Mineral Apposition Rate Research Articles

The development of bone microstructure, metabolism and biomechanics in lumbar vertebra under short-term glucocorticoid exposure

sObjective: This study investigated the characteristics of bone microstructure, metabolism, and biomechanics in rat’s lumbar vertebra undergoing short-term glucocorticoid administration.Methods: Forty 4-month-old female Sprague–Dawley rats were treated with either vehicle (Cont) or prednisone acetate (Pre) at 3.5 mg/kg/day, respectively for periods of 7 days and 21 days. The lumbar vertebras were processed for MicroCT scan, histomorphometry analysis, mechanical compression test, in addition to Dual-Energy X-ray absorptiometry scan, respectively.Results: The connective density (Conn. D) along with trabecular connection nodes decreased while trabecular termini increased in Pre at day 21 when compared to Cont at day 21 as well as Pre at day 0. The mineralizing surface (MS/BS), mineral apposition rate (MAR), bone formation rate (BFR), osteoblast surfaces (Ob.S/BS) were lower in Pre at day 21 than that in Cont at day 21, Pre at day 0 and Pre at day 7. Only the bending stiffness of compression test decreased in Pre group at day 21 compared to age-matched control.Conclusion: The results suggested that excess prednisone significantly inhibited bone formation and slightly depressed bone resorption in the lumbar vertebra of intact rats for the duration of 21 days. Accordingly, the trabecular spatial microstructure made an adjustment yet failed to maintain the anti-compression mechanical property.

Predicting Bone Modeling Parameters in Response to Mechanical Loading

In vivo studies in mechanobiology and mechanotransduction explained the importance of mechanical loading in promoting osteogenesis (new bone formation) and thus, in preventing the bone loss. The literature suggests that the cyclic loading parameters viz. loading cycles, strain and frequency regulate the extent of new bone formation. Nevertheless, the amount of regulation has not been defined. As a result, researchers have been trying a data driven approach to estimate the new bone formation by proposing different empirical models. The models proposed so far have mainly focused on some specific bone modelling parameters such as mineral apposition rate (MAR). The literature, however, suggests that there are equally important bone modelling parameters which are also influenced by the change in cyclic loading parameters. Therefore, the results obtained from earlier computer modelling studies remain incomplete. This paper presents an improved empirical model which attempts to establish a relation between bone modelling parameters mineral apposition rate (MAR) and mineralising surface (MS/BS), and cyclic loading parameters. The results indicate that the proposed model has better accuracy in terms of prediction as compared to the state-of-the-art models involving only one bone modelling parameter i.e., MAR. The model may be useful in designing the optimal loading regimen to induce a desired new bone response. Based on these outcomes, a better bio-mechanical intervention may be developed in future to check bone loss.

Chemical Sympathectomy Impairs Peri-implant Osseointegration in Mice: Role of the Sympathetic Nervous System in Osseointegration.

The possibility that the sympathetic nervous system (SNS) controls bone remodeling has been raised; however, the actual function of the SNS in osseointegration is still unknown. This study aimed to investigate the effect of chemical sympathectomy on peri-implant osseointegration in adult mice. Forty C57BL/6J mice (8-week-old) were divided into two groups: a sympathectomy group and a control group, which were administered 6-hydroxydopamine and saline, respectively, by intraperitoneal injection for 5 days. Then, the mice were exposed to implant surgery. Analyses of serum chemistry, microcomputed tomography, biomechanical test, and bone histomorphometry were employed at 2 and 4 weeks. Compared with the control, the chemical sympathectomy group had a higher serum level of C-terminal collagen I cross-links but lower serum osteocalcin. After 4 weeks, peri-implant trabecular microstructure, including trabecular volume, trabecular thickness, the percentage of osseointegration, and bone-to-implant contact, was lower; however, the trabecular separation was higher in the sympathectomy group mice in comparison with the control group. In addition, the strength of bone-titanium integration measured by the biomechanical resistance test was lower. Furthermore, histomorphologic evidence revealed that the osteoclast counts were higher in the sympathectomy group, while the mineral apposition rate and the bone formation rate per bone surface were significantly lower. Within the limitations of this experimental study, the data showed that chemical sympathectomy has a negative effect in peri-implant osseointegration, suggesting that the SNS may need to be taken into consideration in terms of peri-implant bone healing.

A novel device to prevent osteoporosis by promoting bone metabolism using a newly developed double-loading stimulation with vibration and shaking.

In Japan, 13 million people have osteoporosis, including approximately 9 hundred thousand people who are bedridden owing to bone fractures from falls. Preventing osteoporosis is considered to be an important and effective way of preventing fall-related fractures. Thus, we developed a novel method of locomotor stimulation and analyzed its effectiveness in mice. Specifically, we created a double-loading device that combines vibration and shaking stimulation. The device was used to continuously stimulate ovariectomy-induced decreased bone density mouse models 30 minutes daily for 10 weeks. We then collected femur samples, created undecalcified tissue slices, calculated parameters using bone histomorphomtry, and conducted comparative testing. BS/TV (bone surface/tissue volume), N.Oc/ES (osteoclast number/eroded surface), Oc.S/ES (osteoclast osteoid surface/eroded surface), Omt (osteoid maturation time), Tb.N (trabecular number), Mlt (mineralization lag time) < (p < 0.01), N.Ob (osteoblast number), N.Ob/TV (osteoblast number/tissue volume), sLS (single labeled suface), N.Mu.Oc/ES (multinucle osteoclast number/eroded surface), and N.Mo.Oc/ES (mononucle osteoclast number/eroded surface) (p < 0.05) were significantly higher in the stimulation group than in the non-stimulation group. In addition, BS/BV (bone surface/bone volume), Tb.Sp (trabecular separation), MAR (mineral apposition rate), Aj.Ar (adjusted apposition rate) (p < 0.01), ES (eroded surface ), ES/BS (eroded surface/bone surface), and BRs.R (bone resorption rate) (p < 0.05) were significantly lower in the stimulation group than in the non-stimulation group. These results suggest that stimulation activated osteoblasts and osteoclasts, thereby leading to highly active bone remodeling. We anticipate that bone mineralization will subsequently occur, suggesting that this stimulation technique is effective in preventing osteoporosis by alleviating sudden bone density loss.

Effect of shaking and vibration stimulation on lumbar vertebrae in ovariectomized mice.

Bone fractures affect the activities of daily living and lower quality of life, so investigating preventative measures is important. We developed novel stimulation equipment that combined a vibration stimulus with a shaking stimulus for preventing osteoporosis (one of the causes of bone fractures). We aimed to investigate the effect of this equipment on ovariectomized mice. Oophorectomy of 8-week-old female mice was done. The stimulation group was stimulated for 10 consecutive weeks. The stimulation group showed significantly higher values (p<0.05) for osteoid thickness, osteoid volume-to-bone volume ratio and mineral apposition rate than those in the non-stimulation control group. The stimulation group showed significantly higher values (p<0.05) compared with the non-stimulation for expression of bone morphogenetic protein-2, interleukin-1β, interleukin-6 and myogenic determination gene in quadriceps femoris muscles (QFMs). These data suggest that cytokine secretion by QFMs carried a humoral factor throughout the body via the blood and blood vessels and acted on bone and various organs. Development of this stimulation method and its clinical application, new methods for preventing and treating osteoporosis could ensue.

Osteonecrosis of the jaws caused by bisphosphonate treatment and oxidative stress in mice.

Aging is a significant risk factor for the development of bisphosphonate-related osteonecrosis of the jaws (BRONJ). Accumulating evidence suggests that bone aging is associated with oxidative stress (OS), and OS is associated with osteonecrosis. To elucidate the mechanisms of the onset of BRONJ, the present study focused on OS and the effects of treatment with the pro-oxidant DL-buthionine-(S,R)-sulfoximine (BSO), an oxidative stressor, on healing of a surgically induced penetrating injury of the palate. Six-week-old C57BL/6J mice were randomly divided into four groups (n=5 each) and treated with or without zoledronic acid (ZOL) and with or without BSO (experimental groups: ZOL, BSO, and ZOL+BSO; control group: saline solution). A penetrating injury of the midline palate was surgically created using a root elevator. ZOL (250 µg/kg/day) was injected intraperitoneally every day from 7 days prior to the surgical treatment to 4 days following the surgical treatment. BSO (500 µg/kg/day) was administered 7 days prior to the surgical treatment as a single intraperitoneal injection. The maxillae were harvested at 5 days following the surgical treatment for histological and histochemical studies. The presence of empty osteocyte lacunae in the palatal bone was increased by ZOL and BSO treatment. The highest number of empty osteocyte lacunae was observed in the ZOL+BSO group. The number of tartrate-resistant acid phosphatase-positive cells was decreased by ZOL treatment and increased by BSO treatment. The number of canaliculi per osteocyte lacuna was significantly decreased by BSO treatment. The mineral apposition rate was significantly lower in the treatment groups than the control group. Bisphosphonates and OS suppressed bone turnover. The present study has demonstrated that BSO treatment affects osteocytes, and OS in osteocytes exacerbates impairment of the osteocytic canalicular networks. As a result, bisphosphonates and OS may induce osteonecrosis following invasive dentoalveolar surgery. OS has been identified as an additional risk factor for the development of BRONJ.

Pro-inflammatory Cytokines: Cellular and Molecular Drug Targets for Glucocorticoid-induced-osteoporosis via Osteocyte.

Glucocorticoids are widely used to treat varieties of allergic and autoimmune diseases, however, long-term application results in glucocorticoid-induced osteoporosis (GIOP). Inflammatory cytokines: tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) play important regulatory roles in bone metabolism, but their roles in GIOP remain largely unknown. Osteocytes can modulate the formation and function of both osteoblasts and osteoclasts, directly via gap junctions, or indirectly by transferring molecule signaling. Apoptotic osteocytes release RANKL, HMGB1 and pro-inflammatory cytokines to stimulate osteoclastogenesis. Moreover, osteocytes can secrete FGF23 to regulate bone metabolism. Exposure to high levels of GCs can drive osteocyte apoptosis and influence gap junctions, leading to bone loss. GCs treatment is regarded to produce more FGF23 to inhibit bone mineralization. GCs also disrupt the vascular to decrease osteocyte feasibility and mineral appositional rate, resulting in a decline in bone strength. Apoptotic bodies from osteocytes induced by GCs treatment can enhance production of TNF-α and IL-6. On the other hand, TNF-α and IL-6 show synergistic effects by altering osteocytes signaling towards osteoclasts and osteoblasts. In addition, TNF-α can induce osteocyte apoptosis and attribute to a worsened bone quality in GCs. IL-6 and osteocytes may interact with each other. Therefore, we hypothesize that GCs regulate osteocyteogenesis through TNF-α and IL-6, which are highly expressed around osteocyte undergoing apoptosis. In the present review, we summarized the roles of osteocytes in regulating osteoblasts and osteoclasts. Furthermore, the mechanism of GCs altered relationship between osteocytes and osteoblasts/osteoclasts. In addition, we discussed the roles of TNF-α and IL-6 in GIOP by modulating osteocytes. Lastly, we discussed the possibility of using pro-inflammatory signaling pathway as therapeutic targets to develop drugs for GIOP.

Histological Effects of the Combined Administration of Eldecalcitol and a Parathyroid Hormone in the Metaphyseal Trabeculae of Ovariectomized Rats

Intermittent administration of human parathyroid hormone (1-34) (hPTH(1-34)) promotes anabolic action in bone by stimulating bone remodeling, while eldecalcitol, an analog of active vitamin D3, suppresses osteoclastic bone resorption, and forms new bone by minimodeling. We have examined the biological effects of combined administration of eldecalcitol and hPTH(1-34) on 9-week-old Wistar rats that underwent an ovariectomy (OVX) or Sham operation. They were divided into a Sham group, OVX with vehicle (OVX group), OVX with 10 µg/kg/day of hPTH(1-34) (PTH group), OVX with 20 ng/kg/day of eldecalcitol (eldecalcitol group) or OVX with 10 μg/kg/day of hPTH(1-34), and 20 ng/kg/day of eldecalcitol (combined group) for 4 or 8 weeks. As a consequence, the combined group showed a marked increase in bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) than OVX and had the highest bone mineral density (BMD) compared with other groups. OVX and PTH groups exhibited a high osteoblastic surface/bone surface (Ob.S/BS), mineral apposition rate (MAR), and bone formation rate/bone surface (BFR/BS) indices and many TRAP-reactive osteoclasts. Contrastingly, eldecalcitol and combined groups tended to attenuate the indices of osteoclastic surface/bone surface (Oc.S/BS) and Ob.S/BS than that the other groups. The combined group revealed histological profiles of minimodeling- and remodeling-based bone formation. Thus, the combined administration of eldecalcitol and hPTH(1-34) augments their anabolic effects by means of minimodeling and remodeling.

Microbubble-Mediated Ultrasound Outweighs Low-Intensity Pulsed Ultrasound on Osteogenesis and Neovascularization in a Rabbit Model of Steroid-Associated Osteonecrosis.

Microbubbles magnify the acoustic pressure of low-intensity pulsed ultrasound (LIPUS) and may enhance its bioeffect for diagnostic and therapeutic purposes. This study compared the effect of this novel microbubble-mediated ultrasound (MUS) with that of the traditional LIPUS on osteogenesis and neovascularization in a rabbit model of steroid-associated osteonecrosis. We hypothesized that MUS might outweigh LIPUS on promoting osteogenesis and neovascularization in steroid-associated osteonecrosis. The bilateral femoral head necrosis was induced by lipopolysaccharide and methylprednisolone in the rabbits. The indices of bone mineral density (BMD), trabecular number, maximal loading strength, and mineral apposition rate were analyzed, demonstrating that the animal model of steroid-associated osteonecrosis was successfully established. Both the MUS group (GM) and the LIPUS group (GL) were insonated 20 min daily for six weeks. GM received an extra intracapsular injection of microbubbles before insonation every other day. Fluorescence bone labeling, Micro-CT Analysis, biomechanical test, quantitative real-time PCR, Western blot analysis, and histological evaluation were performed for comparing GM with GL. The results demonstrated a 39% higher mineral apposition rate in GM compared with GL. The BMD and the maximal loading strength of femoral head of GM increased by 4.3% and 27.8% compared to those of GL, respectively. The mRNA and protein expression of BMP-2 and VEGF were also significantly higher in GM. The number of blood vessels of GM was 65% greater than that of GL. MUS is more potent than LIPUS in enhancing osteogenesis, neovascularization, and biomechanical strength of femoral head in the animal model of steroid-associated osteonecrosis. Without increasing the intensity of insonation or the risk of tissue damage, MUS is better for inhibiting the process of steroid-associated osteonecrosis.

Interleukin-6 deletion stimulates revascularization and new bone formation following ischemic osteonecrosis in a murine model

Legg-Calvé-Perthes disease (LCPD) is a childhood form of ischemic osteonecrosis of the femoral head which can produce a permanent femoral head deformity and early osteoarthritis. The femoral head deformity results from increased bone resorption and decreased bone formation during repair and remodeling of the necrotic femoral head. A recent study showed that a pro-inflammatory cytokine, interleukin-6 (IL-6), is significantly elevated in the synovial fluid of patients with LCPD. We hypothesized that IL-6 elevation decreases bone formation during the repair process following ischemic osteonecrosis and that IL-6 depletion will increase new bone formation. To test this hypothesis, we surgically induced ischemic osteonecrosis in the wild-type (n = 29) and IL-6 knockout (KO) mice (n = 25). The animals were assessed at 48 h, 2 weeks and 4 weeks following the induction of ischemic osteonecrosis using histologic, histomorphometric and micro-CT methods. IL-6 immunohistochemistry showed high expression of IL-6 in the osteonecrotic side of the wild-type mice at 48 h and 4 weeks following ischemic osteonecrosis, but not in the IL-6 KO mice. We also confirmed an undetectable level of IL-6 expression in the primary osteoblasts of the IL-6 KO mice compared to the readily detectable level in the wild-type mice. Furthermore, we confirmed that IL-6 deletion did not affect the extent of bone necrosis in the IL-6 KO mice compared to the wild-type mice by performing histologic and terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) assessments at 2 weeks following the induction of ischemia. Both groups had the same extent of ischemic osteonecrosis and absence of repair at 2 weeks. At 4 weeks, the necrotic epiphyses showed a significant increase in the extent of revascularization in the IL-6 KO mice compared to the wild-type mice (p = 0.001). In addition, a significantly greater recovery of the hematopoietic bone marrow was observed in the osteonecrotic side of the IL-6 KO mice compared to the wild-type mice (p < 0.01). Vascular endothelial growth factor (VEGF) immunohistochemistry showed regionally increased staining in the areas of repair in the osteonecrosis side of IL-6 KO mice compared to the wild-type mice at 4 weeks following ischemic osteonecrosis. Micro-CT assessment of the wild-type mice at 4 weeks showed a significant decrease in the percent bone volume (p < 0.01) in the osteonecrotic side compared to the control side. In contrast, IL-6 KO mice showed significantly increased bone volume in the osteonecrotic side compared to the osteonecrotic side of WT mice (p < 0.001). No significant difference in the bone volume percentage was found between the control side of the wild-type and the IL-6 KO mice. Histomorphometric analysis at 4 weeks revealed increased osteoblast number/bone surface (p < 0.001), bone formation rate (BFR) (p = 0.0001), and mineral apposition rate (MAR) (p < 0.0001) in the osteonecrotic side of the IL-6 KO mice compared to the wild-type mice. The number of osteoclast/bone surface was also increased in the IL-6 KO mice compared to the wild-type mice (p < 0.0001). No significant difference was observed between the control side of the wild-type and IL-6 KO mice with regards to the number of osteoblast or osteoclast/bone surface, BFR, and MAR. We next obtained primary osteoblasts from IL-6 KO mice and showed they expressed a significantly higher level of RANKL/OPG than wild-type mice (p = 0.001) in hypoxia culture condition. Taken together, the findings indicate that IL-6 deletion stimulates revascularization and new bone formation following ischemic osteonecrosis. This study provides new evidence that therapeutic strategies to block IL-6 may be beneficial for bone healing following ischemic osteonecrosis.

A peptide containing the receptor binding site of insulin-like growth factor binding protein-2 enhances bone mass in ovariectomized rats

Male Igfbp2−/− mice have a significant reduction in bone mass and administration of a peptide that contains the insulin-like growth factor binding protein-2(IGFBP-2) receptor-binding domain stimulates bone formation in these animals. Female Igfbp2−/− mice do not have this phenotype but following ovariectomy (OVX) lose more bone than OVX wild-type mice. This suggests that in the absence of estrogen, IGFBP-2 is required to maintain bone mass. Therefore these studies were undertaken to determine if this peptide could stimulate bone acquisition in OVX rats. OVX rats were divided into seven treatment groups: sham animals, OVX animals, OVX animals receiving a control scrambled peptide, or one of three doses of the active peptide termed PEG-HBD-1 (0.7, 2, and 6 mg·kg-1) and an OVX group receiving parathyroid hormone (PTH) (50 µg·kg-1 per day). The peptides were administered for 8 weeks. DXA revealed a significant reduction in femoral and tibial areal bone mineral density (aBMD) after OVX, whereas treatment with the high-dose peptide increased aBMD by 6.2% ± 2.4% (P < 0.01) compared to control peptide; similar to the increase noted with PTH (5.6% ± 3.0%, P < 0.01). Similar increases were noted with two lower doses of the peptide (3.8% ± 1.5%, P < 0.05 for low dose; 3.1% ± 1.6%, P = 0.07 for middle dose). Micro CT showed that the OVX control peptide animals had reductions of 41% and 64% in femoral trabecular BV/TV and trabecular number, respectively. All three doses of the peptide increased bone volume/total volume (BV/TV) significantly, while the low and middle doses increased trabecular number. Cortical BV/TV and thickness at the midshaft increased significantly with each dose of peptide (18.9% ± 9.8%, P < 0.01 and 14.2% ± 7.9%, P < 0.01 for low dose; 23.7% ± 10.7%, P < 0.001 and 15.8% ± 6.1%, P < 0.001 for middle dose; 19.0% ± 6.9%, P < 0.01 and 16.2% ± 9.7%, P < 0.001 for high dose) and with PTH (25.8% ± 9.2%, P < 0.001 and 19.4% ± 8.8%, P < 0.001). Histomorphometry showed that the lowest dose of peptide stimulated BV/TV, trabecular thickness, mineral apposition rate (MAR), bone formation rate/bone surface (BFR/BS), number of osteoblasts/bone perimeter (N.ob/B.pm), and decreased osteoclast surface/bone perimeter (Oc.S/B.Pm). The highest dose stimulated each of these parameters except MAR and BFR/BS. Thus, the heparin-binding domain receptor region of IGFBP-2 accounts for its anabolic activity in bone. Importantly, this peptide enhances bone mass in estrogen-deficient animals.

R-spondin-2 is a Wnt agonist that regulates osteoblast activity and bone mass

The R-spondin family of proteins are Wnt agonists, and the complete embryonic disruption of Rspo2 results in skeletal developmental defects that recapitulate the phenotype observed with Lrp5/6 deficiency. Previous work has shown that R-spondin-2 (Rspo2, RSPO2) is both highly expressed in Wnt-stimulated pre-osteoblasts and its overexpression induces osteoblast differentiation in the same cells, supporting its putative role as a positive autocrine regulator of osteoblastogenesis. However, the role of Rspo2 in regulating osteoblastogenesis and bone formation in postnatal bone has not been explored. Here we show that limb-bud progenitor cells from Rspo2 knockout mice undergo reduced mineralization during osteoblastogenesis in vitro and have a corresponding alteration in their osteogenic gene expression profile. We also generated the first Rspo2 conditional knockout (Rspo2floxed) mouse and disrupted Rspo2 expression in osteoblast-lineage cells by crossing to the Osteocalcin-Cre mouse line (Ocn-Cre + Rspo2f/f). Ocn-Cre + Rspo2f/f male and female mice at 1, 3, and 6 months were examined. Ocn-Cre + Rspo2f/f mice are decreased in overall body size compared to their control littermates and have decreased bone mass. Histomorphometric analysis of 1-month-old mice revealed a similar number of osteoblasts and mineralizing surface per bone surface with a simultaneous decrease in mineral apposition and bone formation rates. Consistent with this observation, serum osteocalcin in 3-month-old Ocn-Cre + Rspo2f/f was reduced, and bone marrow-mesenchymal stem cells from Ocn-Cre + Rspo2f/f mice undergo less mineralization in vitro. Finally, gene expression analysis and immunohistochemistry of mature bone shows reduced beta-catenin signaling in Ocn-Cre + Rspo2f/f. Overall, RSPO2 reduces osteoblastogenesis and mineralization, leading to reduced bone mass.

Analysis of mineral apposition rates during alveolar bone regeneration over three weeks following transfer of BMP-2/7 gene via in vivo electroporation

Alveolar bone is not spontaneously regenerated following trauma or periodontitis. We previously proposed an animal model for new alveolar bone regeneration therapy based on the non-viral BMP-2/7 gene expression vector and in vivo electroporation, which induced the formation of new alveolar bone over the course of a week. Here, we analysed alveolar bone during a period of three weeks following gene transfer to periodontal tissue. Non-viral plasmid vector pCAGGS-BMP-2/7 or pCAGGS control was injected into palatal periodontal tissue of the first molar of the rat maxilla and immediately electroporated with 32 pulses of 50 V for 50 msec. Over the following three weeks, rats were double bone-stained by calcein and tetracycline every three days and mineral apposition rates (MAR) were measured. Double bonestaining revealed that MAR of alveolar bone was at similar level three days before BMP-2/7 gene transfer as three days after gene transfer. However, from 3 to 6 days, 6 to 9 days, 9 to 12 days, 12 to 15 days, 15 to 18 days, and 18 to 20 days after, MARs were significantly higher than prior to gene transfer. Our proposed gene therapy for alveolar bone regeneration combining nonviral BMP-2/7 gene expression vector and in vivo electroporation could increase alveolar bone regeneration potential in the targeted area for up to three weeks.

Cyclic loading effects on craniofacial strain and sutural growth in pigs.

Current craniofacial growth modification devices use static forces, but cyclic forces are believed by some to be more effective. The latter have not been evaluated in large animal models, and it is not known how such forces are transmitted to distant parts of the skull. In this study, we aimed to (1) develop a portable loading system capable of delivering reliable cyclic loads to the porcine nasofrontal suture (NFS), (2) explore strain transmission to distant sutures, and (3) characterize the sutural growth effects in a small pilot study. After we validated the device, cyclic (2.5Hz) tensile loads were applied unilaterally to the NFS of 6 abattoir pig heads, with strain gauges on multiple sutures. Similar loading was applied to 3-month-old live pigs (Sus scrofa, n=4 and 1 sham) 30minutes per day for 5days. These animals received fluorescent markers of mineralization on loading days 1 and 3. Suture strains were recorded on day 5. Histomorphometric analysis quantified suture width and mineral apposition rate. A wearable loading system was developed to produce an average of +900 microstrain at the targeted NFS. Substantial strains were seen at the contralateral NFS and midline sutures, but bone strains were low. Strain patterns were similar exvivo and invivo, with the latter generally having higher magnitudes. Preliminary evidence demonstrates wider sutures with higher mineral apposition rates in the loaded sutures. Daily spurts of cyclic load caused sutural strain throughout the skull. This regimen most likely enhances sutural growth and may be therapeutically useful.

CMHX008, a Novel Thiazolidinedione-Like PPARγ Partial Agonist, Enhances Insulin Sensitivity with Minor Influences on Bone Loss

Objective: Traditional thiazolidinediones (TZDs), rosiglitazone for instance, are peroxisome proliferator-activated receptor γ (PPARγ) potent agonists and can treat type 2 diabetes but carry unwanted effects including increased risk for fracture. Here we compared insulin-sensitizing efficacies and bone-loss side effects of CMHX008, a novel TZDs-like PPARγ partial agonist, with rosiglitazone. Methods: TR-FRET PPARγ competitive binding assay was used to compare the binding affinity between CMHX0and rosiglitazone. High fat diet fed and ob/ob mice were administered with vehicle, CMHX0or rosiglitazone. Bone marrow mesenchymal stem cells (BMMSCs) were used to examine differences in differentiation into osteoblasts after treated with CMHX0and rosiglitazone. Results: TR-FRET showed lower affinity to PPARγ by CMHX0compared with rosiglitazone. Mice treated with CMHX0for 16 weeks had the comparable effect of insulin sensitization as rosiglitazone, which was related to significant inhibition on PPARγ Ser 273 phosphorylation and improved insulin sensitivity by facilitating phosphorylation insulin receptor and AKT in adipose tissues. Micro-CT and histomorphometric analyses demonstrated that the degree of trabecular bone loss after treated with CMHX0was weaker than rosiglitazone as evidence by consistent changes in BV/TV, Tb.N, Tb.Th, Tb.Sp, and mineral apposition rate. BMMSCs treated with CMHX0showed higher ALP activity and mRNA levels of bone formation markers than those treated by rosiglitazone in osteoblast differentiation test. Conclusion: PPARγ sparing eliminates the skeletal side effects of TZDs while maintaining their insulin-sensitizing properties. CMHX0shared the comparable insulin-sensitizing effects as rosiglitazone with lower risk of bone loss. Disclosure X. Xiao: None. Y. Hou: None. X. Cao: None. J. Li: None.

Effects of Biomineralization on Osseointegration of Pure Titanium Implants in the Mandible of Beagles

The purpose of this study was to investigate the effect of a biologically active dental implant surface (treated with sandblasting and acid etching [SLA] followed by immersion in simulated body fluid [SBF]) on osseointegration. We randomly divided 9 healthy adult male beagles (aged 8months; body weight, 12kg) into 3 groups: machined, SLA, and SLA-biomineralization (SLA-Bio). Six pure titanium implants (diameter of 3.5mm and length of 8mm) were used in the mandible of each dog after observation of the surface morphology, as well as analysis of the composition of the surface elements by scanning electron microscopy-energy dispersive x-ray spectroscopy. At 4, 8, and 12weeks after implantation, animals were euthanized to collect the mandibles so that we could perform the removal torque test to evaluate the implant stability in bone and histomorphometry to analyze the implant-bone osseointegration. Scanning electron microscopy results showed that uniformly distributed sponge-like structures were found on the SLA-treated surface and an apatite layer was observed on the SLA-SBF-treated surface (SLA-Bio group). In the energy dispersive x-ray spectroscopy analysis, the elements titanium, oxygen, carbon, calcium, and phosphorus were found on the surfaces of the SLA-Bio group, whereas titanium was the only element found in the other groups.The removal torque test showed that the peak removal torque values of the 3 groups increased gradually with the passage of time, and the peak removal torque values of the SLA-Bio group were significantly higher than those of the other groups (P<.01) at 4, 8, and 12weeks after implantation. Histomorphometric analysis showed that osseointegration was being enabled more rapidly in the SLA-Bio group, as well as that the mineral apposition rate and percentage of bone-to-implant contact of the SLA-Bio group were higher than those of the remaining groups at 4, 8, and 12weeks after implantation (P<.01). Treating titanium implants with SLA-SBF can improve osseointegration as well as increase the interfacial shear strength.

CMHX008, a PPARγ partial agonist, enhances insulin sensitivity with minor influences on bone loss

Traditional thiazolidinediones (TZDs), such as rosiglitazone, are peroxisome proliferator-activated receptor γ (PPARγ) potent agonists that can be used to treat type 2 diabetes but carry unwanted effects, including increased risk for fracture. The present work aimed to compare the insulin-sensitizing efficacies and bone-loss side effects of CMHX008, a novel TZDs-like PPARγ partial agonist, with those of rosiglitazone. A TR-FRET PPARγ competitive binding assay was used to compare the binding affinity between CMHX008 and rosiglitazone. Mice were administered vehicle, CMHX008 or rosiglitazone for 16 weeks. Mesenchymal stem cells (MSCs) were used to examine differences in differentiation into osteoblasts after compounds treatment. TR-FRET showed lower affinity to PPARγ by CMHX008 compared with rosiglitazone. Mice treated with CMHX008 showed insulin sensitization similar to that of mice treated with rosiglitazone, which was related to the significant inhibition of PPARγ Ser273 phosphorylation and improved insulin sensitivity by facilitating the phosphorylation of insulin receptor and Akt in adipose tissues. Micro-CT and histomorphometric analyses demonstrated that the degree of trabecular bone loss after treatment with CMHX008 was weaker than that observed with rosiglitazone, as evidenced by consistent changes in BV/TV, Tb.N, Tb.Th, Tb.Sp, and the mineral apposition rate. MSCs treated with CMHX008 showed higher ALP activity and mRNA levels of bone formation markers than did cells treated with rosiglitazone in the osteoblast differentiation test. Thus, CMHX008 showed insulin-sensitizing effects similar to those of rosiglitazone with a lower risk of bone loss, suggesting that PPARγ sparing eliminates the skeletal side effects of TZDs while maintaining their insulin-sensitizing properties.

Effects of the calcineurin inhibitors cyclosporine and tacrolimus on bone metabolism in rats.

Immunosuppressive therapy is considered as one of the factors inducing to the onset of osteoporosis after organ transplantation. Chronic immunosuppressive therapy after transplantation is required for organ transplant patients, and it is important to prevent the occurrence of osteoporotic fractures to maintain the quality of life in patients. In this study, we examined the effects of cyclosporine and tacrolimus on bone metabolism in rats. Five-week-old male Wistar rats were treated orally with 15 mg/kg cyclosporine or 1.5 mg/kg tacrolimus daily for 4 weeks. Each of cyclosporine and tacrolimus significantly reduced the bone strength of the femoral mid-diaphysis and bone mineral density of the tibia and femur. Bone histomorphometry showed that the administration of both drugs resulted in a decrease in bone volume, number and thickness of trabeculae, and an increase in trabecular separation. Bone formation parameters such as osteoid volume, osteoblast surface, mineralizing surface, mineral apposition rate, and bone formation rate significantly increased in the cyclosporine-treated group. Bone resorption parameters such as eroded surface, osteoclast surface, and osteoclast number significantly increased in both the cyclosporine- and the tacrolimus- treated groups. These results showed that cyclosporine increases both bone formation and bone resorption, leading to a high-turnover bone loss, and that tacrolimus increases bone resorption without affecting bone formation, leading to bone loss.

Enhancement of surface bioactivity on carbon fiber-reinforced polyether ether ketone via graphene modification.

Background and objectiveThe modulus of carbon fiber-reinforced polyether ether ketone (CFR-PEEK), a composite containing layers of carbon fiber sheets, can be precisely controlled to match bone. However, CFR-PEEK is biologically inert and cannot promote bone apposition. The objective of this study was to investigate whether graphene modification could enhance the bioactivity of CFR-PEEK.Methods and resultsIn vitro, the proliferation and differentiation of rat bone marrow stromal cells on scaffolds were quantified via cell-counting kit-8 assay and Western blotting analysis of osteoblast-specific proteins. Graphene modification significantly promoted bone marrow stromal cell proliferation and accelerated induced differentiation into osteogenic lineages compared to cells seeded onto nongraphene-coated CFR-PEEK. An in vivo rabbit extraarticular graft-to-bone healing model was established. At 4, 8, and 12 weeks after surgery, microcomputed tomography analyses and histological observations revealed significantly better microstructural parameters and higher average mineral apposition rates for graphene-modified CFR-PEEK implants than CFR-PEEK implants (P<0.05). van Gieson staining indicated more new bone was formed around graphene-modified CFR-PEEK implants than CFR-PEEK implants.ConclusionGraphene may have considerable potential to enhance the bioactivity and osseointegration of CFR-PEEK implants for clinical applications.

Strontium-releasing fluorapatite glass-ceramic scaffolds: Structural characterization and in vivo performance

There is increasing interest in biodegradable ceramic scaffolds for bone tissue engineering capable of in situ delivery of ionic species favoring bone formation. Strontium has been shown to be osteogenic, but strontium-containing drugs such as strontium ranelate, used in Europe for the treatment of osteoporosis, are now restricted due to clinical evidence of systemic effects. By doping fluorapatite-based glasses with strontium, we developed ceramic scaffolds with fully interconnected macroporosity and cell size similar to that of cancellous bone, that are also capable of releasing strontium. The crystallization behavior, investigated by XRD and SEM, revealed the formation of akermanite and fluorapatite at the surface of strontium-free glass-ceramic scaffolds, and strontium-substituted fluorapatite at the surface of the strontium-doped scaffolds. At 8 weeks after implantation in a rat calvarial critical size defect, scaffolds doped with the highest amount of strontium led to the highest mineral apposition rate. A significantly higher amount of newly-formed bone was found with the strontium-free glass-ceramic scaffold, and possibly linked to the presence of akermanite at the scaffold surface. We demonstrate by energy dispersive XRF analyses of skull sections that strontium was present in newly formed bone with the strontium-doped scaffolds, while a significant amount of fluorine was incorporated in newly formed bone, regardless of composition or crystallization state. Statement of SignificanceThe present work demonstrates the in vivo action of strontium-containing glass-ceramic scaffolds. These bone graft substitutes are targeted at non load-bearing bone defects. Results show that strontium is successfully incorporated in newly formed bone. This is associated with a significantly higher Mineral Apposition Rate. The benefits of in situ release of strontium are demonstrated. The broader scientific impact of this works builds on the concept of resorbable ceramic scaffolds as reservoirs of ionic species capable of enhancing bone regeneration.