Increased Osteoblast GαS Promotes Ossification by Suppressing Cartilage and Enhancing Callus Mineralization During Fracture Repair in Mice.

GαS, the stimulatory G protein α-subunit that raises intracellular cAMP levels by activating adenylyl cyclase, plays a vital role in bone development, maintenance, and remodeling. Previously, using transgenic mice overexpressing GαS in osteoblasts (GS-Tg), we demonstrated the influence of osteoblast GαS level on osteogenesis, bone turnover, and skeletal responses to hyperparathyroidism. To further investigate whether alterations in GαS levels affect endochondral bone repair, a postnatal bone regenerative process that recapitulates embryonic bone development, we performed stabilized tibial osteotomy in male GS-Tg mice at 8 weeks of age and examined the progression of fracture healing by micro-CT, histomorphometry, and gene expression analysis over a 4-week period. Bone fractures from GS-Tg mice exhibited diminished cartilage formation at the time of peak soft callus formation at 1 week post-fracture followed by significantly enhanced callus mineralization and new bone formation at 2 weeks post-fracture. The opposing effects on chondrogenesis and osteogenesis were validated by downregulation of chondrogenic markers and upregulation of osteogenic markers. Histomorphometric analysis at times of increased bone formation (2 and 3 weeks post-fracture) revealed excess fibroblast-like cells on newly formed woven bone surfaces and elevated osteocyte density in GS-Tg fractures. Coincident with enhanced callus mineralization and bone formation, GS-Tg mice showed elevated active ß-catenin and Wntless proteins in osteoblasts at 2 weeks post-fracture, further substantiated by increased mRNA encoding various canonical Wnts and Wnt target genes, suggesting elevated osteoblastic Wnt secretion and Wnt/ß-catenin signaling. The GS-Tg bony callus at 4 weeks post-fracture exhibited greater mineral density and decreased polar moment of inertia, resulting in improved material stiffness. These findings highlight that elevated GαS levels increase Wnt signaling, conferring an increased osteogenic differentiation potential at the expense of chondrogenic differentiation, resulting in improved mechanical integrity. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

ß-Arrestin 2 knockout prevents bone loss in response to continuous parathyroid hormone stimulation in male and female mice.

BACKGROUND: ß-Arrestin 2 (ß-arr2) binds activated parathyroid hormone (PTH) receptors stimulating internalization. PTH stimulates both anabolic and catabolic effect on bone depending on the way it is administered. Intermittent PTH stimulation increases trabecular bone formation in mice, but this is decreased in mice lacking ß-arr 2, suggesting a role for ß-arr 2 in the anabolic effects of PTH. The role of ß-arr 2 in the catabolic effects of continuous PTH (cPTH) treatment is not known. OBJECTIVE: To assess the effects of cPTH administration on bone in mice lacking ß-arr 2 compared to wild-type (WT). METHODS: Groups of male and female WT or ß-arr2 knockout (KO) mice were administered either PTH or phosphate-buffered saline by osmotic pumps for 2 weeks. Following treatment, serum calcium and phosphate levels were measured, bone structure and mineral density were measured by microcomputed tomography, and bone cells measured by static and dynamic histomorphometry. RESULTS: ß-arr2 KO had no effects on skeletal development in mice of either sex. PTH treatment caused hypercalcemia and hypophosphatemia and decreased trabecular and cortical bone only in male WT mice. ß-arr2 KO in male mice completely abrogated the effects of PTH on bone, while in female ß-arr2 KO mice, PTH treatment increased trabecular bone with no effects on cortical bone. CONCLUSIONS: These results demonstrate a profound sex effect on skeletal responses to cPTH treatment, suggesting a protective effect of estrogen on bone loss. ß-arr2 plays a role in restraining the anabolic effects of PTH in both male and female mice.

Increased osteoblast GαS level determines bone response to hyperparathyroidism in female mice.

GS, the stimulatory heterotrimeric G protein, is an essential regulator of osteogenesis and bone turnover. To determine if increasing GαS in osteoblasts alters bone responses to hyperparathyroidism, we used a transgenic mouse line overexpressing GαS in osteoblasts (GS-Tg mice). Primary osteoblasts from GS-Tg mice showed increased basal and parathyroid hormone (PTH)-stimulated cAMP and greater responses to PTH than cells from WT mice. Skeletal responses to 2-week continuous PTH administration (cPTH) in female mice resulted in trabecular bone loss in WT mice but 74% and 34% increase in trabecular bone mass in long bones and vertebrae, respectively, in GS-Tg mice. Vertebral biomechanical strength was compromised by cPTH treatment in WT mice but not in GS-Tg. Increased peritrabecular fibrosis was greatly increased by cPTH in Gs-Tg compared to WT mice and corresponded with greater increases in Wnt pathway proteins in trabecular bone. Cortical bone responded negatively to cPTH in WT and Gs-Tg mice with large increases in porosity, decreased cortical thickness and compromised biomechanical properties. These results demonstrate that hyperparathyroidism can increase trabecular bone when GS expression and cAMP stimulation in osteoblasts are increased but this is not the case in cortical bone where increased GS expression exacerbates cortical bone loss.

Irregular porous titanium enhances implant stability and bone ingrowth in an intra-articular ovine model.

Two commercially available porous coatings, Gription and Porocoat, were compared for the first time in a challenging intra-articular, weight-bearing, ovine model. Gription has evolved from Porocoat and has higher porosity, coefficient of friction, and microtextured topography, which are expected to enhance bone ingrowth. Cylindrical implants were press-fit into the weight-bearing regions of ovine femoral condyles and bone ingrowth and fixation strength evaluated 4, 8, and 16 weeks postoperatively. Biomechanical push-out tests were performed on lateral femoral condyles (LFCs) to evaluate the strength of the bone-implant interface. Bone ingrowth was assessed in medial femoral condyles (MFCs) as well as implants retrieved from LFCs following biomechanical testing using backscattered electron microscopy and histology. By 16 weeks, Gription-coated implants exhibited higher force (2455 ± 1362 vs. 1002 ± 1466 N; p = 0.046) and stress (12.60 ± 6.99 vs. 5.14 ± 7.53 MPa; p = 0.046) at failure, and trended towards higher stiffness (11,510 ± 7645 vs. 5010 ± 8374 N/mm; p = 0.061) and modulus of elasticity (591 ± 392 vs. 256 ± 431 MPa; p = 0.061). A strong, positive correlation was detected between bone ingrowth in LFC implants and failure force (r = 0.93, p < 10-13 ). By 16 weeks, bone ingrowth in Gription-coated implants in MFCs was 10.50 ± 6.31% compared to 5.88 ± 2.77% in Porocoat (p = 0.095). Observations of the bone-implant interface, made following push-out testing, showed more bony material consistently adhered to Gription compared to Porocoat at all three time points. Gription provided superior fixation strength and bone ingrowth by 16 weeks.

B cell acute lymphoblastic leukemia cells mediate RANK-RANKL-dependent bone destruction.

Although most children survive B cell acute lymphoblastic leukemia (B-ALL), they frequently experience long-term, treatment-related health problems, including osteopenia and osteonecrosis. Because some children present with fractures at ALL diagnosis, we considered the possibility that leukemic B cells contribute directly to bone pathology. To identify potential mechanisms of B-ALL-driven bone destruction, we examined the p53 -/-; Rag2 -/-; Prkdcscid/scid triple mutant (TM) mice and p53 -/-; Prkdcscid/scid double mutant (DM) mouse models of spontaneous B-ALL. In contrast to DM animals, leukemic TM mice displayed brittle bones, and the TM leukemic cells overexpressed Rankl, encoding receptor activator of nuclear factor κB ligand. RANKL is a key regulator of osteoclast differentiation and bone loss. Transfer of TM leukemic cells into immunodeficient recipient mice caused trabecular bone loss. To determine whether human B-ALL can exert similar effects, we evaluated primary human B-ALL blasts isolated at diagnosis for RANKL expression and their impact on bone pathology after their transplantation into NOD.Prkdcscid/scidIl2rgtm1Wjl /SzJ (NSG) recipient mice. Primary B-ALL cells conferred bone destruction evident in increased multinucleated osteoclasts, trabecular bone loss, destruction of the metaphyseal growth plate, and reduction in adipocyte mass in these patient-derived xenografts (PDXs). Treating PDX mice with the RANKL antagonist recombinant osteoprotegerin-Fc (rOPG-Fc) protected the bone from B-ALL-induced destruction even under conditions of heavy tumor burden. Our data demonstrate a critical role of the RANK-RANKL axis in causing B-ALL-mediated bone pathology and provide preclinical support for RANKL-targeted therapy trials to reduce acute and long-term bone destruction in these patients.

Achieving enhanced bone regeneration using monetite granules with bone anabolic drug conjugates (C3 and C6) in rat mandibular defects.

Bone grafting procedures are commonly used to manage bone defects in the craniofacial region. Monetite is an excellent biomaterial option for bone grafting, however, it is limited by lack of osteoinduction. Several molecules can be incorporated within the monetite matrix to promote bone regeneration. The aim was to investigate whether incorporating bone forming drug conjugates (C3 and C6) within monetite can improve their ability to regenerate bone in bone defects. Bilateral bone defects were created in the mandible of 24 Sprague-Dawley rats and were then packed with monetite control, monetite+C3 or monetite+C6. After 2 and 4 weeks, post-mortem samples were analyzed using microcomputed tomography, histology and back-scattered electron microscopy to calculate the percentages of bone formation and remaining graft material. At 2 and 4 weeks, monetite with C3 and C6 demonstrated higher bone formation than monetite control, while monetite+C6 had the highest bone formation percentage at 4 weeks. There were no significant differences in the remaining graft material between the groups at 2 or 4 weeks. Incorporating these anabolic drug conjugates within the degradable matrix of monetite present a promising bone graft alternative for bone regeneration and repair in orthopedic as well as oral and maxillofacial applications.

Improved bone regeneration using bone anabolic drug conjugates (C3 and C6) with deproteinized bovine bone mineral as a carrier in rat mandibular defects.

BACKGROUND: Deproteinized bovine bone mineral (DBBM) has been extensively studied and used for bone regeneration in oral and maxillofacial surgery. However, it lacks an osteoinductive ability. We developed two novel bone anabolic conjugated drugs, known as C3 and C6, of an inactive bisphosphonate and a bone activating synthetic prostaglandin agonist. The aim was to investigate whether these drugs prebound to DBBM granules have the potential to achieve rapid and enhanced bone regeneration. METHODS: Bilateral defects (4.3 mm diameter circular through and through) were created in mandibular angles of 24 Sprague-Dawley rats were filled with DBBM Control, DBBM with C3 or DBBM with C6 (n = 8 defects per group/ each timepoint). After 2 and 4 weeks, postmortem samples were analyzed by microcomputed tomography followed by backscattering electron microscopy and histology. RESULTS: DBBM grafts containing the C3 and C6 conjugated drugs showed significantly more bone formation than DBBM control at 2 and 4 weeks. The C6 containing DBBM demonstrated the highest percentage of new bone formation at 4 weeks. There was no significant difference in the percentage of the remaining graft between the different groups at 2 or 4 weeks. CONCLUSIONS: DBBM granules containing conjugated drugs C3 and C6 induced greater new bone volume generated and increased the bone formation rate more than the DBBM controls. This is expected to allow the development of clinical treatments that provide more predictable and improved bone regeneration for bone defect repair in oral and maxillofacial surgery.

In Vivo Bone Effects of a Novel Bisphosphonate-EP4a Conjugate Drug (C3) for Reversing Osteoporotic Bone Loss in an Ovariectomized Rat Model.

Pathological bone loss is a regular feature of postmenopausal osteoporosis, and the microstructural changes along with the bone loss make the individual prone to getting hip, spine, and wrist fractures. We have developed a new conjugate drug named C3, which has a synthetic, stable EP4 agonist (EP4a) covalently linked to an inactive alendronate (ALN) that binds to bone and allows physiological remodeling. After losing bone for 12 weeks, seven groups of rats were treated for 8 weeks via tail-vein injection. The groups were: C3 conjugate at low and high doses, vehicle-treated ovariectomy (OVX) and sham, C1 (a similar conjugate, but with active ALN at high dose), inactive ALN alone, and a mixture of unconjugated ALN and EP4a to evaluate the conjugation effects. Bone turnover was determined by dynamic and static histomorphometry; µCT was employed to determine bone microarchitecture; and bone mechanical properties were evaluated via biomechanical testing. Treatment with C3 significantly increased trabecular bone volume and vertebral BMD versus OVX controls. There was also significant improvement in the vertebral load-bearing abilities and stimulation of bone formation in femurs after C3 treatment. This preclinical research revealed that C3 resulted in significant anabolic effects on trabecular bone, and EP4a and ALN conjugation components are vital to conjugate anabolic efficacy. A combined therapy using an EP4 selective agonist anabolic agent linked to an inactive ALN is presented here that produces significant anabolic effects, allows bone remodeling, and has the potential for treating postmenopausal osteoporosis or other diseases where bone strengthening would be beneficial. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Growth Hormone Increases Bone Toughness and Decreases Muscle Inflammation in Glucocorticoid-Treated Mdx Mice, Model of Duchenne Muscular Dystrophy.

The absence of functional dystrophin with mutations of the dystrophin-encoding gene in Duchenne muscular dystrophy (DMD) results in muscle inflammation and degeneration, as well as bone fragility. Long-term glucocorticoid therapy delays the muscular disease progression but suppresses growth hormone secretion, resulting in short stature and further deleterious effects on bone strength. This study evaluated the therapeutic potential of daily growth hormone therapy in growing mdx mice as a model of DMD. Growth hormone treatment on its own or in combination with glucocorticoids significantly improved muscle histology and function and decreased markers of inflammation in mdx mice. Glucocorticoid treatment thinned cortical bone and decreased bone strength and toughness. Despite the minimal effects of growth hormone on bone microarchitecture, it significantly improved biomechanical properties of femurs and vertebrae, even in the presence of glucocorticoid treatment. Together these studies suggest that the use of growth hormone in DMD should be considered for improvements to muscle and bone health. © 2019 American Society for Bone and Mineral Research.

Chlorthalidone Is Superior to Potassium Citrate in Reducing Calcium Phosphate Stones and Increasing Bone Quality in Hypercalciuric Stone-Forming Rats.

BACKGROUND: The pathophysiology of genetic hypercalciuric stone-forming rats parallels that of human idiopathic hypercalciuria. In this model, all animals form calcium phosphate stones. We previously found that chlorthalidone, but not potassium citrate, decreased stone formation in these rats. METHODS: To test whether chlorthalidone and potassium citrate combined would reduce calcium phosphate stone formation more than either medication alone, four groups of rats were fed a fixed amount of a normal calcium and phosphorus diet, supplemented with potassium chloride (as control), potassium citrate, chlorthalidone (with potassium chloride to equalize potassium intake), or potassium citrate plus chlorthalidone. We measured urine every 6 weeks and assessed stone formation and bone quality at 18 weeks. RESULTS: Potassium citrate reduced urine calcium compared with controls, chlorthalidone reduced it further, and potassium citrate plus chlorthalidone reduced it even more. Chlorthalidone increased urine citrate and potassium citrate increased it even more; the combination did not increase it further. Potassium citrate, alone or with chlorthalidone, increased urine calcium phosphate supersaturation, but chlorthalidone did not. All control rats formed stones. Potassium citrate did not alter stone formation. No stones formed with chlorthalidone, and rats given potassium citrate plus chlorthalidone had some stones but fewer than controls. Rats given chlorthalidone with or without potassium citrate had higher bone mineral density and better mechanical properties than controls, whereas those given potassium citrate did not. CONCLUSIONS: In genetic hypercalciuric stone-forming rats, chlorthalidone is superior to potassium citrate alone or combined with chlorthalidone in reducing calcium phosphate stone formation and improving bone quality.

A Novel Anabolic Conjugate (C3) in the Matrix of Dicalcium Phosphate Onlay Block Grafts for Achieving Vertical Bone Augmentation: An Experimental Study on Rabbit Calvaria.

PURPOSE: Achieving successful and predictable alveolar ridge augmentation in the vertical dimension is extremely challenging. Several materials have been investigated to achieve vertical ridge augmentation; however, the results are highly unpredictable. The collaborative team presenting this research has developed brushite- and monetite-based grafts that incorporate in their matrix a novel bone anabolic conjugate (C3) of a bisphosphonate and a potent bone-activating EP4 receptor agonist. The study objective was to investigate the potential of these graft formulations to achieve rapid, enhanced, and clinically significant bone regeneration in the vertical dimension. MATERIALS AND METHODS: Brushite and monetite grafts were fabricated and characterized for phase purity, porosity, compressive strength, and microstructural morphology. They were implanted in 12 rabbit calvaria for 12 weeks. Each group (n = 6): brushite control, brushite with C3, monetite control, and monetite with C3. Postmortem samples were retrieved and processed for analysis. The percentage bone volume, vertical bone height gained, and graft resorption were calculated and assessed. RESULTS: The brushite and monetite grafts containing C3 integrated well onto the calvarial bone surface, with new bone extending through the graft area (36% and 80%, respectively), while the C3 lacking grafts showed decreased surface integration and bone infiltration (28% and 38%, respectively). The C3 containing brushite and monetite grafts demonstrated bone growth vertically (1.8 mm and 2.7 mm, respectively) and improved graft resorption. CONCLUSION: The brushite- and monetite-based grafts loaded with the C3 conjugate resulted in greater de novo bone formation in the vertical dimension when compared with the grafts without the drug. However, the monetite grafts produced much more and predictable vertical height gain than was achieved with brushite grafts. The advantages of this new graft drug formulation in future would be to provide more predictable vertical bone regeneration, which will ultimately benefit patients undergoing dental implant placement.

Use of backscattered scanning electron microscopy to quantify the bone tissues of midthoracic human ribs.

OBJECTIVES: Novel information on apartheid health conditions may be obtained through the study of recent skeletal collections. Using a backscattered scanning electron microscopy (BSE-SEM) approach, this study aims to produce bone quality and tissue mineralization data for an understudied South African population from the Western Cape province. METHODS: Using BSE-SEM imaging, cortical porosity (Ct.Po), osteocyte lacunar density (Ot.Lc.Dn), and the degree of tissue mineralization were quantified in midthoracic ribs from the Kirsten Skeletal Collection. Individuals ( n female = 75, n male = 68, and mean age = 46.3 years) were predominantly from the South Africa Colored (SAC) population group ( n SAC = 103, 72%). Full cross-sectional images of each rib were manually stitched together in Adobe Photoshop. Photomontages were imported into MATALB (Mathworks, Natick, MA) for image processing and analysis. Age-related changes in histomorphometric parameters and sex differences were examined using correlation analysis, as well as linear and nonlinear regressions. RESULTS: Young adult men have significantly less mineralized bone and fewer osteocyte lacunae, compared to women. Only men demonstrate a significant negative relationship between Ot.Lc.Dn and age. Average tissue mineralization decreases with age in women, while Ct.Po increases. Pore area (Po.Ar) does not vary with age, but pore density (Po.Dn) is highest in the perimenopause, when accelerated rates of bone turnover are first anticipated. Ct.Po is highest in the years following the predicted age of menopause, but levels off in the final decades of life. CONCLUSIONS: Men and women display disparate patterns of bone aging. Systemic disenfranchisement of non-white population groups affected bone health in South Africa, and may continue to do so today. Indicators of poor bone quality are evident in the full study sample, indicating that osteoporosis and fracture risk are not just of concern to the aged white female population.

Pre-treatment with Pamidronate Improves Bone Mechanical Properties in Mdx Mice Treated with Glucocorticoids.

Duchenne muscular dystrophy (DMD) is an X-linked disease of progressive muscle deterioration and weakness. Patients with DMD have poor bone health which is partly due to treatment with glucocorticoids, a standard therapy to prolong muscle function that also induces bone loss. Bisphosphonates are used to treat adults at risk of glucocorticoid-induced osteoporosis but are not currently used in DMD patients until after they sustain fractures. In this study, C57BL/10ScSn-mdx mice, a commonly used DMD animal model, received continuous glucocorticoid, prednisone treatment (0.083 mg/day) from 5 to 10 weeks of age. Pre-treatment with the bisphosphonate pamidronate started at 4 weeks of age over a period of 2 weeks or 6 weeks (cumulative dose 8 mg/kg for both) to assess the effectiveness of the two dosing regimens in ameliorating glucocorticoid-induced bone loss. Mdx mice treated with prednisone had improved muscle function that was not changed by pamidronate treatment. Glucocorticoid treatment caused cortical bone loss and decreased cortical bone strength. Both 2 and 6 week pamidronate treatment increased cortical thickness and bone area compared to prednisone-treated Mdx mice, however, only 2 week pamidronate treatment improved the strength of cortical bone compared to that of glucocorticoid-treated Mdx mice. In the trabecular bone, both pamidronate treatments significantly increased the amount of bone, and increased the ultimate load but not the energy to fail. These results highlight the importance of when and how much bisphosphonate is administered prior to glucocorticoid exposure.

Effect of 25-HydroxyVitamin D Deficiency and Its Interaction with Prednisone Treatment on Musculoskeletal Health in Growing Mdx Mice.

Duchenne muscular dystrophy (DMD) results from genetic mutations of the gene encoding dystrophin, leading to muscle inflammation and degeneration that is typically treated with glucocorticoids. DMD and its treatment with glucocorticoids result in poor bone health and high risk of fractures. Insufficient levels of 25-hydroxyvitamin D (25-hydroxy D) that may contribute to weakened bone are routinely found in DMD patients. To determine the effect of 25-hydroxy D deficiency, this study examined the effects of low vitamin D dietary intake with and without glucocorticoids on the musculoskeletal system of the Mdx mouse model of DMD. At 10 weeks of age, Mdx mice on control diet had low trabecular bone mineral density of distal femurs and lumbar vertebrae with increased osteoclast numbers compared to wild-type mice. Low vitamin D intake resulted in 25-hydroxy D deficiency but had no effect on trabecular or cortical bone. Cortical bone loss and bone weakness were induced by glucocorticoids while they improved muscle grip strength in Mdx mice. 25-hydroxy D deficiency did not result in any significant effects on growing bone or muscle in the Mdx mice. In combination with glucocorticoid treatment, low 25-hydroxy D resulted in no change in cortical bone mineral density but bone ductility was significantly increased suggesting lower bone mineralization.

Targeting therapeutics to bone by conjugation with bisphosphonates.

Bisphosphonates target and bind avidly to the mineral (hydroxyapatite) found in bone. This targeting ability has been exploited to design and prepare bisphosphonate conjugate prodrugs to deliver a wide variety of drug molecules selectively to bones. It is important that conjugates be stable in the blood stream and that conjugate that is not taken up by bone is eliminated rapidly. The prodrugs should release active drug at a rate appropriate so as to provide efficacy. Radiolabelling is the best method to quantify and evaluate pharmacokinetics, tissue distribution, bone uptake and release of the active drug(s). Recent reports have described bisphosphonate conjugates derived from the antiresorptive drug, alendronic acid and anabolic prostanoid drugs that effectively deliver prostaglandins and prostaglandin EP4 receptor agonists to bone and show enhanced anabolic efficacy and tolerability compared to the drugs alone. These conjugate drugs can be dosed infrequently (weekly or bimonthly) whereas the free drugs must be dosed daily.

Sex-specific patterns in cortical and trabecular bone microstructure in the Kirsten Skeletal Collection, South Africa.

OBJECTIVES: The purpose of this study was to provide bone histomorphometric reference data for South Africans of the Western Cape who likely dealt with health issues under the apartheid regime. METHODS: The 206 adult individuals (n female = 75, n male = 131, mean = 47.9 ± 15.8 years) from the Kirsten Skeletal Collection, U. Stellenbosch, lived in the Cape Town metropole from the late 1960s to the mid-1990s. To study age-related changes in cortical and trabecular bone microstructure, photomontages of mid-thoracic rib cross-sections were quantitatively examined. Variables include relative cortical area (Rt.Ct.Ar), osteon population density (OPD), osteon area (On.Ar), bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp). RESULTS: All cortical variables demonstrated significant relationships with age in both sexes, with women showing stronger overall age associations. Peak bone mass was compromised in some men, possibly reflecting poor nutritional quality and/or substance abuse issues throughout adolescence and early adulthood. In women, greater predicted decrements in On.Ar and Rt.Ct.Ar suggest a structural disadvantage with age, consistent with postmenopausal bone loss. Age-related patterns in trabecular bone microarchitecture are variable and difficult to explain. Except for Tb.Th, there are no statistically significant relationships with age in women. Men demonstrate significant negative correlations between BV/TV, Tb.N, and age, and a significant positive correlation between Tb.Sp and age. CONCLUSIONS: This research highlights sex-specific differences in patterns of age-related bone loss, and provides context for discussion of contemporary South African bone health. While the study sample demonstrates indicators of poor bone quality, osteoporosis research continues to be under-prioritized in South Africa.

Overexpression of GαS in Murine Osteoblasts In Vivo Leads to Increased Bone Mass and Decreased Bone Quality.

GαS is a heterotrimeric G protein that transduces signals from activated G protein-coupled receptors on the cell surface to stimulate adenylyl cyclase/cyclic adenosine monophosphate (AMP) signaling. GαS plays a central role in mediating numerous growth and maintenance processes including osteogenesis and bone turnover. Decreased GαS expression or activating mutations in GαS both affect bone, suggesting that modulating GαS protein levels may be important for bone health and development. To examine the effects of increased osteoblastic GαS expression on bone development in vivo, we generated transgenic mice with GαS overexpression in osteoblasts (HOM-Gs mice) driven by the 3.6-kilobase (kb) Col1A1 promoter. Both male and female HOM-Gs mice exhibit increased bone turnover with overactive osteoblasts and osteoclasts, resulting in a high bone mass phenotype with significantly reduced bone quality. At 9 weeks of age, HOM-Gs mice have increased trabecular number, volumetric BMD (vBMD), and bone volume; however, the bone was woven and disorganized. There was also increased cortical bone volume despite an overall reduction in size in HOM-Gs mice along with increased cortical porosity and brittleness. The skeletal phenotype of HOM-Gs mice progressed into maturity at 26 weeks of age with further accrual of trabecular bone, whereas WT mice lost trabecular bone at this age. Although cortical bone volume and geometry were similar between mature HOM-Gs and WT mice, increased porosity persisted and the bone was weaker. At the cellular level, these alterations were mediated by an increase in bone resorption by osteoclasts and an overwhelmingly higher increase in bone formation by osteoblasts. In summary, our findings demonstrate that high osteoblastic GαS expression results in aberrant skeletal development in which bone production is favored at the cost of bone quality. © 2017 American Society for Bone and Mineral Research.

Ubiquitin ligase RNF146 coordinates bone dynamics and energy metabolism.

Cleidocranial dysplasia (CCD) is an autosomal dominant human disorder characterized by abnormal bone development that is mainly due to defective intramembranous bone formation by osteoblasts. Here, we describe a mouse strain lacking the E3 ubiquitin ligase RNF146 that shows phenotypic similarities to CCD. Loss of RNF146 stabilized its substrate AXIN1, leading to impairment of WNT3a-induced ß-catenin activation and reduced Fgf18 expression in osteoblasts. We show that FGF18 induces transcriptional coactivator with PDZ-binding motif (TAZ) expression, which is required for osteoblast proliferation and differentiation through transcriptional enhancer associate domain (TEAD) and runt-related transcription factor 2 (RUNX2) transcription factors, respectively. Finally, we demonstrate that adipogenesis is enhanced in Rnf146-/- mouse embryonic fibroblasts. Moreover, mice with loss of RNF146 within the osteoblast lineage had increased fat stores and were glucose intolerant with severe osteopenia because of defective osteoblastogenesis and subsequent impaired osteocalcin production. These findings indicate that RNF146 is required to coordinate ß-catenin signaling within the osteoblast lineage during embryonic and postnatal bone development.

RANKL coordinates multiple osteoclastogenic pathways by regulating expression of ubiquitin ligase RNF146.

Bone undergoes continuous remodeling due to balanced bone formation and resorption mediated by osteoblasts and osteoclasts, respectively. Osteoclasts arise from the macrophage lineage, and their differentiation is dependent on RANKL, a member of the TNF family of cytokines. Here, we have provided evidence that RANKL controls the expression of 3BP2, an adapter protein that is required for activation of SRC tyrosine kinase and simultaneously coordinates the attenuation of ß-catenin, both of which are required to execute the osteoclast developmental program. We found that RANKL represses the transcription of the E3 ubiquitin ligase RNF146 through an NF-κB-related inhibitory element in the RNF146 promoter. RANKL-mediated suppression of RNF146 results in the stabilization of its substrates, 3BP2 and AXIN1, which consequently triggers the activation of SRC and attenuates the expression of ß-catenin, respectively. Depletion of RNF146 caused hypersensitivity to LPS-induced TNF-α production in vivo. RNF146 thus acts as an inhibitory switch to control osteoclastogenesis and cytokine production and may be a control point underlying the pathogenesis of chronic inflammatory diseases.

Calcium polyphosphate particulates for bone void filler applications.

This study investigates the characteristics of porous calcium polyphosphate particulates (CPPp) formed using two different processing treatments as bone void fillers in non- or minimally load-bearing sites. The two calcium polyphosphate particulate variants (grades) were formed using different annealing conditions during particulate preparation to yield either more slowly degrading calcium polyphosphate particulates (SD-CPPp) or faster degrading particulates (FD-CPPp) as suggested by a previous degradation study conducted in vitro (Hu et al., Submitted for publication 2016). The two CPPp grades were compared as bone void fillers in vivo by implanting particulates in defects created in rabbit femoral condyle sites (critical size defects). The SD-CPPp and FD-CPPp were implanted for 4- and 16-week periods. The in vivo study indicated a significant difference in amount of new bone formed in the prepared sites with SD-CPPp resulting in more new bone formation compared with FD-CPPp. The lower bone formation characteristic of the FD-CPPp was attributed to its faster degradation rate and resulting higher local concentration of released polyphosphate degradation products. The study results indicate the importance of processing conditions on preparing calcium polyphosphate particulates for potential use as bone void fillers in nonload-bearing sites. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 874-884, 2017.