Bone Mineral Apposition Research Articles

In vitro and in vivo characterization of anodised zirconium as a potential material for biomedical applications

In vitro studies offer the insights for the understanding of the mechanisms at the tissue–implant interface that will provide an effective functioning in vivo. The good biocompatibility of zirconium makes a good candidate for biomedical applications and the attractive in vivo performance is mainly due to the presence of a protective oxide layer. The aim of this study is to evaluate by in vitro and in vivo approach, the influence of surface modification achieved by anodisation at 30 and 60V on zirconium implants on the first steps of the osseointegration process. In this study cell attachment, proliferation and morphology of mouse myoblast C2C12-GFP and in mouse osteoprogenitor MC3T3-E1 cells was evaluated. Also, together with the immune system response, osteoclast differentiation and morphology with RAW 264.7 murine cell line were analysed. It was found that anodisation treatment at 60V enhanced cell spreading and the osteoblastic and osteoclastic cells morphology, showing a strong dependence on the surface characteristics. In vivo tests were performed in a rat femur osteotomy model. Dynamical and static histological and histomorphometric analyses were developed 15 and 30days after surgery. Newly formed bone around Zr60V implants showed a continuous newly compact and homogeneous bone just 15 after surgery, as judged by the enhanced thickness and mineralization rate. The results indicate that anodising treatment at 60V could be an effective improvement in the osseointegration of zirconium by stimulating adhesion, proliferation, morphology, new bone thickness and bone mineral apposition, making zirconium an emerging candidate material for biomedical applications.

Intraosseous Injection of Simvastatin in Poloxamer 407 Hydrogel Improves Pedicle-Screw Fixation in Ovariectomized Minipigs.

Osteoporosis leads to poor osseointegration and reduces implant stability. Statins have been found to stimulate bone formation, but the bioavailability from oral administration is low. Local application may be more effective at augmenting bone formation and enhancing implant stability. This study was performed to evaluate the efficacy of an intraosseous injection of simvastatin in thermosensitive poloxamer 407 hydrogel to enhance pedicle-screw fixation in calcium-restricted ovariectomized minipigs. Nine mature female Guangxi Bama minipigs underwent bilateral ovariectomy and were fed a calcium-restricted diet for 18 months. Simvastatin (0, 0.5, or 1 mg) in thermosensitive poloxamer 407 hydrogel was injected into the lumbar vertebrae (L4-L6), and titanium alloy pedicle screws were implanted. Bone mineral density (BMD) measurements of the lumbar vertebrae were determined by dual x-ray absorptiometry (DXA) before and 3 months after treatment. The lumbar vertebrae were harvested and analyzed with use of microcomputed tomography, biomechanical pull-out testing, histological analysis, and Western blot analysis for bone morphogenetic protein (BMP)-2 and vascular endothelial growth factor (VEGF) expression. Evaluation over a 3-month study period demonstrated that the BMD of the vertebrae injected with 0.5 and 1.0 mg of simvastatin had increased by 31.25% and 31.09%, respectively, compared with vehicle-only injection (p ≤ 0.00014 for both) and increased by 32.12% and 28.16%, respectively, compared with the pre-treatment levels (p < 0.0001 for both). A single injection of simvastatin in poloxamer 407 increased trabecular volume fraction, thickness, and number and decreased trabecular separation (p ≤ 0.002). The bone formation and mineral apposition rates significantly increased (p ≤ 0.023). The percentage of osseointegration in the simvastatin 0.5 and 1-mg groups was 46.54% and 42.63% greater, respectively, than that in the vehicle-only group (p ≤ 0.006), and the maximum pull-out strength was 45.75% and 51.53% greater, respectively, than in the vehicle-only group (p ≤ 0.0005). BMP-2 and VEGF expressions were higher than for the vehicle-only injection. A single intraosseous injection of simvastatin in thermosensitive poloxamer 407 hydrogel stimulated bone formation, increased BMD, improved bone microstructure, promoted osseointegration, and significantly enhanced the stability of pedicle screws in calcium-restricted ovariectomized minipigs. These results provide rationale for evaluating intraosseous injection of simvastatin in poloxamer 407 to enhance implant fixation in patients with osteoporosis.

Only minor differences in renal osteodystrophy features between wild-type and sclerostin knockout mice with chronic kidney disease

Renal osteodystrophy affects the majority of patients with advanced chronic kidney disease (CKD) and is characterized by progressive bone loss. This study evaluated the effects of sclerostin knockout on bone in a murine model of severe, surgically induced CKD in both sclerostin knockout and wild-type mice. Mice of both genotypes with normal kidney function served as controls. Tibiae were analyzed using micro-computed tomography, and lumbar vertebrae were analyzed by histomorphometry. Results were tested for statistical significance by 2-way ANOVA to investigate whether bone of the knockout mice reacted differently to CKD compared with bone of wild-type mice. In the tibiae, there was no difference after creation of CKD between wild-type and knockout animals for cortical thickness or cross-sectional moment of inertia. Increases in cortical porosity induced by CKD differed significantly between genotypes in the tibial metaphysis but not in the diaphysis. In the trabecular compartment, no difference in reaction to CKD between genotypes was found for bone volume, trabecular number, trabecular thickness, and trabecular separation. In the lumbar vertebrae, significant differences in response to CKD between wild-type and knockout mice were seen for both bone volume and trabecular thickness. Osteoblast parameters did not differ significantly, whereas osteoclast numbers significantly increased in the wild-type but significantly decreased in knockout mice with CKD. No differences in response to CKD between genotypes were found for bone formation rate or mineral apposition rate. Thus, complete absence of sclerostin has only minor effects on CKD-induced bone loss in mice.

Repair of segmental radial defects in dogs using tailor-made titanium mesh cages with plates combined with calcium phosphate granules and basic fibroblast growth factor-binding ion complex gel.

Repair of large segmental defects of long bones are a tremendous challenge that calls for a novel approach to supporting immediate weight bearing and bone regeneration. This study investigated the functional and biological characteristics of a combination of a tailor-made titanium mesh cage with a plate (tTMCP) with tetrapod-shaped alpha tricalcium phosphate granules (TB) and basic fibroblast growth factor (bFGF)-binding ion complex gel (f-IC gel) to repair 20-mm segmental radial defects in dogs. The defects were created surgically in 18 adult beagle dogs and treated by implantation of tTMCPs with TB with (TB-gel group) or without (TB group) f-IC gel. Each tTMCP fitted the defect well, and all dogs could bear weight on the affected limb immediately after surgery. Dogs were euthanized 4, 8 and 24weeks after implantation. Histomorphometry showed greater infiltration of new vessels and higher bone union rate in the TB-gel group than in the TB group. The lamellar bone volume and mineral apposition rate did not differ significantly between the groups, indicating that neovascularization may be the primary effect of f-IC gel on bone regeneration. This combination method which is tTMCP combined with TB and f-IC gel, would be useful for the treatment of segmental long bone defects.

Feasibility of Electroporation in Bone and in the Surrounding Clinically Relevant Structures: A Preclinical Investigation.

Skeletal metastases are a common cause of severe morbidity, reduction in quality of life and often early mortality. Consequently, improvements in therapies are necessary. Electroporation uses electric energy to alter cancer cell membrane permeability and enhance the local uptake of chemotherapeutics, thus leading to local tumor control. The aim of this study was to investigate the feasibility and safety of delivering electric field protocols causing electroporation of healthy bone and structures of clinical relevance using small and large animal models. Reversible electroporation was used in the rabbit sciatic nerve by applying 2 series of 8 pulses 100ms long at 1000 V/cm. Irreversible electroporation was used in rabbit distal femur condyles and in sheep vertebral body by applying 120 pulses 100ms long at 1750 V/cm. Any effect on surrounding sensitive structures was investigated. Reversible electroporation of sciatic nerve was associated with transient foot functional deficit that completely recovered at 30 days. Irreversible electroporation removed cells from trabeculae in the femurs of rabbits and in the vertebral body of sheep. After irreversible protocol, histology and microtomography demonstrated that the trabecular structure was maintained, the presence of new bone marrow cells, osteoblasts, and mineral apposition characterized by new trabeculae thinner than controls (P = .005) and a significant reduction in the ablated areas (-225%, P = .0219). Spinal cord, vertebral pedicles and spinal nerves showed transient edema in the absence of functional or structural alterations. Collectively, these results show that electroporation can be safely applied to bone even in the proximity of neuronal structures.

A neoflavonoid dalsissooal isolated from heartwood of Dalbergia sissoo Roxb. has bone forming effects in mice model for osteoporosis

Dalbergia sissoo Roxb. is a well known medicinal plant of India, enriched with various flavonoids used for treating multiple diseases. Earlier, we have shown that extract of Dalbergia sissoo Roxb. leaves mitigate ovariectomy induced bone loss and pure compounds (neoflavonoids) isolated from it, promote osteoblastogenesis in primary calvarial osteoblasts cells in vitro. Here, we hypothesize that dalsissooal (DSL), a novel neoflavonoid isolated from the heartwood of Dalbergia sissoo Roxb. is an important constituent of the extract that imparts bone forming effects. Treatment with DSL enhanced trabecular bone micro-architecture parameters, biomechanical strength, increased bone formation rate and mineral apposition rate in OVx mice comparable to 17β-estradiol. It increased bone formation by enhancing osteoblast gene expression and reduced bone turnover by decreasing osteoclastic gene expressions. Interestingly, we observed that DSL has no uterine estrogenic effects. At cellular levels, DSL promoted differentiation of bone marrow cells as well as calvaria osteoblast cells towards osteoblast lineage by enhancing differentiation and mineralizing ability to form mineralizing nodules via stimulating BMP-2 and RunX-2 expressions. Overall, our data suggest that oral supplementation of a novel neoflavonoid dalsissooal isolated from heartwood of Dalbergia sissoo Roxb. exhibited bone anabolic action by improving structural property of bone, promoting new bone formation and reducing bone turnover rate in post-menopausal model for osteoporosis with no uterine hyperplasia.

Constitutively Elevated Blood Serotonin Is Associated with Bone Loss and Type 2 Diabetes in Rats.

Reduced peripheral serotonin (5HT) in mice lacking tryptophan hydroxylase (TPH1), the rate limiting enzyme for 5HT synthesis, was reported to be anabolic to the skeleton. However, in other studies TPH1 deletion either had no bone effect or an age dependent inhibition of osteoclastic bone resorption. The role of 5HT in bone therefore remains poorly understood. To address this issue, we used selective breeding to create rat sublines with constitutively high (high-5HT) and low (low-5HT) platelet 5HT level (PSL) and platelet 5HT uptake (PSU). High-5HT rats had decreased bone volume due to increased bone turnover characterized by increased bone formation and mineral apposition rate, increased osteoclast number and serum C-telopeptide level. Daily oral administration of the TPH1 inhibitor (LX1032) for 6 weeks reduced PSL and increased the trabecular bone volume and trabecular number of the spine and femur in high-5HT rats. High-5HT animals also developed a type 2 diabetes (T2D) phenotype with increased: plasma insulin, glucose, hemoglobin A1c, body weight, visceral fat, β-cell pancreatic islets size, serum cholesterol, and decreased muscle strength. Serum calcium accretion mediated by parathyroid hormone slightly increased, whereas treatment with 1,25(OH)2D3 decreased PSL. Insulin reduction was paralleled by a drop in PSL in high-5HT rats. In vitro, insulin and 5HT synergistically up-regulated osteoblast differentiation isolated from high-5HT rats, whereas TPH1 inhibition decreased the number of bone marrow-derived osteoclasts. These results suggest that constitutively elevated PSL is associated with bone loss and T2D via a homeostatic interplay between the peripheral 5HT, bone and insulin.

Deletion of the membrane complement inhibitor CD59a drives age and gender-dependent alterations to bone phenotype in mice

Degenerative joint diseases such as osteoarthritis are characterised by aberrant region-specific bone formation and abnormal bone mineral content. A recent study suggested a role for the complement membrane attack complex in experimental models of osteoarthritis. Since CD59a is the principal regulator of the membrane attack complex in mice, we evaluated the impact of CD59a gene deletion upon maintenance of bone architecture.In vivo bone morphology analysis revealed that male CD59a-deficient mice have increased femur length and cortical bone volume, albeit with reduced bone mineral density. However, this phenomenon was not observed in female mice. Histomorphometric analysis of the trabecular bone showed increased rates of bone homeostasis, with both increased bone resorption and mineral apposition rate in CD59a-deficient male mice. When bone cells were studied in isolation, in vitro osteoclastogenesis was significantly increased in male CD59a-deficient mice, although osteoblast formation was not altered.Our data reveal, for the first time, that CD59a is a regulator of bone growth and homeostasis. CD59a ablation in male mice results in longer and wider bones, but with less density, which is likely a major contributing factor for their susceptibility to osteoarthritis. These findings increase our understanding of the role of complement regulation in degenerative arthritis.

Conditional Deletion of Prolyl Hydroxylase Domain-Containing Protein 2 (Phd2) Gene Reveals Its Essential Role in Chondrocyte Function and Endochondral Bone Formation.

The hypoxic growth plate cartilage requires hypoxia-inducible factor (HIF)-mediated pathways to maintain chondrocyte survival and differentiation. HIF proteins are tightly regulated by prolyl hydroxylase domain-containing protein 2 (Phd2)-mediated proteosomal degradation. We conditionally disrupted the Phd2 gene in chondrocytes by crossing Phd2 floxed mice with type 2 collagen-α1-Cre transgenic mice and found massive increases (>50%) in the trabecular bone mass of long bones and lumbar vertebra of the Phd2 conditional knockout (cKO) mice caused by significant increases in trabecular number and thickness and reductions in trabecular separation. Cortical thickness and tissue mineral density at the femoral middiaphysis of the cKO mice were also significantly increased. Dynamic histomorphometric analyses revealed increased longitudinal length and osteoid surface per bone surface in the primary spongiosa of the cKO mice, suggesting elevated conversion rate from hypertrophic chondrocytes to mineralized bone matrix as well as increased bone formation in the primary spongiosa. In the secondary spongiosa, bone formation measured by mineralizing surface per bone surface and mineral apposition rate were not changed, but resorption was slightly reduced. Increases in the mRNA levels of SRY (sex determining region Y)-box 9, osterix (Osx), type 2 collagen, aggrecan, alkaline phosphatase, bone sialoprotein, vascular endothelial growth factor, erythropoietin, and glycolytic enzymes in the growth plate of cKO mice were detected by quantitative RT-PCR. Immunohistochemistry revealed an increased HIF-1α protein level in the hypertrophic chondrocytes of cKO mice. Infection of chondrocytes isolated from Phd2 floxed mice with adenoviral Cre resulted in similar gene expression patterns as observed in the cKO growth plate chondrocytes. Our findings indicate that Phd2 suppresses endochondral bone formation, in part, via HIF-dependent mechanisms in mice.

Neoflavonoid dalbergiphenol from heartwood of Dalbergia sissoo acts as bone savior in an estrogen withdrawal model for osteoporosis.

Dalbergiphenol (DGP) is a neoflavonoid isolated from heartwood of Dalbergia sissoo. Effects of DGP on skeletal health remain to be elucidated. The objective of the present study was to investigate the biological effects of DGP on bone loss in ovariectomized mice. Adult BALB/c mice were ovariectomized and administered DGP (1 and 5 mg/kg/d) or 17β-estradiol (E2) orally for 6 weeks. The sham group and the ovariectomy (OVX) + vehicle group served as controls. Eight female BALB/c mice were taken for each group. Uterine estrogenicity, bone microarchitecture, biomechanical strength, new bone formation (based on bone formation rate and mineral apposition rate), and skeletal expression of osteogenic and resorptive gene markers were studied. OVX resulted in a marked increase in body weight and a decrease in femoral and vertebral trabecular bone volume that were prevented by DGP or E2 treatment. DGP treatment increased bone biomechanical strength and new bone formation rate in ovariectomized mice, comparable with E2 treatment. However, increase in uterine weight and estrogenicity were observed in E2-treated ovariectomized mice, but not in response to DGP treatment. Treatment with DGP increased messenger RNA expression of runt-related transcription factor 2, osterix, and collagen type I, and decreased messenger RNA expression of tartrate-resistant acid phosphatase and the osteoprotegerin-to-receptor activator of nuclear factor-κB ligand ratio in the femur of ovariectomized mice. Overall findings suggest that DGP treatment can effectively prevent OVX-induced increase in bone loss and decrease in bone strength possibly by increasing osteoblastic activities and by decreasing osteoclastic activities.

Skeletal maturation substantially affects elastic tissue properties in the endosteal and periosteal regions of loaded mice tibiae

Although it is well known that the bone adapts to changes in the mechanical environment by forming and resorbing the bone matrix, little is known about the influence of mechanical loading on tissue material properties of the pre-existing and newly formed bone. In this study, we analyzed the newly formed and pre-existing tissue after two weeks of controlled in vivo axial compressive loading in tibia of young (10week-old) and adult (26week-old) female mice and compared to the control contralateral limb, by means of scanning acoustic microscopy. Additionally, we used quantitative backscattered electron imaging to determine the bone mineral density distribution within the newly formed and pre-existing bone of young mice. No significant differences were found in tissue stiffness or mineral density in the pre-existing bone tissue as a result of external loading. In the endosteal region, 10 and 26week loaded animals showed a 9% reduction in bone tissue stiffness compared to control animals. An increase of 200% in the mineral apposition rate in this region was observed in both age groups. In the periosteal region, the reduction in bone tissue stiffness and the increase in bone mineral apposition rate as a result of loading were two times higher in the 10 compared to the 26week old animals. These data suggest that, during growth and skeletal maturation, the response of bone to mechanical loading is a deposition of new bone matrix, where the tissue amount but not its mineral or elastic properties are influenced by animal age.

Surface chemistry and effects on bone regeneration of a novel biomimetic synthetic bone filler.

The paper presents results of physico-chemical and biological investigations of a surface-engineered synthetic bone filler. Surface analysis confirms that the ceramic phosphate granules present a collagen nanolayer to the surrounding environment. Cell cultures tests show that, in agreement with literature reports, surface-immobilized collagen molecular cues can stimulate progression along the osteogenic pathway of undifferentiated human mesenchymal cells. Finally, in vivo test in a rabbit model of critical bone defects shows statistically significant increase of bone volume and mineral apposition rate between the biomimetic bone filler and collagen-free control. All together, obtained data confirm that biomolecular surface engineering can upgrade the properties of implant device, by promoting more specific and targeted implant-host cells interactions.

Death Receptor 3 (TNFRSF25) Increases Mineral Apposition by Osteoblasts and Region Specific New Bone Formation in the Axial Skeleton of Male DBA/1 Mice

Objectives. Genome wide association studies identified TNFSF member TNF-like protein 1A (TL1A, TNFSF15) as a potential modulator of ankylosing spondylitis (AS). TL1A is the only confirmed TNFSF ligand of death receptor 3 (DR3, TNFRSF25); however, its role in disease pathology is not characterised. We evaluated DR3's role in controlling osteoblast- (OB-) dependent bone formation in vitro and in vivo. Methods. Osteoprogenitor cells and OB were cultured from male DR3-deficient (DR3ko) and wild-type (DR3wt) DBA/1 mice. DR3 and RANKL expression were tested by flow cytometry. Alkaline phosphatase and mineralization were quantified. Osteopontin, osteoprotegerin, and pro MMP-9 were measured by ELISA. A fluorescent probe (BoneTag) was used to measure in vivo mineralization in 10-month-old mice. Results. DR3 was expressed on osteoprogenitors and OB from DR3wt mice. Alkaline phosphatase, osteopontin, and mineral apposition were significantly elevated in DR3wt cultures. Levels of RANKL were comparable whilst osteoprotegerin was significantly increased in DR3wt cultures. In vivo incorporation of BoneTag was significantly lower in the thoracic vertebrae of 10-month-old DR3ko mice. Conclusions. These data identify new roles for DR3 in regulating OB-dependent bone mineral apposition. They potentially begin to explain the atypical pattern of new bone formation observed in the axial skeleton of grouped, aging DBA/1 mice.

Single-Dose Local Simvastatin Injection Improves Implant Fixation via Increased Angiogenesis and Bone Formation in an Ovariectomized Rat Model

BackgroundStatins have been reported to promote bone formation. However, taken orally, their bioavailability is low to the bones. Implant therapies require a local repair response, topical application of osteoinductive agents, or biomaterials that promote implant fixation.Material/MethodsThe present study evaluated the effect of a single local injection of simvastatin on screw fixation in an ovariectomized rat model of osteoporosis.ResultsDual-energy X-ray absorptiometry, micro-computed tomography, histology, and biomechanical tests revealed that 5 and 10 mg simvastatin significantly improved bone mineral density by 18.2% and 22.4%, respectively (P<0.05); increased bone volume fraction by 51.0% and 57.9%, trabecular thickness by 16.4% and 18.9%, trabeculae number by 112.0% and 107.1%, and percentage of osseointegration by 115.7% and 126.3%; and decreased trabeculae separation by 34.1% and 36.6%, respectively (all P<0.01). Bone mineral apposition rate was significantly increased (P<0.01). Furthermore, implant fixation was significantly increased (P<0.05), and bone morphogenetic protein 2 (BMP2) expression was markedly increased. Local injection of a single dose of simvastatin also promoted angiogenesis. Vessel number, volume, thickness, surface area, and vascular volume per tissue volume were significantly increased (all P<0.01). Vascular endothelial growth factor (VEGF), VEGF receptor-2, von Willebrand factor, and platelet endothelial cell adhesion molecule-1 expression were enhanced.ConclusionsA single local injection of simvastatin significantly increased bone formation, promoted osseointegration, and enhanced implant fixation in ovariectomized rats. The underlying mechanism appears to involve enhanced BMP2 expression and angiogenesis in the target bone.

Role of WNT16 in the regulation of periosteal bone formation in female mice.

In this study, we evaluated the role of WNT16 in regulating bone size, an important determinant of bone strength. Mice with targeted disruption of the Wnt16 gene exhibited a 24% reduction in tibia cross-sectional area at 12 weeks of age compared with that of littermate wild-type (WT) mice. Histomorphometric studies revealed that the periosteal bone formation rate and mineral apposition rate were reduced (P < .05) by 55% and 32%, respectively, in Wnt16 knockout (KO) vs WT mice at 12 weeks of age. In contrast, the periosteal tartrate resistant acid phosphatase-labeled surface was increased by 20% in the KO mice. Because mechanical strain is an important physiological regulator of periosteal bone formation (BF), we determined whether mechanical loading-induced periosteal BF is compromised in Wnt16 KO mice. Application of 4800-μe strain to the right tibia using a 4-point bending loading method for 2 weeks (2-Hz frequency, 36 cycles per day, 6 days/wk) produced a significant increase in cross-sectional area (11% above that of the unloaded left tibia, P < .05, n = 6) in the WT but not in the KO mice (-0.2% change). Histomorphometric analyses revealed increases in the periosteal bone formation rate and mineral apposition rate in the loaded bones of WT but not KO mice. Wnt16 KO mice showed significant (20%-70%) reductions in the expression levels of markers of canonical (β-catenin and Axin2) but not noncanonical (Nfatc1 and Tnnt2) WNT signaling in the periosteum at 5 weeks of age. Our findings suggest that WNT16 acting via canonical WNT signaling regulates mechanical strain-induced periosteal BF and bone size.

Evaluation of morphological parameters of bone formation in Sprague–Dawley rats of different ages by in vivo fluorochrome labeling

Our study focuses on the morphological parameters of bone growth and bone formation in Sprague–Dawley (SD) rats of different ages, using fluorochrome labeling in vivo. We concluded that bone longitudinal growth rate (LGR) and bone mineral apposition rate (MAR) were significantly increased in both the 1- and 3-month-old rats compared with the 6- and 14-month-old rats, and that of the parameters were significantly decreased in both the 6- and 14-month-old rats. Meanwhile, bone formation rate (BFR) and percent labeled perimeter (%L.Pm) peaked in 3-month-old rats, and were followed by remarkable decrease with aging. In the present study, static parameters including percent trabecular area (%Tb.Ar), trabecular thickness (Tb.Th) and trabecular number (Tb.N), contrary to previous reports, progressively increased, whereas trabecular separation (Tb.Sp) reduced with increasing age. Furthermore, bone mass increased for 14-month-old rats due to the significantly decreased bone turnover rate.

Systemic Injection of CK2.3, a Novel Peptide Acting Downstream of Bone Morphogenetic Protein Receptor BMPRIa, Leads to Increased Trabecular Bone Mass

Bone Morphogenetic Protein 2 (BMP2) regulates bone integrity by driving both osteogenesis and osteoclastogenesis. However, BMP2 as a therapeutic has significant drawbacks. We have designed a novel peptide CK2.3 that blocks the interaction of Casein Kinase 2 (CK2) with Bone Morphogenetic Protein Receptor type Ia (BMPRIa), thereby activating BMP signaling pathways in the absence of ligand. Here, we show that CK2.3 induced mineralization in primary osteoblast cultures isolated from calvaria and bone marrow stromal cells (BMSCs) of 8 week old mice. Further, systemic tail vein injections of CK2.3 in 8 week old mice resulted in increased bone mineral density (BMD) and mineral apposition rate (MAR). In situ immunohistochemistry of the femur found that CK2.3 injection induced phosphorylation of extracellular signal-related kinase (ERK), but not Smad in osteocytes and osteoblasts, suggesting that CK2.3 signaling occurred through Smad independent pathway. Finally mice injected with CK2.3 exhibited decreased osteoclast differentiation and osteoclast activity. These data indicate that the novel mimetic peptide CK2.3 activated BMPRIa downstream signaling to enhance bone formation without the increase in osteoclast activity that accompanies BMP 2 stimulation.

Bisphosphonate-associated atypical sub-trochanteric femur fractures: Paired bone biopsy quantitative histomorphometry before and after teriparatide administration

ObjectivesBisphosphonate-associated atypical sub-trochanteric femur fractures (ASFF) may be seen with long-term bisphosphonate use, though these fractures are also seen in patients never exposed to bisphosphonates. One theory for the mechanism of action whereby bisphosphonates may induce these ASFF is over-suppression of bone turnover. Bisphosphonates suppress bone turnover, but in bisphosphonate clinical trials, over-suppression defined whether by maintaining the biochemical markers of bone turnover below the defined reference range or by quantitative bone histomorphometry, has not been observed. MethodsWe studied 15 clinic patients referred to The Colorado Center for Bone Research (CCBR) after they had a bisphosphonate-associated ASFF and performed quantitative bone histomorphometry both before and after 12 months of teriparatide (20µg SQ/day). All patients had been on long-term alendronate (mean = 7 years, range: 6–11 years) and had already had intramedullary rods placed when first seen (6 weeks to 7 months after rod placement). Alendronate had been discontinued in all patients at the time of their first clinic visit to CCBR. All of the fractures fulfilled The American Society for Bone and Mineral Research major radiological criteria for ASFF. ResultsThree key dynamic histomorphometric features show that 7 of the 15 patients had unmeasurable bone formation, mineralizing surface, and mineral apposition, while the other 8 patients had measurable dynamic parameters; although for all 15 patients, the mean values for all 3 dynamic parameters was far below the average for the published normal population. Administration of teriparatide was associated with an increase in all 3 dynamic histomorphometric parameters. Baseline bone turnover markers did not correlate with the baseline histomorphometry. While there is heterogeneity in the bone turnover in patients with bisphosphonate ASFF, there is a large portion in this uncontrolled series that had absent bone turnover at the standard biopsy site (iliac crest). Discontinuation of the bisphosphonate and administration of the anabolic agent, teriparatide was associated with improvement in bone turnover. ConclusionsWhile our study does not establish causality or address the ability of teriparatide to prevent progression of early stress fracture to displaced fractures, it does suggest that teriparatide may improve bone formation in these patients. Our study should stimulate other investigations using larger sample sizes and early stress fractures to see if anabolic agents can reverse these fractures from becoming displaced.

D-36 Free Communication/Poster - Ultrasound

PURPOSE: The molecular mechanism of bone strengthening with exercise remains unclear. We have identified a molecule, follistatin-like 3 (FSTL3), that may drive mechanical load-dependent bone remodeling. METHODS: Female Sprague Dawley rats (12-14 wks, n=10), wild-type (Fstl3+/+, 10-12 wks, n=10) or homozygous Fstl3-/- C57Bl/6 mice (10-12 wks, n=10) were subjected to treadmill walking (EX) for 45 min/day. After 0, 2, 5 or 15 days of EX, gene expression was analyzed by quantitative real time polymerase chain reaction, Western blots, or immunohistochemistry of the trabecular bone/bone marrow at distal femur ends. Bone mineral apposition rates were assessed by administration of Calcein and Alizarin complexones on day 3 and 12. Statistical analysis was performed by one-way ANOVA with Tukey’s post hoc or T-test. RESULTS: Gene expression analysis revealed that EX stimulated Fstl3 mRNA expression by 6 fold on day 2, and mRNA declined to basal levels by day 5. Minimal FSTL3 protein expression was exhibited in the control non-EX trabecular bone, but a robust increase in FSTL3 expression was observed in EX rat femurs at days 2 and 5. Non-EX mice exhibited a significantly narrow distance between the Calcein and Alizarin incorporation and limited bone deposition. EX Fstl3+/+ and Fstl3+/- mice demonstrated a significant increase in bone deposition on the endosteal surface of the femurs not observed in Fstl3-/- mice. Representative stress-strain curves of the cortical bone of the Fstl3+/+ and Fstl3 -/- femurs revealed that strain at ultimate strength of femurs from Fstl3+/+ mice was approximately 25% > than that of Fstl3 -/- mice. Furthermore, the yield strain and yield energy were significantly upregulated by EX in Fstl3+/+, but not in Fstl3-/- mice. CONCLUSION: FSTL3 is a mechanoresponsive protein that may provide a new paradigm for investigating EX-driven bone formation as well as a marker to monitor the effects of EX on bone in health and disease.

Effects of dietary fat levels and feeding durations on musculoskeletal health in female rats

This study evaluates the effects of dietary fat levels and feeding durations on body composition, bone properties, and endocrine factors in female rats. Forty-eight 3-month-old female rats were assigned into a 2 (low-fat diet vs. high-fat diet) × 2 (4-month vs. 8-month feeding duration) factorial design. The body composition, bone matrix and microstructure, and endocrine factors were examined. Compared to the low-fat diet, the high-fat diet (i) increased the % fat mass, femoral mineral density, trabecular and cortical formation and erosion, osteoblast and osteoclast numbers, insulin-like growth factor-I, and leptin and (ii) decreased the % fat-free mass, bone strength, and trabecular and cortical bone volume and thickness. Relative to the 4-month feeding duration, the 8-month feeding duration (i) increased the femoral mineral density, trabecular separation, formation and erosion, endocortical formation rate, and osteoclast number and (ii) decreased the trabecular bone volume, thickness, and number, osteoblast number, and adiponectin. Interactions between dietary fat level and feeding duration were observed in bone strength, trabecular thickness and formation rate, as well as cortical bone volume and mineral apposition rate. Both dietary fat level and feeding duration affect the bone mass and microstructure in female rats, possibly through the modulation of body composition and endocrine factors.