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.

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.

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.

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.

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.

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.

Elevated Gα11 expression in osteoblast lineage cells promotes osteoclastogenesis and leads to enhanced trabecular bone accrual in response to pamidronate.

Osteoblastic cells indirectly induce osteoclastogenesis in the bone microenvironment by expressing paracrine factors such as RANKL and M-CSF, leading to increased bone resorption. These cytokines can be regulated by a variety of intracellular pathways, which include G protein-coupled receptor signaling. To explore how enhanced signaling of the Gαq/11 pathway in osteoblast lineage cells may mediate osteoclast formation, we cocultured wild-type (WT) preosteoclasts with BMSCs derived from either WT or transgenic mice with osteoblast-specific overexpression of Gα11 (G11-Tg). G11-Tg cocultures had elevated osteoclast numbers with greater resorptive capacity and increased expression of Rankl, Rankl:Opg (osteoprotegerin), and M-csf compared with cocultures with WT BMSCs. As well, cocultures with G11-Tg BMSCs required a higher concentration of OPG to inhibit osteoclast formation and less angiotensin II to increase osteoclast size. These indicate that G11-Tg osteoblasts drive the increased osteoclast formation and osteopenia seen in G11-Tg mice. Pamidronate treatment of G11-Tg mice restored the trabecular bone loss phenotype, as bone mineral density, bone volume, trabecular number, separation, and expressions of osteoblastic and osteoclastic genes were comparable with WT parameters. These changes were characterized by enhanced accumulation of calcified cartilage in trabecular bone, demonstrating that resorption of the cartilaginous intermediate by osteoclasts is more affected by bisphosphonate treatment in G11-Tg mice. In conclusion, overexpression of Gα11 in osteoblastic cells promotes osteoclastogenesis by upregulation of Rankl and M-csf and bone loss by increased osteoclast resorption of the trabecular bone and cartilaginous matrix.

Overexpression of Gα11 in Osteoblast Lineage Cells Suppresses the Osteoanabolic Response to Intermittent PTH and Exercise.

Intermittent parathyroid hormone (iPTH) treatment and mechanical loading are osteoanabolic stimuli that are partially mediated through actions on G protein-coupled receptors (GPCRs). GPCR signaling can be altered by heterotrimeric G protein Gα subunits levels, which can therefore lead to altered responses to such stimuli. Previous studies have suggested that enhanced signaling through the Gαq/11 pathway inhibits the osteoanabolic actions of PTH. The influence of Gαq/11 signaling on mechanotransduction, however, has not been reported in vivo. Using transgenic mice that specifically overexpress Gα11 in osteoblast lineage cells (G11-Tg mice), we investigated the skeletal effects of elevated Gα11 levels on iPTH and mechanical loading by treadmill exercise. Both regimens increased trabecular and cortical bone in Wild-Type (WT) mice as a result of increased bone formation. In G11-Tg mice, there was no change in trabecular or cortical bone and no increase in bone formation in response to iPTH or exercise. While exercise reduced osteoclast parameters in WT mice, these changes were diminished in G11-Tg mice as expression of M-csf and Trap remained increased. Collectively, our results suggest that osteoblastic upregulation of Gα11 is inhibitory to osteoanabolic actions of both PTH and exercise, and that its suppression may be a promising target for treating bone loss.

Long-term effects of castration on the skeleton of male rhesus monkeys (Macaca mulatta).

While osteopenia (OPE) and osteoporosis (OPO) have been studied in various species of aging nonhuman primates and extensively in ovariectomized rhesus and cynomolgus macaques, there is virtually no information on the effects of castration on the skeleton of male nonhuman primates. Most information on castrated male primates comes from a few studies on the skeletons of eunuchs. This report used a subset of the Caribbean Primate Research Center's (CPRC) Cayo Santiago (CS) rhesus macaque skeletal collection to qualitatively and quantitatively compare the bone mineral density (BMD) of castrated and age-matched intact males and, thereby, determine the long-term effects of castration (orchidectomy) on bone. Lumbar vertebrae, femora, and crania were evaluated using dual-energy X-ray absorptiometry (DEXA or DXA) and digital radiography augmented, when fresh tissues were available, with autoradiography and histology. Results confirmed physical examinations of long bones that castration causes changes in the skeleton of male rhesus macaques similar to those found in eunuchs, including OPE and OPO of the vertebrae and femora, thinning of the skull, and vertebral fractures and kyphosis of the spine more severe than that caused by normal aging alone. Also like eunuchs, some castrated CS male rhesus monkeys had a longer life span than intact males or females. Based on these results and the effects of castration on other tissues and organs of eunuchs, on behavior, hormone profiles and possibly on cognition and visual perception of human and nonhuman primates, and other mammals, castrated male rhesus macaques should be used with caution for laboratory studies and should be considered a separate category from intact males. Despite these caveats, the castrated male rhesus macaque should make an excellent animal model in which to test hormone replacement therapies for boys and men orchidectomized for testicular and prostate cancer.

Effect of Potassium Citrate on Calcium Phosphate Stones in a Model of Hypercalciuria.

Potassium citrate is prescribed to decrease stone recurrence in patients with calcium nephrolithiasis. Citrate binds intestinal and urine calcium and increases urine pH. Citrate, metabolized to bicarbonate, should decrease calcium excretion by reducing bone resorption and increasing renal calcium reabsorption. However, citrate binding to intestinal calcium may increase absorption and renal excretion of both phosphate and oxalate. Thus, the effect of potassium citrate on urine calcium oxalate and calcium phosphate supersaturation and stone formation is complex and difficult to predict. To study the effects of potassium citrate on urine supersaturation and stone formation, we utilized 95th-generation inbred genetic hypercalciuric stone-forming rats. Rats were fed a fixed amount of a normal calcium (1.2%) diet supplemented with potassium citrate or potassium chloride (each 4 mmol/d) for 18 weeks. Urine was collected at 6, 12, and 18 weeks. At 18 weeks, stone formation was visualized by radiography. Urine citrate, phosphate, oxalate, and pH levels were higher and urine calcium level was lower in rats fed potassium citrate. Furthermore, calcium oxalate and calcium phosphate supersaturation were higher with potassium citrate; however, uric acid supersaturation was lower. Both groups had similar numbers of exclusively calcium phosphate stones. Thus, potassium citrate effectively raises urine citrate levels and lowers urine calcium levels; however, the increases in urine pH, oxalate, and phosphate levels lead to increased calcium oxalate and calcium phosphate supersaturation. Potassium citrate induces complex changes in urine chemistries and resultant supersaturation, which may not be beneficial in preventing calcium phosphate stone formation.

Bone Marrow Stress Decreases Osteogenic Progenitors.

Age-related bone loss may be a result of declining levels of stem cells in the bone marrow. Using the Col2.3Δtk (DTK) transgenic mouse, osteoblast depletion was used as a source of marrow stress in order to investigate the effects of aging on osteogenic progenitors which reside in the marrow space. Five-month-old DTK mice were treated with one or two cycles of ganciclovir to conditionally ablate differentiated osteoblasts, whereas controls were saline-treated. Treatment cycles were two weeks in length followed by four weeks of recovery. All animals were sacrificed at 8 months of age; bone marrow stromal cells (BMSCs) were harvested for cell culture and whole bones were excised for bone quality assessment. Colony-forming unit (CFU) assays were conducted to investigate the osteogenic potential of BMSC in vitro, and RNA was extracted to assess the expression of osteoblastic genes. Bone quality assessments included bone histomorphometry, TRAP staining, microcomputed tomography, and biomechanical testing. Osteoblast depletion decreased CFU-F (fibroblast), CFU-ALP (alkaline phosphatase), and CFU-VK (von Kossa) counts and BMSC osteogenic capacity in cell culture. Ex vivo, there were no differences in bone mineral density of vertebrae or femurs between treatment groups. Histology showed a decrease in bone volume and bone connectivity with repeated osteoblast depletion; however, this was accompanied by an increase in bone formation rate. There were no notable differences in osteoclast parameters or observed bone marrow adiposity. We have developed a model that uses bone marrow stress to mimic age-related decrease in osteogenic progenitors. Our data suggest that the number of healthy BMSCs and their osteogenic potential decline with repeated osteoblast depletion. However, activity of the remaining osteoblasts increases to compensate for this loss in progenitor osteogenic potential.

1,25(OH)2D3 induces a mineralization defect and loss of bone mineral density in genetic hypercalciuric stone-forming rats.

Genetic hypercalciuric stone-forming (GHS) rats, bred to maximize urine (u) calcium (Ca) excretion, demonstrate increased intestinal Ca absorption, increased bone Ca resorption, and reduced renal Ca reabsorption, all leading to elevated uCa compared to the parental Sprague-Dawley (SD) rats. GHS rats have increased numbers of vitamin D receptors (VDRs) at each site, with normal levels of 1,25(OH)2D3 (1,25D), suggesting their VDR is undersaturated with 1,25D. We have shown that 1,25D induces a greater increase in uCa in GHS than SD rats. To examine the effect of the increased VDR on the osseous response to 1,25D, we fed GHS and SD rats an ample Ca diet and injected either 1,25D [low dose (LD) 12.5 or high dose (HD) 25 ng/100 g body weight/day] or vehicle (veh) daily for 16 days. Femoral areal bone mineral density (aBMD, by DEXA) was decreased in GHS+LD and GHS+HD relative to GHS+veh, while there was no effect on SD. Vertebral aBMD was lower in GHS compared to SD and further decreased in GHS+HD. Both femoral and L6 vertebral volumetric BMD (by µCT) were lower in GHS and further reduced by HD. Histomorphometry indicated a decreased osteoclast number in GHS+HD compared to GHS+veh or SD+HD. In tibiae, GHS+HD trabecular thickness and number increased, with a 12-fold increase in osteoid volume but only a threefold increase in bone volume. Bone formation rate was decreased in GHS+HD relative to GHS+veh, confirming the mineralization defect. The loss of BMD and the mineralization defect in GHS rats contribute to increased hypercalciuria; if these effects persist, they would result in decreased bone strength, making these bones more fracture-prone. The enhanced effect of 1,25D in GHS rats indicates that the increased VDRs are biologically active.

Collagen modifications in postmenopausal osteoporosis: advanced glycation endproducts may affect bone volume, structure and quality.

The classic model of postmenopausal osteoporosis (PM-OP) starts with the depletion of estrogen, which in turn stimulates imbalanced bone remodeling, resulting in loss of bone mass/volume. Clinically, this leads to fractures because of structural weakness. Recent work has begun to provide a more complete picture of the mechanisms of PM-OP involving oxidative stress and collagen modifications known as advanced glycation endproducts (AGEs). On one hand, AGEs may drive imbalanced bone remodeling through signaling mediated by the receptor for AGEs (RAGE), stimulating resorption and inhibiting formation. On the other hand, AGEs are associated with degraded bone material quality. Oxidative stress promotes the formation of AGEs, inhibits normal enzymatically derived crosslinking and can degrade collagen structure, thereby reducing fracture resistance. Notably, there are multiple positive feedback loops that can exacerbate the mechanisms of PM-OP associated with oxidative stress and AGEs. Anti-oxidant therapies may have the potential to inhibit the oxidative stress based mechanisms of this disease.

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.

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.

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.

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.

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.

Measuring and interpreting age-related loss of vertebral bone mineral density in a medieval population.

This study investigates the age- and sex-related patterns in vertebral bone mineral density (BMD) and the relationship between BMD and vertebral osteophytosis (VO), using a specialized peripheral densitometer in a skeletal sample excavated from the British medieval village Wharram Percy. A total of 58 individuals were divided by sex into three broad age categories (18-29, 30-49, 50+ years.). Each fourth intact vertebral centra was scored for VO and 5-mm thick coronal sections scanned in a specialized peripheral densitometer (GE Lunar Piximus DXA). Changes in BMD associated with age, sex, and VO severity were examined in the whole vertebral section, a strictly trabecular region, and a primarily cortical region of bone separately. Significant change in vertebral BMD was found to occur by middle age with little or no statistical change in BMD between middle and old age. Females appear to suffer greater bone loss at an earlier age with no change in BMD between middle and old age, whereas males show a more steady loss of BMD across the age groups. The bone mineral content and BMD of the cortical region is higher in individuals with pronounced/severe osteophytosis. The unusual age- and sex-related patterns of change in vertebral BMD at Wharram Percy are compared with the patterns of age-related change from recent longitudinal population-based studies. The results emphasize the different pattern of bone loss in young adulthood seen in trabecular regions of the skeleton and highlight the importance of consideration of degenerative joint disease in BMD studies. The influence of lifestyle factors on vertebral BMD in this medieval population is also discussed.