, and human bone. The isolation and staining protocols can be used with either cell sorting or flow cytometry. Our method allows for the enumeration and collection of hematopoietic and mesenchymal lineage cells in the bone microenvironment combined with bone-residing stromal cells. Thus, we have established a multi-fluorochrome bone marrow cell-typing methodology. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Partial digestion for murine long bone stromal cell isolation Alternate Protocol 1: Partial digestion for primate vertebrae stromal cell isolation Alternate Protocol 2: Murine vertebrae crushing for bone stromal cell isolation Basic Protocol 2: Staining of bone stromal cells Support Protocol 1: Fluorescence minus one control, isotype control, and antibody titration Basic Protocol 3: Cell sorting of bone stromal cells Alternate Protocol 3: Flow cytometry analysis of bone stromal cells Support Protocol 2: Preparing compensation beads.

CSF-1 in Osteocytes Inhibits Nox4-mediated Oxidative Stress and Promotes Normal Bone Homeostasis.

CSF-1 is a key factor in regulating bone remodeling; osteocytes express CSF-1 and its receptor. Viable osteocytes are essential for bone remodeling through cell-cell contact and secretion of factors that regulate osteoblasts and osteoclasts. Increased oxidative stress contributes to osteocyte death and correlates with bone loss during aging. The NADPH oxidase Nox4 is a major source of ROS in bone. CSF-1 decreases Nox4, suggesting that CSF-1 protects against oxidative stress. Here, we show that osteocyte apoptosis previously reported in our global CSF-1KO mice is associated with increased Nox4, as well as 4-HNE expression in osteocytes. Osteocytes isolated from CSF-1KO mice were less viable and showed increased intracellular ROS, elevated NADPH oxidase activity/Nox4 protein, activation of mTOR/S6K, and downstream apoptosis signals compared with WT osteocytes. Nox4 expression was also increased in CSF-1KO osteocytes and colocalized with MitoTracker Red in mitochondria. Notably, CSF-1 inhibited Nox4 expression and apoptosis cascade signals. In additional studies, shNox4 decreased these signals in CSF-1KO osteocytes, whereas overexpression of Nox4 in WT osteocytes activated the apoptosis pathway. To determine the role of CSF-1 in osteocytes, DMP1Cre-CSF-1cKO (CSF-1cKO) mice that lack CSF-1 in osteocytes/late osteoblasts were developed. Osteocyte defects in CSF-1cKO mice overlapped with those in CSF-1KO mice, including increased apoptosis, Nox4, and 4-HNE-expressing osteocytes. CSF-1cKO mice showed unbalanced cancellous bone remodeling with decreased bone formation and resorption. Continued exposure to high Nox4/ROS levels may further compromise bone formation and predispose to bone loss and skeletal fragility. Taken together, our findings suggest a novel link between CSF-1, Nox4-derived ROS, and osteocyte survival/function that is crucial for osteocyte-mediated bone remodeling. Results reveal new mechanisms by which CSF-1/oxidative stress regulate osteocyte homeostasis, which may lead to therapeutic strategies to improve skeletal health in aging. © 2018 American Society for Bone and Mineral Research.

Healthcare disparities in adolescent idiopathic scoliosis: the impact of socioeconomic factors on Cobb angle.

STUDY DESIGN: Retrospective chart review. OBJECTIVES: The aim of this study is to assess the role of insurance type, geographic socioeconomic status, and ethnicity in AIS disease severity in a state with mandated scoliosis screenings. Early detection of adolescent idiopathic scoliosis (AIS) is associated with reduced curve progression, surgical treatment, and long-term sequelae. Type of insurance, ethnicity, and socioeconomic status are important determinants in healthcare access. METHODS: Data were obtained for 561 AIS patients aged 10-18 years, living within a single county, and presenting to a single healthcare system for initial evaluation of AIS between 2010 and 2016 that met inclusion criteria. Demographic data including gender, age, self-reported ethnicity, insurance, and zip code were collected. Outcome measures included Cobb angle, curve severity, and referral delay. A single fellowship-trained pediatric orthopedic surgeon calculated presenting Cobb angle for each case. Zip code was used as a proxy for household income level. Independent sample t tests, analysis of variance and covariance, and χ2 analysis were used to determine the significant differences and correlations. RESULTS: Female patients (n = 326, CA = 22.4°) had significantly greater Cobb angle measurements compared with male patients (n = 117, CA = 18.1°). Patients with government-supported insurance had significantly higher Cobb angles (CA = 22.1°) than privately insured patients (CA = 19.2°) but were both classified within the "mild" range clinically, and are likely not clinically significant. There was no correlation between income level and Cobb angle. Referral delay and Cobb angle severity did not vary by age, income, or insurance. A χ2 analysis showed no association between Cobb angle and race. CONCLUSIONS: Cobb angle severity was not influenced by SES factors, including ethnicity and household income. LEVEL OF EVIDENCE: Level-II.

Connexin 43 hemichannels protect bone loss during estrogen deficiency.

Estrogen deficiency in postmenopausal women is a major cause of bone loss, resulting in osteopenia, osteoporosis, and a high risk for bone fracture. Connexin 43 (Cx43) hemichannels (HCs) in osteocytes play an important role in osteocyte viability, bone formation, and remodeling. We showed here that estrogen deficiency reduced Cx43 expression and HC function. To determine if functional HCs protect osteocytes and bone loss during estrogen deficiency, we adopted an ovariectomy model in wild-type (WT) and two transgenic Cx43 mice: R76W (dominant-negative mutant inhibiting only gap junction channels) and Cx43 Δ130-136 (dominant-negative mutant compromising both gap junction channels and HCs). The bone mineral density (BMD), bone structure, and histomorphometric changes of cortical and trabecular bones after ovariectomy were investigated. Our results showed that the Δ130-136 transgenic cohort had greatly decreased vertebral trabecular bone mass compared to WT and R76W mice, associated with a significant increase in the number of apoptotic osteocyte and empty lacunae. Moreover, osteoclast surfaces in trabecular and cortical bones were increased after ovariectomy in the R76W and WT mice, respectively, but not in ∆130-136 mice. These data demonstrate that impairment of Cx43 HCs in osteocytes accelerates vertebral trabecular bone loss and increase in osteocyte apoptosis, and further suggest that Cx43 HCs in osteocytes protect trabecular bone against catabolic effects due to estrogen deficiency.

Validity of the Hispanic version of the American Academy of Orthopaedic Surgeons Foot and Ankle Outcomes Questionnaire in patients with traumatic foot and ankle injuries.

BACKGROUND: Hispanics represent the largest minority group in the United States and are projected to represent 29% of the US population by 2060. Enrolling Hispanic patients in clinical outcome trials is critical to study a representative sample of the general population. Lack of translated and validated survey tools has been identified as a major barrier to enrolling Spanish speaking patients. The purpose of this validation study was to study the correlation between the Spanish translation of the American Academy of Orthopaedic Surgeons Foot and Ankle Outcomes questionnaire (AAOS-FAOQ) and the Spanish versions of the Foot Function Index (FFI) and the Foot Health Status Questionnaire (FHSQ) in Hispanics from Mexican lineage with traumatic foot and ankle injuries. METHODS: A cross-sectional validation study in 36 Hispanic patients from Mexican lineage with foot and ankle injuries was performed. The Hispanic version of the AAOS-FAOQ and the Spanish translations of the FAOQ, FHSQ, FFI, and the Short-Form 36 questionnaire (SF-36) were distributed among all patients. Subsequent statistical analysis correlating the Hispanic version of the AAOS-FAOQ to the FFI, FHSQ, and SF-36 was performed. Additional analysis on the Hispanic AAOS-FAOQ included test-retest reliability and internal consistency. RESULTS: The Hispanic AAOS-FAOQ Global Foot and Ankle subscale showed statistically significant (P < .05) correlations with 5 of 8 subscales of the FHSQ, the FFI, and the Physical Component Summary subscale of the SF-36. The AAOS-FAOQ Global Foot & Ankle Scale also demonstrated a test-retest reliability of 0.736 and a strong internal consistency. CONCLUSIONS: This study further validates AAOS-FAOQ in Mexican Hispanics by showing strong correlations with the validated Spanish versions of the FFI and FHSQ.

Ethnic differences in patients' perceptions towards isolated orthopedic injuries: a pilot study.

BACKGROUND: Patients' perceptions of their healthcare have been reported to influence clinical outcomes following orthopedic trauma. Findings across clinical outcomes have demonstrated significant differences in perceptions towards healthcare between Hispanics and non-Hispanic whites. However, ethnic disparities in perceptions towards orthopedic injuries have not been examined in the literature. AIM OF STUDY: The aim of this pilot study is to explore whether Hispanic patients with isolated orthopedic injuries will demonstrate different perceptions towards their injury as compared to non-Hispanic white patients. The pilot data will be used to inform a subsequent larger clinical investigation and interventional study. METHODS: A total of 43 patients (31 Hispanics and 12 non-Hispanic whites) with isolated orthopedic injuries requiring surgical treatment were enrolled in this cross-sectional observational pilot study. Outcome measures included the Questionnaire of Perceived Injustice (QPI), Short-Form 36 Health Survey (SF-36v2), Pain Catastrophizing Scale, and Consumer Assessment of Healthcare Providers and Systems (CAHPS) Cultural Competence (CC) item set. RESULTS: The CAHPS was completed by 34 patients, and the remaining scoring systems were completed by all 43 subjects enrolled in this study. Hispanic patients trended towards higher QPI scores indicating poorer outcomes than non-Hispanic whites (mean difference [MD] 5.4, 95%; confidence interval [CI] - 4.4, 15.2). The mental component summary score of the SF-36 trended lower in Hispanics as compared to non-Hispanic white (MD - 6.8, 95%; CI - 15.0, 1.4). Hispanic patients also expressed less trust in their doctor on a scale from 0 to 10 (MD - 1.0, 95%; CI - 1.9, - 0.1). CONCLUSIONS: Our study suggests ethnic differences in patients' perceptions towards isolated orthopedic injuries. These results must be interpreted cautiously given the limited number of subjects in this pilot examination. We collected sufficient data to allow a sample size calculation for a subsequent larger clinical investigation. Future clinical investigations may determine the influence of ethnic differences in patients' perceptions towards orthopedic injuries, identify their impact on the functional outcomes, and establish intervention strategies.

Publication Productivity of Orthopaedic Surgery Chairs.

BACKGROUND: As academic leaders, orthopaedic chairs represent role models for scholarly activities. Despite the importance of journal publications as a measure of scholarly activity, data on the publication productivity of orthopaedic chairs remain limited. The goals of this study were to record the publication productivity of orthopaedic chairs and evaluate the extent to which they maintained their scholarly activity while serving as chairs. METHODS: The chairs of all orthopaedic residency programs in the United States were identified through the Accreditation Council for Graduate Medical Education (ACGME) web site, and were confirmed by information found on the web site of each orthopaedic program that was included in the study. University and non-university chairs were defined based on affiliation of the program with a medical school. The publication records of the program chairs were retrieved through the Scopus database. RESULTS: During the 7 years prior to their appointment to chair, the mean number of total publications was significantly higher for university chairs (n = 58.6, range 0 to 217) than for non-university chairs (n = 29.1, range 0 to 13) (p = 0.003). The mean number of publications per year during the 7 years leading up to the chair position was 4.66 (range, 0 to 25) for the university chairs, and 2.29 (range, 0 to 10.9) for the non-university group (p = 0.02). While serving as chair, the mean number of publications per year significantly decreased among the university chairs to 3.75 (range, 0 to 32.8; p = 0.015), whereas no significant change was observed among non-university chairs. The mean percentage of first authorships was not significantly different between university and non-university chairs. Both groups showed significant declines in first authorships while serving as chair. CONCLUSIONS: At the time of becoming chair, the average university chair had published approximately 60 manuscripts, whereas the average non-university chair had published approximately 30 manuscripts. While serving as chair, the number of publications per year significantly decreased for university chairs. Among all chairs, the percentage of first authorships significantly decreased while serving as chair.

Spanish Translation, Cross-Cultural Adaptation, and Validation of the American Academy of Orthopaedic Surgeons Foot and Ankle Outcomes Questionnaire in Mexican-Americans With Traumatic Foot and Ankle Injuries.

BACKGROUND: Hispanics represent the largest minority group within the US population accounting for an estimated 55.4 million individuals. Enrolling Hispanics into clinical outcome studies is important in order for study populations to be externally valid and representative of the US population. Inclusion of Mexican-Americans in clinical studies is frequently limited by the lack of validated outcome measures. The goal of this study was to validate a Spanish version of the American Academy of Orthopaedic Surgeons Foot and Ankle Outcomes Questionnaire (AAOS-FAOQ) in Mexican-Americans with traumatic foot and ankle injuries. METHODS: The translation and cross-cultural adaptation procedure was performed by a committee of bilingual speakers using the following steps: (1) forward translation and adaptation, (2) synthesis, (3) back translation, (4) committee review, and (5) pilot testing. The validation was performed in 100 Mexican-Americans with traumatic foot and ankle injuries. RESULTS: A total of 41 females and 59 males were enrolled in this study. The mean age was 42.98 years (range 18-88). The Spanish version of the Global Foot and Ankle Scale of the AAOS-FAOQ showed statistically significant correlations with all 8 subscales of the Spanish SF-36 as well as the Physical Component Summary scale and the Mental Component Summary scale (P < 0.05). The Global Foot and Ankle scale of the Spanish AAOS-FAOQ demonstrated a test-retest reliability of 0.68. CONCLUSION: We provide a Spanish translation and cross-cultural adaptation of the AAOS-FAOQ. The instrument demonstrates appropriate psychometric properties in Mexican-Americans with traumatic foot and ankle injuries.

Is Diabetic Skeletal Fragility Associated with Microvascular Complications in Bone?

PURPOSE OF REVIEW: The objective of this literature review is to determine whether there are indications that microvascular complications occur in diabetic bone. Evidence definitively linking diabetic skeletal fragility with microvascular complications in bone remains elusive. RECENT FINDINGS: Circumstantial evidence, some recent and some lost to time, suggests that atherosclerotic vascular diseases such as peripheral arterial disease cause poor blood perfusion of bone and subsequent hypoxia and contribute to low bone density and high cortical porosity, patterns similar to some recently observed in diabetic subjects. Evidence also exists to suggest that potentially anti-angiogenic conditions, such as impaired vascular endothelial growth factor (VEGF) signaling, predominate in diabetic bone. Microvascular complications may contribute, in part, to diabetic skeletal fragility but data supporting this interpretation are primarily circumstantial at this time. This review highlights gaps in our knowledge and hopefully spurs further discussions and research on this topic.

Capsaicin-sensitive Innervation Modulates the Development of Apical Periodontitis.

INTRODUCTION: Nociceptive neurons play a critical role in the detection of stimuli evoking actual or potential tissue injury. In addition, they are involved in neurogenic inflammation by the peripheral release of neuropeptides such as calcitonin gene-related peptide (CGRP). The dental pulp and periradicular tissues are innervated by capsaicin-sensitive neurons known to release CGRP. However, the role of these capsaicin-sensitive neurons in the development of apical periodontitis is largely unknown. The aim of this study was to evaluate the contribution of peptidergic neurons to the development of apical periodontitis. METHODS: Neonatal Sprague-Dawley rats were injected with vehicle (control group) or a single subcutaneous capsaicin dose to cause the selective ablation of peptidergic neurons (neonatal capsaicin group). Ablation of capsaicin-sensitive neurons was verified with confocal microscopy, capsaicin-induced eye-wipe nocifensive behavior test, and by measurement of immunoreactive CGRP levels in the dental pulp. Five weeks after ablation, standardized pulp exposures were made in the mandibular left first molars. Mandibles were harvested at 7, 14, 21, and 28 days after pulp exposure and imaged with micro-computed tomography (µCT) to quantify apical lesion volume. Data were analyzed by using 2-way ANOVA analysis with Bonferroni post hoc test. RESULTS: Rats in the control group displayed a robust capsaicin-induced nocifensive behavior, which was nearly abolished in the neonatal capsaicin group. In addition, the neonatal capsaicin group showed a significant depletion of susceptible neurons and CGRP in the dental pulp compared with control. Importantly, micro-computed tomography analysis showed larger periradicular lesions at 7 and 14 days after pulp exposure in the neonatal capsaicin group when compared with control. CONCLUSIONS: Results identify a protective role for capsaicin-sensitive neurons in the initial phase of apical periodontitis. Thus, interventions or disorders that alter activity of capsaicin-sensitive fibers are likely to alter the development of apical periodontitis.

Maturation stage enamel malformations in Amtn and Klk4 null mice.

Amelotin (AMTN) and kallikrein-4 (KLK4) are secreted proteins specialized for enamel biomineralization. We characterized enamel from wild-type, Amtn(-/-), Klk4(-/-), Amtn(+/-)Klk4(+/-) and Amtn(-/-)Klk4(-/-) mice to gain insights into AMTN and KLK4 functions during amelogenesis. All of the null mice were healthy and fertile. The mandibular incisors in Amtn(-/-), Klk4(-/-) and Amtn(-/-)Klk4(-/-) mice were chalky-white and chipped. No abnormalities except in enamel were observed, and no significant differences were detected in enamel thickness or volume, or in rod decussation. Micro-computed tomography (µCT) maximum intensity projections localized the onset of enamel maturation in wild-type incisors distal to the first molar, but mesial to this position in Amtn(-/-), Klk4(-/-) and Amtn(-/-)Klk4(-/-) mice, demonstrating a delay in enamel maturation in Amtn(-/-) incisors. Micro-CT detected significantly reduced enamel mineral density (2.5 and 2.4gHA/cm(3)) in the Klk4(-/-) and Amtn(-/-)Klk4(-/-) mice respectively, compared with wild-type enamel (3.1gHA/cm(3)). Backscatter scanning electron microscopy showed that mineral density progressively diminished with enamel depth in the Klk4(-/-) and Amtn(-/-)Klk4(-/-) mice. The Knoop hardness of the Amtn(-/-) outer enamel was significantly reduced relative to the wild-type and was not as hard as the middle or inner enamel. Klk4(-/-) enamel hardness was significantly reduced at all levels, but the outer enamel was significantly harder than the inner and middle enamel. Thus the hardness patterns of the Amtn(-/-) and Klk4(-/-) mice were distinctly different, while the Amtn(-/-)Klk4(-/-) outer enamel was not as hard as in the Amtn(-/-) and Klk4(-/-) mice. We conclude that AMTN and KLK4 function independently, but are both necessary for proper enamel maturation.

Estrogen and estrogen receptor alpha promotes malignancy and osteoblastic tumorigenesis in prostate cancer.

The role of estrogen signaling in regulating prostate tumorigenesis is relatively underexplored. Although, an increasing body of evidence has linked estrogen receptor beta (ERß) to prostate cancer, the function of estrogen receptor alpha (ERα) in prostate cancer is not very well studied. We have discovered a novel role of ERα in the pathogenesis of prostate tumors. Here, we show that prostate cancer cells express ERα and estrogen induces oncogenic properties in prostate cancer cells through ERα. Importantly, ERα knockdown in the human prostate cancer PacMetUT1 cells as well as pharmacological inhibition of ERα with ICI 182,780 inhibited osteoblastic lesion formation and lung metastasis in vivo. Co-culture of pre-osteoblasts with cancer cells showed a significant induction of osteogenic markers in the pre-osteoblasts, which was attenuated by knockdown of ERα in cancer cells suggesting that estrogen/ERα signaling promotes crosstalk between cancer and osteoblastic progenitors to stimulate osteoblastic tumorigenesis. These results suggest that ERα expression in prostate cancer cells is essential for osteoblastic lesion formation and lung metastasis. Thus, inhibition of ERα signaling in prostate cancer cells may be a novel therapeutic strategy to inhibit the osteoblastic lesion development as well as lung metastasis in patients with advanced prostate cancer.

Connexin 43 channels are essential for normal bone structure and osteocyte viability.

Connexin (Cx) 43 serves important roles in bone function and development. Targeted deletion of Cx43 in osteoblasts or osteocytes leads to increased osteocyte apoptosis, osteoclast recruitment, and reduced biomechanical properties. Cx43 forms both gap junction channels and hemichannels, which mediate the communication between adjacent cells or between cell and extracellular environments, respectively. Two transgenic mouse models driven by a DMP1 promoter with the overexpression of dominant negative Cx43 mutants were generated to dissect the functional contribution of Cx43 gap junction channels and hemichannels in osteocytes. The R76W mutant blocks the gap junction channel, but not the hemichannel function, and the Δ130-136 mutant inhibits activity of both types of channels. Δ130-136 mice showed a significant increase in bone mineral density compared to wild-type (WT) and R76W mice. Micro-computed tomography (µCT) analyses revealed a significant increase in total tissue and bone area in midshaft cortical bone of Δ130-136 mice. The bone marrow cavity was expanded, whereas the cortical thickness was increased and associated with increased bone formation along the periosteal area. However, there is no significant alteration in the structure of trabecular bone. Histologic sections of the midshaft showed increased apoptotic osteocytes in Δ130-136, but not in WT and R76W, mice which correlated with altered biomechanical and estimated bone material properties. Osteoclasts were increased along the endocortical surface in both transgenic mice with a greater effect in Δ130-136 mice that likely contributed to the increased marrow cavity. Interestingly, the overall expression of serum bone formation and resorption markers were higher in R76W mice. These findings suggest that osteocytic Cx43 channels play distinctive roles in the bone; hemichannels play a dominant role in regulating osteocyte survival, endocortical bone resorption, and periosteal apposition, and gap junction communication is involved in the process of bone remodeling.

Correlation of ameloblastin with enamel mineral content.

In enamel formation, the deposition of minerals as crystallites starts when the mineralization front first forms at the start of the secretory stage. During maturation, the enamel layer accumulates significant amounts of new mineral as the crystallites grow in volume. Inversely related to mineral gain is loss of protein and water from the forming enamel. Both ameloblastin (Ambn) and enamelin are essential components for formation of a functional enamel layer. The aim of this study was to quantify the proportion of mineral and non-mineral material present in developing enamel relative to Ambn concentration using Ambn mutant mice mated with others overexpressing full-length Ambn from the mouse amelogenin promoter at lower (+), similar (++) or higher (+++) concentration than normal. Mandibular incisors (age: 7 weeks, n = 8) were imaged by micro-computed tomography and the enamel was analyzed from the apical region to the incisal edge in sequential 1.0 mm volumes of interest. Mineral density was determined using a series of hydroxyapatite (HA) phantoms to calibrate enamel density measurements. At the site where the mandibular incisor emerged into the oral cavity, the enamel volume, mineral weight, and mineral density were reduced when Tg Ambn was expressed at lower or higher levels than normal. While in wild-type the % mineral was >95%, it was negligible in Ambn-/-, 22.3% in Ambn-/-, Tg(+), 75.4% in Ambn-/-, Tg(++), and 45.2% in Ambn-/-, Tg(+++). These results document that the deposition of mineral and removal of non-mineral components are both very sensitive to expressed Ambn concentrations.

Caspase-2 maintains bone homeostasis by inducing apoptosis of oxidatively-damaged osteoclasts.

Osteoporosis is a silent disease, characterized by a porous bone micro-structure that enhances risk for fractures and associated disabilities. Senile, or age-related osteoporosis (SO), affects both men and women, resulting in increased morbidity and mortality. However, cellular and molecular mechanisms underlying senile osteoporosis are not fully known. Recent studies implicate the accumulation of reactive oxygen species (ROS) and increased oxidative stress as key factors in SO. Herein, we show that loss of caspase-2, a cysteine aspartate protease involved in oxidative stress-induced apoptosis, results in total body and femoral bone loss in aged mice (20% decrease in bone mineral density), and an increase in bone fragility (30% decrease in fracture strength). Importantly, we demonstrate that genetic ablation or selective inhibition of caspase-2 using zVDVAD-fmk results in increased numbers of bone-resorbing osteoclasts and enhanced tartrate-resistant acid phosphatase (TRAP) activity. Conversely, transfection of osteoclast precursors with wild type caspase-2 but not an enzymatic mutant, results in a decrease in TRAP activity. We demonstrate that caspase-2 expression is induced in osteoclasts treated with oxidants such as hydrogen peroxide and that loss of caspase-2 enhances resistance to oxidants, as measured by TRAP activity, and decreases oxidative stress-induced apoptosis of osteoclasts. Moreover, oxidative stress, quantified by assessment of the lipid peroxidation marker, 4-HNE, is increased in Casp2-/- bone, perhaps due to a decrease in antioxidant enzymes such as SOD2. Taken together, our data point to a critical and novel role for caspase-2 in maintaining bone homeostasis by modulating ROS levels and osteoclast apoptosis during conditions of enhanced oxidative stress that occur during aging.

A review of rodent models of type 2 diabetic skeletal fragility.

Evidence indicating that adult type 2 diabetes (T2D) is associated with increased fracture risk continues to mount. Unlike osteoporosis, diabetic fractures are associated with obesity and normal to high bone mineral density, two factors that are typically associated with reduced fracture risk. Animal models will likely play a critical role in efforts to identify the underlying mechanisms of skeletal fragility in T2D and to develop preventative treatments. In this review we critically examine the ability of current rodent models of T2D to mimic the skeletal characteristics of human T2D. We report that although there are numerous rodent models of T2D, few have undergone thorough assessments of bone metabolism and strength. Further, we find that many of the available rodent models of T2D have limitations for studies of skeletal fragility in T2D because the onset of diabetes is often prior to skeletal maturation and bone mass is low, in contrast to what is seen in adult humans. There is an urgent need to characterize the skeletal phenotype of existing models of T2D, and to develop new models that more closely mimic the skeletal effects seen in adult-onset T2D in humans.

Conditional deletion of cytochrome p450 reductase in osteoprogenitor cells affects long bone and skull development in mice recapitulating antley-bixler syndrome: role of a redox enzyme in development.

NADPH-cytochrome P450 oxidoreductase (POR) is the primary electron donor for cytochromes P450, dehydrocholesterol reductase, heme oxygenase, and squalene monooxygenase. Human patients with specific mutations in POR exhibit severe developmental malformations including disordered steroidogenesis, sexual ambiguities and various bone defects, similar to those seen in patients with Antley-Bixler syndrome (ABS). To probe the role of POR during bone development, we generated a conditional knockout mouse (CKO) by cross breeding Por (lox/lox) and Dermo1 Cre mice. CKO mice were smaller than their littermate controls and exhibited significant craniofacial and long bone abnormalities. Differential staining of the CKO mice skull bases shows premature fusion of the sphenooccipital and basioccipital-exoccipital synchondroses. Class III malocclusion was noted in adult knockout mice with an unusual overgrowth of the lower incisors. Shorter long bones were observed along with a reduction in the bone volume fraction, measured by microCT, in the Por-deleted mice compared to age- and sex-matched littermate controls. Concerted up- or down-regulation of proteins in the FGF signaling pathway observed by immunohistochemistry in the tibia samples of CKO mice compared to wild type controls shows a decrease in the FGF signaling pathway. To our knowledge, this is the first report of a mouse model that recapitulates both skull and long bone defects upon Por deletion, offering an approach to study the sequelae of POR mutations. This unique model demonstrates that P450 metabolism in bone itself is potentially important for proper bone development, and that an apparent link exists between the POR and FGF signaling pathways, begging the question of how an oxidation-reduction flavoprotein affects developmental and cellular signaling processes.