Serum TRACP Research Articles

Zoledronic Acid Prevents Bone Resorption Caused by the Combination of Radium-223, Abiraterone Acetate, and Prednisone in an Intratibial Prostate Cancer Mouse Model.

An increased risk of non-pathological fractures in patients with prostate cancer and bone metastases has been associated with combination treatment with radium-223, abiraterone, and prednisone/prednisolone in the absence of bone-protecting agents. Here, we investigated possible mechanisms leading to this outcome using an intratibial LNCaP model mimicking prostate cancer bone metastases. Male NOD.scid mice were inoculated intratibially with LNCaP prostate cancer cells and treated with vehicle, radium-223, abiraterone, prednisone, zoledronic acid, or their combinations for 28 days. Serum TRACP 5b and PSA levels were measured. Bone structure, quality, and formation rate of non-tumor-bearing and tumor-bearing tibiae were analyzed by microCT, 3-point bending assay, and dynamic histomorphometry, respectively. Radium-223 incorporation into bone was also measured. Radium-223/abiraterone/prednisone combination treatment induced a transient increase in bone resorption indicated by elevated TRACP 5b levels, which was inhibited by concurrent treatment with zoledronic acid. Furthermore, radium-223/abiraterone/prednisone combination reduced periosteal and trabecular new bone formation and the number of osteoblasts, but bone structure or biomechanical quality were not affected. The abiraterone/prednisone treatment decreased radium-223 incorporation into tumor-bearing bone, possibly explaining the lack of additional antitumor efficacy. In conclusion, radium-223/abiraterone/prednisone combination increased bone resorption, which may have been one of the mechanisms leading to an increased fracture risk in patients with mCRPC.

A CXCR4 inhibitor (balixafortide) enhances docetaxel-mediated antitumor activity in a murine model of prostate cancer bone metastasis.

Prostate cancer (PCa) bone metastases have been shown to be more resistant to docetaxel than soft tissue metastases. The proinflammatory chemokine receptor CXCR4 has been shown to confer resistance to docetaxel (DOC) in PCa cells. Balixafortide (BLX) is a protein epitope mimetic inhibitor of CXCR4. Accordingly, we hypothesized that BLX would enhance DOC-mediated antitumor activity in PCa bone metastases. PC-3 luciferase-labeled cells were injected into the tibia of mice to model bone metastases. Four treatment groups were created: vehicle, DOC (5 mg/kg), BLX (20 mg/kg), and combo (receiving both DOC and BLX). Mice were injected twice daily subcutaneously with either vehicle or BLX starting on Day 1 and weekly intraperitoneally with DOC starting on Day 1. Tumor burden was measured weekly via bioluminescent imaging. At end of study (29 days), radiographs were taken of the tibiae and blood was collected. Serum levels of TRAcP, IL-2, and IFNγ levels were measured using ELISA. Harvested tibiae were decalcified and stained for Ki67, cleaved caspase-3, and CD34 positive cells or microvessels were quantified. Tumor burden was lower in the combo group compared to the DOC alone group. Treatment with the combination had no impact on the number of mice with osteolytic lesions, however the area of osteolytic lesions was lower in the combo group compared to the vehicle and BLX groups, but not the DOC group. Serum TRAcP levels were lower in the combo compared to vehicle group, but not the other groups. No significant difference in Ki67 staining was found among the groups; whereas, cleaved caspase-3 staining was lowest in the Combo group and highest in the BLX group. The DOC and combo groups had more CD34+ microvessels than the control and BLX groups. There was no difference between the treatment groups for IL-2, but the combo group had increased levels of IFNγ compared to the DOC group. Our data demonstrate that a combination of BAL and DOC has greater antitumor activity in a model of PCa bone metastases than either drug alone. These data support further evaluation of this combination in metastatic PCa.

Effect of Magnetic Nanoparticles on Hormone Level Changes During Perimenopausal Period and Regulation of Bone Metabolism.

This work aimed to explore the effect of nerve magnetic stimulation based on superparamagnetic Fe3O4 nanoparticle (NP) on bone metabolism during the perimenopausal period. First, the multifunctional water-soluble polymer PTMP-PMAA was utilized as the ligand. PTMP-MAA@ Fe3O4 NP with high magnetization was prepared by the co-precipitation method, and NPX diffraction pattern analysis and in vitro stability analysis were implemented. Then, NPs were co-cultured with 293T cells, and the cytotoxicity was detected by the CCK-8 method. Subsequently, 3-month-old female young SD rats and 11~15-month-old natural menopausal SD rats were taken as the research objects. According to the vaginal smear, the rats were randomly rolled into a young control, perimenopausal period model, estrogen treatment, and osteoporosis prevention groups. Rats in the estrogen treatment group were given Premarin suspension by gavage. Rats in the osteoporosis prevention group were injected stereotaxically with PTMP-MAA@ Fe3O4 NP suspension, and a rotating magnetic field was applied to the brain for nerve magnetic stimulation. The rats were sacrificed three days after treatment and brain tissues were taken for pathological analysis. Rat humerus was weighted and dual-energy X-ray was utilized to determine bone density and bone mineral content. Serum was collected and radioimmunoassay and ELISA were employed to detect estradiol (E2), osteocalcin (Boneglaprotein, BGP), oxytocin (OT), bone alkaline phosphatase (BALP), type I collagen carboxy-terminated cross-linked peptide (CTX-I), and tartrate-resistant acid phosphatase (TRACP-5b) in the serum of rats in each group. The results showed that PTMP-MAA@ Fe3O4 NP had good biocompatibility, and the CCK-8 test results showed that PTMP-MAA@ Fe3O4 NP had low cytotoxicity. Compared with the young control group, the humeral dry weight, wet weight, bone density, and bone mineral content, serum E2, OT, and BGP content in the perimenopausal period model group were reduced, while the serum BALP, CTX-I, and TRACP -5b content was increased (P<0.05). It was verified that nerve magnetic stimulation based on PTMP-MAA@ Fe3O4 NP increased the serum estrogen level of female rats during the perimenopausal period, increased the bone density of rats, promoted bone formation, and regulated bone metabolism.

Effect of moxibustion of "Ganshu"(BL18) and "Shenshu" (BL23) on musculoskeletal interaction in osteoporosis rats

To observe the effect of moxibustion of "Ganshu" (BL18)and "Shenshu" (BL23) on bone mineral density (BMD), biomechanics, bone metabolism and musculoskeletal morphology of osteoporosis (OP) rats, so as to explore its mechanism underlying treatment of OP via bone-muscle interaction. Thirty-two female SD rats were randomly divided into sham operation, OP model, moxibustion and medication (nilestriol) groups, with 8 rats in each group. For rats of the sham operation group, a small amount of fat around the ovaries was removed, and those of the other 3 groups received removal of bila-teral ovaries for establishing OP model. Moxibustion was applied to bilateral BL18 and BL23 for 30 min, once every other day, 3 times a week for 12 weeks. Rats of the medication group received gavage of nilestriol (1 mg/kg) once a week for 12 weeks.After the interventions, a dual-energy absorptiometry was used to determine the BMD and bone mineral content of the rats' right femur and the fourth lumbar vertebrae under anesthesia, and three-point bending test used to detect the biomechanical properties (including load, displacement, stiffness) of the right femur. The levels of serum type I collagen C-terminal peptide (CTX-I), acid-resis-tant tartrate phosphatase (TRACP) and estradiol (E2) were determined using enzyme-linked immunosorbent assay, and histopathological changes of the left femur and the quadriceps observed after hematoxylin-eosin (H.E.) staining. Compared with the sham operation group, the BMD and bone mineral contents of the right femur and the fourth lumbar vertebra, the load and stiffness of the right femur, and concentration of serum E2 were significantly decreased (P<0.01, P<0.05), and the displacement of the right femur, and concentrations of serum CTX-Ⅰ and TRACP notably increased in the model group (P<0.01). After the interventions, the decreased levels of BMD and bone mineral contents, the load and stiffness and concentration of serum E2, and the increased levels of the displacement, and concentrations of serum CTX-Ⅰ and TRACP were all reversed by both moxibustion (except the bone mineral content of the fourth lumbar vertebra) and medication (P<0.01,P<0.05). No signi-ficant differences were found between moxibustion and medication in up-regulating the levels of BMD and bone mineral contents, the load and stiffness (except serum E2) and down-regulating the levels of the displacement, and concentrations of serum CTX-Ⅰ and TRACP (P>0.05). H.E. staining revealed that rats in the sham operation group showed mild thinness of the bone cortex, uneven thickness of trabecular bone, with distortion, fracture and osteoporosis of the left femur, and different size of rhabdomyocytes in the right quadriceps femoris muscle, with obvious proliferation of interstitial fibrous tissue and inflammatory cell infiltration, which were relatively and clearly milder in both moxibustion and medication groups. Moxibustion of BL18 and BL23 can increase the BMD and bone mineral content, improve biomechanical performance, adjust bone metabolism, and mitigate bone and the attached muscle histopathological changes in OP rats, suggesting that modulating interaction between bones and muscles is probably one of the ideas in the treatment of OP.

Serum biomarkers for bisphosphonate-related osteonecrosis of the jaw: a prospective clinical study.

Bisphosphonate-related osteonecrosis of the jaw (ONJ) is an adverse effect of long-term bisphosphonate therapy. This study aimed to identify bone biomarkers for ONJ risk assessment and diagnosis. This prospective study included patients with histories of bisphosphonate therapy without current ONJ who were in need of dentoalveolar surgery of the jaw area. Serum levels of 12 possible bone markers, selected based on their involvement in ONJ pathogenesis, were compared between ONJ and control groups before dentoalveolar surgery (T0), at 8 postoperative weeks (T1), and at 4months after diagnosis(T2). Seventy-six patients who met the inclusion criteria were included in the study; 33 were assigned to the ONJ group, and 43 patients without ONJ signs or symptoms after dentoalveolar surgery were assigned to the control group. In the ONJ group, at both T0 and T1, the mean tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) levels were significantly lower and the mean Dickkopf-related protein 1 (DKK1) levels were significantly higher than the corresponding values for the control group. Linear mixed model analysis revealed significant group effects over time for serum TRACP 5b and DKK1 after adjusting for demographic, pharmacological, and diagnostic variables. Lower serum levels of TRACP 5b under a specified cut-off value (≤ 2.899 U/L) at T0 indicated a 20.40-fold increased risk of ONJ development. Patients with abnormally low serum levels of TRACP 5b and high serum levels of DKK1 should be monitored closely before and after dentoalveolar surgery for the prevention and early diagnosis of ONJ.

Phase 2 Study of Carfilzomib and Bone Metabolism in Patients with Relapsed Multiple Myeloma

Background: Carfilzomib is an epoxyketone proteasome inhibitor (PI) that binds selectively and irreversibly to the constitutive proteasome and immunoproteasome. In addition to its direct cytotoxicity, PIs possess bone anabolic effect, by facilitating osteoblast differentiation, which in turn, inhibits multiple myeloma (MM) growth. However, the relative contribution of carfilzomib on direct tumor killing and bone formation has been difficult to discern when it is used in combination with other agents such as steroids, chemotherapeutics, or immunomodulatory agents (iMiDs); most of which increase bone loss.Objective: We explored the effect of single agent carfilzomib on bone metabolism and disease response in patients with relapsed MM.Methods: We conducted a phase II study of single-agent carfilzomib at 56 mg/m2 day 1, 2, 8, 9, 15, 16 of a 28 day cycle in patients with relapsed MM. Serum markers of bone resorption including carboxyterminal cross-linked telopeptide of type I collagen (ICTP) and tartrate resistant acid phosphatase 5b (TRACP 5b) and bone formation markers including N-terminal type 1 procollagen (P1NP) and osteocalcin (OC) were analyzed at baseline and 6 months after therapy, and compared using paired t-tests. Imaging using 18F-NaF PET-CT scans to monitor bone response and 8F-FDG PET-CT to measure localized tumor burden were done at baseline and 6 months post treatment. For each patient, the three sites with the highest baseline SUVs and an iliac crest (representing an uninvolved site) were selected for quantitative comparisons using paired t-test.Results: Ten patient were accrued. Average age was 70.1 +/- 6.79 (mean +/- SD) years. Median prior lines of therapy was 2. 9 were evaluable for response. Overall response rate was 77.8%: VGPR (1/9: 11%), partial response (6/9: 67%), stable disease (2/9: 22%). Median PFS was 11.5 months. Overall survival was 21.9 months. Common treatment adverse events (AE) were comparable to previous reports including diarrhea (40%), fever (40%), dyspnea (30%) and fatigue (30%). Grade 3/4 AEs, occurring in 10% each included neutropenia, thrombocytopenia, dyspnea, fatigue, vomiting, ataxia, heart failure and pulmonary edema.Serum ICTP and TRACP were elevated at baseline in 80% of subjects; means (+/- SD) of serum ICTP (14.4 +/- 3.1 ug/L) and TRACP (8.2 +/- 4.2 U/L), respectively. Both markers declined significantly after therapy (means +/- SD of serum ICTP 8.4 +/- 3.1 ug/L and TRACP (5.2 +/- 4.2 U/L); p<0.01), suggesting osteoclast suppression. Serum PINP and OC varied among subjects; 5 patients with low levels and 4 normal levels and 1 with elevated level: mean (+/- SD) of PINP 25 +/- 23 ug/L and OC (18 +/- 12 ug/L). Neither post treatment mean PINP nor OC differed significantly from baseline overall (p=0.16). However, all subjects who achieve PR or better had a more than 25% increase in PINP and OC, suggesting increased new bone formation.8F-FDG and 18F-NaF were positive in all subjects. Number of active sites and SUVs vary largely among subjects. 18F-NaF images mirrored the 8F-FDG signals, appearing as rings around the tumor sites, suggesting a bone reaction to the localized tumors. 6 of 9 subjects had post treatment PET/CTs for paired comparison. Post treatment 8F-FDG SUV of representative sites decreased significantly, while 18F-NaF SUVs varied; 58% of lesions had a decline in SUV, while 32% were unchanged and 10% were brighter, suggesting heterogeneous bone response. 18F-NaF SUVs of iliac sites decreased post treatment, suggesting a lower bone turnover overall. CT scans showed increased sclerosis at the rim of lytic lesions without significant change in lesion size, at this early time point.Conclusion: Carfilzomib at 56 mg/m2 dosing strongly inhibits bone resorption and demonstrates bone anabolic effects, in addition to cytotoxicity, in relapsed MM. Single agent use allows bone response analysis without the confounding effects of other anti-MM agents used in combination therapy. Response rates and side effects are favorable to prior reports. Serum markers of bone remodeling are useful for an assessment of overall bone metabolism while the 18F-NaF PET/CT allows assessment of both local and systemic changes but lacks specificity. Studies of osteoblast differentiation from subject's bone marrow stromal cells pre and post treatment are ongoing. Whether carfilzomib can heal bone lesions requires longer follow up. DisclosuresSuvannasankha:Celgene: Research Funding; Bristol-Myers Squibb: Research Funding; Takeda: Research Funding; Janssen Pharmaceuticals: Research Funding. Abonour:Celgene: Other: Steering committee, Research Funding; Prothena: Research Funding; Takeda: Other: Steering committee, Research Funding. Roodman:PharmaMar: Research Funding.

Abstract 6286: The effects of prednisone, abiraterone acetate and radium-223 dichloride on bone in the intratibial LNCaP prostate cancer model

Abstract Radium-223 dichloride (Ra-223) is a targeted alpha therapy that binds to newly formed bone matrix in bone metastases and induces double-stranded DNA breaks in cancer cells, osteoblasts, and osteoclasts. Ra-223 showed survival benefit in patients with bone-metastatic castration-resistant prostate cancer in a pivotal phase 3 trial. Recently, in a phase 3 trial of Ra-223 in combination with abiraterone acetate (AA) and prednisone/prednisolone (P), an increased risk of fractures was observed, which could be largely diminished by concurrent administration of bone resorption-inhibiting bisphosphonates or denosumab. The aim of this study was to investigate the effects of vehicle, Ra-223, AA, P, AA/P, and Ra-223/AA/P on bone using a mouse model mimicking osteoblastic prostate cancer bone metastases. Male NOD.scid mice were injected intratibially with LNCaP cells. Following stratification based on serum PSA and total bone volume of the tumor-bearing tibia, mice were treated for 28 days. Serum TRACP 5b and PSA levels were measured at multiple time points. At sacrifice, hind limbs were collected to analyze healthy and tumor-bearing bone structure, quality and formation rate by microCT, 3-point bending assay and dynamic histomorphometry, respectively. Also, Ra-223 incorporation was measured. Treatment with AA/P reduced Ra-223 incorporation to bone. PSA levels were similarly reduced at sacrifice upon Ra-223, AA, AA/P, and Ra-223/AA/P treatment indicating the absence of triple combination-specific additive anti-tumor effects. Bone resorption marker TRACP 5b levels increased transiently during the first two weeks of Ra-223/AA/P treatment. Neither bone structure or bone biomechanical quality in healthy or tumor-bearing tibia were affected by Ra-223/AA/P. Regarding dynamic bone formation, trabecular mineralizing surface (MS) was reduced in all groups containing Ra-223, while periosteal MS was reduced in all groups receiving P. However, only Ra-223/AA/P reduced both trabecular and periosteal MS. In line with the inhibition of dynamic bone formation, Ra-223/AA/P treatment decreased the number of osteoblasts. The AA/P treatment-induced reduction of Ra-223 incorporation may mediate the lack of additive anti-tumor effects in the Ra-223/AA/P group. Regarding bone health, Ra-223/AA/P specifically induced a transient increase in bone resorption and inhibited both periosteal and trabecular new bone formation but did not lead to structural or biomechanical bone changes. This preclinical mechanism may nevertheless contribute to the increased fragility observed in the clinics and may explain the beneficial clinical effects of bone resorption-inhibiting bisphosphonates and denosumab. However, several species- and model-related factors may limit the translational relevance of these findings. Citation Format: Mari I. Suominen, Matias Knuuttila, Birgitta Sjöholm, Esa Alhoniemi, Dominik Mumberg, Jussi M. Halleen, Sanna-Maria Käkönen, Arne Scholz. The effects of prednisone, abiraterone acetate and radium-223 dichloride on bone in the intratibial LNCaP prostate cancer model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6286.

Chondroitin Sulfate Prevents STZ Induced Diabetic Osteoporosis through Decreasing Blood Glucose, AntiOxidative Stress, Anti-Inflammation and OPG/RANKL Expression Regulation.

Chondroitin sulfate (CS) has antioxidative, anti-inflammatory, anti-osteoarthritic and hypoglycemic effects. However, whether it has antidiabetic osteoporosis effects has not been reported. Therefore, in this study, we established a STZ-induced diabetic rat model; CS (500 mg kg−1 d−1) was orally administrated for eight weeks to study its preventive effects on diabetic osteoporosis. The results showed that eight weeks of CS treatment improved the symptoms of diabetes; the CS-treated group has increased body weight, decreased water or food intake, decreased blood glucose, increased bone-mineral density, repaired bone morphology and decreased femoral osteoclasts and tibia adipocytes numbers. After CS treatment, bone histomorphometric parameters returned to normal, the levels of serum inflammatory cytokines (IL-1β, IL-6 and TNF-α) decreased significantly, serum SOD, GPX and CAT activities increased and MDA level increased. In the CS-treated group, the levels of serum ALP, CTX-1, TRACP 5b, osteocalcin and RANKL decreased and the serum RUNX 2 and OPG levels increased. Bone immunohistochemistry results showed that CS can effectively increase the expression of OPG and RUNX2 and reduce the expression of RANKL in diabetic rats. All of these indicate that CS could prevent STZ induced diabetic osteoporosis—mainly through decreasing blood glucose, antioxidative stress, anti-inflammation and regulation of OPG/RANKL expression. CS can therefore effectively prevent bone loss caused by diabetes.

LyP-1-Modified Oncolytic Adenoviruses Targeting Transforming Growth Factor β Inhibit Tumor Growth and Metastases and Augment Immune Checkpoint Inhibitor Therapy in Breast Cancer Mouse Models.

We report here the development of oncolytic adenoviruses (Ads) that have reduced toxicity, enhanced tumor tropism, produce strong antitumor response, and can overcome resistance to immune checkpoint inhibitor therapy in breast cancer. We have shown that LyP-1 receptor (p32) is highly expressed on the surface of breast cancer cells and tumors from cancer patients, and that increased stromal expression of transforming growth factor β-1 (TGFβ-1) is associated with triple-negative breast cancer. Therefore, we constructed oncolytic Ads, AdLyp.sT and mHAdLyp.sT, in which the p32-binding LyP-1 peptide was genetically inserted into the adenoviral fiber protein. Both AdLyp.sT and mHAdLyp.sT express sTGFβRIIFc, a TGFβ decoy that can inhibit TGFβ pathways. mHAdLyp.sT is an Ad5/48 chimeric hexon virus in which hypervariable regions (HVRs 1-7) of Ad5 are replaced with the corresponding Ad48 HVRs. AdLyp.sT and mHAdLyp.sT exhibited better binding, replication, and produced higher sTGFβRIIFc protein levels in breast cancer cell lines compared with Ad.sT or mHAd.sT control viruses without LyP-1 peptide modification. Systemic delivery of mHAdLyp.sT in mice resulted in reduced hepatic/systemic toxicity compared with Ad.sT and AdLyp.sT. Intravenous delivery of AdLyp.sT and mHAdLyp.sT elicited a strong antitumor response in a human MDA-MB-231 bone metastasis model in mice, as indicated by bioluminescence imaging, radiographic tumor burden, serum TRACP 5b and calcium, and body weight analyses. Furthermore, intratumoral delivery of AdLyp.sT in 4T1 model in immunocompetent mice inhibited tumor growth and metastases, and augmented anti-PD-1 and anti-CTLA-4 therapy. Based on these studies, we believe that AdLyp.sT and mHAdLyp.sT can be developed as potential targeted immunotherapy agents for the treatment of breast cancer.

Prevention of Osteoporosis in the Ovariectomized Rat by Oral Administration of a Nutraceutical Combination That Stimulates Nitric Oxide Production.

Osteoporosis represents an imbalance between bone formation and bone resorption. As a result of low estrogen levels, it is markedly prevalent during menopause, thus making such patients susceptible to fractures. Both bone formation and resorption are modulated by nitric oxide (NO). Currently, there are no risk-free pharmaceutical prevention therapies for osteoporosis. COMB-4, a nutraceutical combination of Paullinia cupana, Muira puama, ginger, and L-citrulline, known to activate the NO-cGMP pathway, was reported to accelerate fracture healing in the rat. To determine whether COMB-4 could be effective in preventing menopausal osteoporosis, it was compared to estradiol (E2) in an ovariectomized (OVX) rat osteoporosis model. Nine-month-old female Sprague Dawley rats were divided into SHAM, OVX, OVX+E2, and OVX+COMB-4. After 100 days of treatment, bone mineral density (BMD) and bone mineral content (BMC) were measured by DXA scan. TRAP staining was performed in the femur and lumbar vertebrae. TRACP 5b and osteocalcin levels were assayed in the serum. MC3T3-E1 cells were differentiated into osteoblasts and treated with COMB-4 for one week in order to evaluate calcium deposition by Alizarin staining, cGMP production by ELISA, and upregulation of the nitric oxide synthase (NOS) enzymes by RT-PCR. OVX resulted in a decrease in BMD, BMC, and serum osteocalcin and an increase in serum TRACP 5b. Except for an increase in BMC with COMB-4, both E2 and COMB-4 reverted all bone and serum markers, as well as the number of osteoclasts in the vertebrae, to SHAM levels. Incubation of MC3T3-E1 cells with COMB-4 demonstrated an increase in the three NOS isoforms, cGMP, and calcium deposition. COMB-4 increased BMD in OVX rats by inhibiting bone resorption and increasing calcium deposition presumably via activation of the NO-cGMP pathway. It remains to be determined whether COMB-4 could be a potential nutraceutical therapy for the prevention of premenopausal bone loss.

Icariin Prevents Diabetes-Induced Bone Loss in Rats by Reducing Blood Glucose and Suppressing Bone Turnover.

Diabetic Osteoporosis (DOP) is a common metabolic bone disease, characterized by decreased bone mineral density (BMD) and destruction of bone microstructure. It has been reported that icariin is beneficial for estrogen deficiency-induced osteoporosis, and alcohol-induced osteoporosis; whether icariin has protective effects on diabetes-induced osteoporosis has not been reported. In this study, a rat model of diabetic osteoporosis was established by streptozotocin injection, the bone protective effects and potential mechanism of icariin on diabetes-induced bone loss was observed. Thirty 8-week-old female Sprague Dawley rats were divided into control group (vehicle treatment), T1DM (diabetic) group and T1DM-icariin (ICA) group (diabetic rats treated with icariin), 10 rats in each group. The bone histomorphometry parameters, bone mineral density (BMD), serum bone turnover markers, and bone marrow adipogenesis were analyzed after 8 weeks of icariin administration. The results showed consumption of icariin at a doses of 100 mg kg−1 decreased blood glucose, and increased the BMD of diabetic rats. Icariin effectively decreased serum bone turnover marker levels, including CTX-1, ALP, TRACP 5b, osteocalcin, and PINP. Meanwhile, the bone histomorphometry parameters, the number of osteoclasts per bone perimeter were turned to be normal level, and the icariin treatment suppressed bone marrow adipogenesis. The runt-related transcription factor 2 (RUNX 2), as well as the osteoprotegerin (OPG)/receptor activator of nuclear factor-κ B ligand (RANKL) ratio in serum and bone tissues were increased significantly after icariin treatment in diabetic rats. All of the above indicate that oral administration of icariin can prevent diabetic osteoporosis; the effect is mainly related to its ability to reduce blood glucose, inhibit bone turnover and bone marrow adipogenesis, as well as up-regulate bone RUNX 2, and OPG expression.

Different Types of Low-frequency Electromagnetic Fields Resist Bone Loss Caused by Weightlessness

Objective To compare the effects of 50-Hz 0.6-mT low-frequency pulsed electromagnetic fields(PEMFs) and 50-Hz 1.8-mT sinusoidal alternating electromagnetic fields(SEMFs) in preventing bone loss in tail-suspended rats,with an attempt to improve the prevention and treatment of bone loss caused by weightlessness.Methods Tail-suspension rat models were used to simulate microgravity on the ground. Forty rats were randomly divided into four groups[control group,hindlimb-suspended(HLS) group,HLS+PEMFs group,and HLS+SEMFs group],with 10 rats in each group. In the PEMFs treatment group and SEMFs treatment group,the intervention was 90 min per day. Rats were sacrificed after four weeks. Bone mineral density(BMD) of femur and vertebra was measured by dual-energy X-ray absorptiometry and biomechanical strength by AG-IS biomechanical instrument. Serum osteocalcin(OC),tartrate-resistant acid phosphatase 5b(Tracp 5b),parathyroid hormone(PTH),and cyclic adenosine monophosphate(cAMP) were detected by ELISA. The microstructure of bone tissue was observed by Micro-CT and HE staining.Results The BMD of the femur(P=0.000) and vertebrae(P=0.001) in the HLS group was significantly lower than in the control group;the BMD of the femurs(P=0.001) and vertebrae(P=0.039) in the HLS+PEMFs group was significantly higher than in the HLS group;the BMD of the femurs in the HLS+SEMFs group was significantly higher than in the HLS group(P=0.003),but the BMD of the vertebrae showed no significant difference(P=0.130). There was no significant difference in the BMD of the femur(P=0.818) and vertebrae(P=0.614) between the HLS+PEMFs group and the HLS+SEMFs group. The maximum load(P=0.000,P=0.009) and elastic modulus(P=0.015,P=0.009) of the femurs and vertebrae in the HLS group were significantly lower than those in the control group;the maximum load of the femur(P=0.038) and vertebrae(P=0.087) in the HLS+PEMFs group was significantly higher than that in the HLS group,but the elastic modulus was not significantly different from that in the HLS group(P=0.324,P=0.091). The maximum load(P=0.190,P=0.222) and elastic modulus(P=0.512,P=0.437) of femurs and vertebrae in the HLS+SEMFs group were not significantly different from those in the HLS group. There were no significant differences in the maximum load and elastic modulus of femurs(P=0.585,P=0.948) and vertebrae(P=0.668,P=0.349) between the HLS+PEMFs group and the HLS+SEMFs group. The serum OC level in the HLS group was significantly lower than that in the control group(P=0.000),and the OC level in HLS+PEMFs group(P=0.000) and HLS+SEMFs group(P=0.006) were significantly higher than that in the HLS group. The serum Tracp 5b concentration in the HLS group was significantly higher than that in the control group(P=0.011). There was no significant difference between the HLS+PEMFs group(P=0.459) and the HLS+SEMFs group(P=0.469) compared with the control group.Serum Tracp 5b concentrations in the HLS+PEMFs group(P=0.056) and the HLS+SEMFs group(P=0.054) were not significantly different from those in the HLS group. The PTH(P=0.000) and cAMP concentrations(P=0.000) in the HLS group were significantly lower than those in the control group. The PTH(P=0.000,P=0.000) and cAMP concentrations(P=0.000,P=0.000) in the HLS+PEMFs group and the HLS+SEMFs group were significantly higher than in the HLS group. The femoral cancellous bone of the HLS group was very sparse and small compared with the control group. The density and volume of the cancellous bone were similar among the control group,HLS+PEMFs group,and HLS+SEMFs group. Compared with the control group,the HLS group had lower BMD(P=0.000),bone volume (BV)/tissue volume(TV)(P=0.000),number of trabecular bone (Tb.N)(P=0.000),and trabecular thickness(Tb.Th)(P=0.000) and higher trabecular bone dispersion(Tb.Sp)(P=0.000) and bone surface area(BS)/BV(P=0.000). Compared with the HLS group,the HLS+PEMFs group and the HLS+SEMFs group had significantly lower Tb.Sp(P=0.000,P=0.000) and BS/BV(P=0.000,P=0.000) and significantly increased BMD(P=0.000,P=0.000),BV/TV(P=0.001,P=0.004),Tb.Th(P=0.000,P=0.001),and Tb.N(P=0.000,P=0.001). The trabecular thickness significantly differed between the HLS+PEMFs group and the HLS+SEMFs group(P=0.024). The HLS group(P=0.000),HLS+PEMFs group(P=0.000),and HLS+SEMFs group(P=0.000) had the significantly lower osteoblast density on the trabecular bone surface than the control group;however,it was significantly higher in the HLS+SEMFs group(P=0.000) and the HLS+PEMFs group(P=0.000) than in the HLS group. The HLS group had significantly lower density of osteoblasts in the endothelium than the control group(P=0.000);however,the density of osteoblasts was significantly higher in HLS+PEMFs group(P=0.000) and HLS+SEMFs group(P=0.000) than HLS group and was significantly higher in HLS+PEMFs group than in HLS+SEMFs group(P=0.041). Compared with the control group,a large number of fatty cavities were produced in the bone marrow cavity in the HLS group,but the fat globules remarkably decreased in the treatment groups,showing no significant difference from the control group. The number of adipose cells per mm 2 bone marrow in the HLS group was 4 times that of the control group(P=0.000);it was significantly smaller in the HLS+PEMFs group(P=0.000) and HLS+SEMFs group(P=0.000) than in the HLS group,whereas the difference between the HLS+PEMFs group and the HLS+SEMFs group was not statistically significant(P=0.086). Conclusions 50-Hz 0.6-mT PEMFs and 50-Hz 1.8-mT SEMFs can effectively increase bone mineral density and biomechanical values in tail-suspended rats,increase the concentration of bone formation markers in rat blood,activate the cAMP pathway by affecting PTH levels,and thus further increase the content of osteoblasts to prevent the deterioration of bone micro-structure. In particular,PEMFs can prevent the reduction of bone mineral density and maximum load value by about 50% and increase the bone mass of tail-suspended rats by promoting bone formation.

Isomeric flavonoid aglycones derived from Epimedii Folium exerted different intensities in anti-osteoporosis through OPG/RANKL protein targets

Two Epimedium-derived isomeric flavonoids, CIT and IT, had the therapeutic effect in osteopenic rats. However, it is difficult to expound their activity differences in anti-osteoporosis. This paper contrasted their anti-osteoporosis activity from the perspective of their affinity to OPG/RANKL protein targets. Molecular docking indicated that both of CIT and IT could interact with the hydrophobic pockets of OPG/RANKL, while CIT was easier and more stable to combine with RANKL. On the contrary, compared with CIT, IT was more inclined to combine with OPG and stay away from combining with RANKL. Subsequently, whether the interaction between isomeric flavonoids and OPG/RANKL targets promoted or suppressed bone resorption was undefined and which was validated by zebrafish embryo and ovariectomized rats in this paper. Compared with IT, the staining area and cumulative optical density of zebrafish skeleton were significantly increased after the treatment of CIT (0.1 μM, p < 0.05). Furthermore, CIT mainly reflected a more significant role in upregulating OPG (p < 0.05), downregulating RANKL (p < 0.05), reducing serum AKP and TRACP level (p < 0.05), enhancing bone biomechanical properties (p < 0.05), increasing bone mineral density (p < 0.05) and improving trabecular bone microarchitecture (p < 0.05) in osteoporotic rats. In conclusion, the combination of isomeric flavonoids (CIT/IT) and OPG/RANKL targets attenuated the excitation effects of OPG or RANKL on RANKL. Because CIT was more firmly combined with RANKL than IT, CIT had stronger anti-osteoporosis effect by inhibiting bone resorption.

The Src family kinase inhibitor dasatinib delays pain-related behaviour and conserves bone in a rat model of cancer-induced bone pain

Pain is a severe and debilitating complication of metastatic bone cancer. Current analgesics do not provide sufficient pain relief for all patients, creating a great need for new treatment options. The Src kinase, a non-receptor protein tyrosine kinase, is implicated in processes involved in cancer-induced bone pain, including cancer growth, osteoclastic bone degradation and nociceptive signalling. Here we investigate the role of dasatinib, an oral Src kinase family and Bcr-Abl tyrosine kinase inhibitor, in an animal model of cancer-induced bone pain. Daily administration of dasatinib (15 mg/kg, p.o.) from day 7 after inoculation of MRMT-1 mammary carcinoma cells significantly attenuated movement-evoked and non-evoked pain behaviour in cancer-bearing rats. Radiographic - and microcomputed tomographic analyses showed significantly higher relative bone density and considerably preserved bone micro-architecture in the dasatinib treated groups, suggesting a bone-preserving effect. This was supported by a significant reduction of serum TRACP 5b levels in cancer-bearing rats treated with 15 mg/kg dasatinib. Furthermore, immunoblotting of lumbar spinal segments showed an increased activation of Src but not the NMDA receptor subunit 2B. These findings support a role of dasatinib as a disease modifying drug in pain pathologies characterized by increased osteoclast activity, such as bone metastases.

Osteoprotective Effect of Radix Scutellariae in Female Hindlimb-Suspended Sprague-Dawley Rats and the Osteogenic Differentiation Effect of Its Major Constituent.

A number of medicinal herbs have demonstrated therapeutic effects for the prevention and treatment of disuse-induced osteoporosis. As a common ingredient in proprietary traditional Chinese medicines, the anti-osteoporosis effects of Radix Scutellariae extract (RSE, 50 mg/kg/day) were evaluated in a hindlimb suspended rat model. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry, and the micro-architecture observed by MicroCT assay with bone biomechanical properties evaluated by a three-point bending test. To elucidate potential mechanisms, the osteogenic differentiation effect of baicalin as the most abundant ingredient in RSE was investigated in rat bone marrow derived mesenchymal stem cells (rBMSC). After drug administration for 42 days, tibia-BMD was significantly increased to 0.176 ± 0.007 and 0.183 ± 0.011 g/cm2 and f-BMD was enhanced to 0.200 ± 0.017 and 0.207 ± 0.021 g/cm2 for RSE and ALE treatment, respectively, whereas tibia-BMD and femur-BMD of the HLS group were 0.157 ± 0.009 and 0.176 ± 0.008 g/cm2. Deterioration of bone trabecula microstructure was improved by RSE and ALE with increased morphological parameters such as bone volume fraction, trabecular thickness, and trabecular number, as well as connectivity density compared to the HLS group (p < 0.01). A three-point bending test suggested that bone mechanical strength was also enhanced by RSE and ALE treatments with increased maximum stress, young’s modulus, maximum load, and stiffness compared to those of the HLS group (p < 0.05). Besides, serum TRACP levels were significantly suppressed by RSE and ALE treatments. Furthermore, in vitro studies demonstrated that baicalin significantly increased ALP activities and the formation of mineralized nodules in rBMSC. Conclusively, supplementation of RSE could significantly prevent weightlessness induced osteoporosis, which might attribute to the osteogenic differentiation enhancement effect of baicalin.

Serum N-terminal telopeptide of type I collagen as an early marker of fracture nonunion in rabbits.

The aim of the present study was to establish an experimental animal model of fracture nonunion, and to investigate the changes in serum biomarker concentrations in fracture nonunion. A total of 20 purebred New Zealand rabbits were divided into two group: A bone defect group and a bone fracture group. In the bone defect group, a 15-mm section of bone (including the periosteum) was removed from the mid-radius, and the medullary cavities were closed with bone wax. In the bone fracture group, the mid-radius was fractured. X-rays were taken and blood samples were collected preoperatively and at 2, 3, 4, 5, 6, 7, 8, 10 and 12 weeks after the surgical procedure. The serum concentrations of osteocalcin (OC) and bone-specific alkaline phosphatase (BSAP) served as markers of bone formation, and those of C-terminal telopeptide of type I collagen (CTX), N-terminal telopeptide of type I collagen (NTX) and tartrate-resistant acid phosphatase 5b (TRACP 5b) served as markers of bone resorption. The concentration levels of the markers were measured using a biotin double-antibody sandwich enzyme-linked immunosorbent assay. In the bone defect group, bone callus was observed on X-ray at 2 weeks in three rabbits and the bone calluses stabilized at 5 weeks; however, none of the bones had healed at 8 weeks. In the bone fracture group, the fracture line was distorted at 2 weeks and bone calluses formed at 6–8 weeks. In the bone defect group, the serum BSAP and TRACP 5b concentrations increased following the surgical procedure, peaked at 4 weeks, began to decrease at 5 weeks and stabilized after 6 weeks. The serum OC concentrations did not change significantly following the surgical procedure. The serum CTX concentrations fluctuated during the first 4 weeks, peaked at 5 weeks, then decreased and stabilized after 6 weeks. The serum NTX concentrations fluctuated during the first 4 weeks, were significantly lower at 5 weeks compared with the other time points and stabilized after 6 weeks. These results suggested that a bone nonunion model can be established in New Zealand rabbits by resecting a 15-mm section of bone from the mid-radius prior to bone wax blocking. Measurement of the serum BSAP, CTX, NTX, and TRACP 5b concentrations may be useful for the early detection of bone nonunion. The serum NTX concentrations changed significantly in rabbits with bone nonunion. Further studies are required in order to determine the feasibility of using serum NTX concentrations for the early diagnosis of bone nonunion.

Tartrate-Resistant Acid Phosphatase 5b in Young Patients With Sickle Cell Disease and Trait Siblings: Relation to Vasculopathy and Bone Mineral Density.

Bone involvement is a frequent cause of acute morbidity in sickle cell disease (SCD). Tartrate-resistant acid phosphatase 5b (TRACP 5b), a bone resorption marker, is produced specifically by activated osteoclasts. We assessed bone mineral density (BMD) in 30 young patients with SCD and 17 asymptomatic patients with sickle cell trait (SCT) compared with 32 healthy controls and determined TRACP 5b levels in relation to vascular complications. Serum ferritin, alkaline phosphatase (ALP), and TRACP 5b were measured. Echocardiography was performed with assessment of BMD using dual energy X-ray absorptiometry (DXA). The BMD was decreased in patients with SCD compared with SCT and controls (P = .005), with no significant difference between the latter 2 groups. Patients with SCD had higher incidence of bone complications than SCT group and controls (P = .03). The SCD group with abnormal DXA scan had higher ferritin and ALP than normal BMD. Serum TRACP 5b was significantly higher in patients with SCD than SCT and controls (P = .003). The TRACP 5b levels were associated with severe vaso-occlusive crisis (P = .022). Patients treated with hydroxyurea and those on chelation therapy had lower TRACP 5b levels than untreated patients. The TRACP 5b level was positively correlated with lactate dehydrogenase, while there was no relation with ferritin, ALP, or BMD. We suggest that bone complications frequently occur in SCD as reflected by low BMD and high ALP and TRACP 5b. Hemolysis and iron overload may be involved in the occurrence of these complications. The lack of correlation between abnormal DXA scan and high TRACP 5b suggests that bone disease in SCD is multifactorial.

Effects of icariin total flavonoids capsule on bone mineral density and bone histomorphometry in growing rats

Objective: To investigate the effect of icariin total flavonoids capsules (ITFC) on bone mineral density (BMD) and bone histomorphometry in growing rats and its anti-osteoporosis mechanism. Methods: Thirty female SD rats were randomly divided into 3 groups:normal control group, ITFC-1 group and ITFC-2 group. Rats in ITFC-1 group and ITFC-2 group were fed with 50 mg·kg-1·d-1 or 100 mg·kg-1·d-1 ITFC, respectively, and those in normal control group were fed with equal volume of distilled water. The whole body BMD was measured after 4, 8 and 12 weeks, and BMDs of the right femur and lumbar vertebrae were measured after 12 weeks. The serum levels of tartaric acid phosphatase 5b (TRACP 5b) and bone alkaline phosphatase (BALP) were measured by ELISA. Bone morphometry was performed on the right tibia. Results: There were no significant differences in the body weight increase between normal control group and two ITFC groups (all P>0.05). There were also no significant differences in whole body BMDs after 4 and 8 weeks between normal control group and ITFC groups (all P>0.05). After 12 weeks, the whole body BMD, BMD of bone in vitro, serum BALP level and trabecular area in ITFC-1 group and ITFC-2 group were significantly higher, trabecular separation was significantly lower than that in normal control group (all P<0.05); and the trabecular width and the number in ITFC-2 group were also significantly higher, and serum TRACP 5b level was significantly lower than that in normal control group (all P<0.05). The BMD of bone in vitro, serum BALP level, trabecular number and area in ITFC-2 group were significantly higher, and serum TRACP 5b level was significantly lower than that in ITFC-1 group (all P<0.05). Conclusion: ITFC can prevent osteoporosis by increasing bone density and bone formation, decreasing bone resorption and improving microstructure of bone.

Potential effects of valproate and oxcarbazepine on growth velocity and bone metabolism in epileptic children- a medical center experience.

BackgroundChildren with longstanding use of antiepileptic drugs (AEDs) are susceptible to developing low bone mineral density and an increased fracture risk. However, the literature regarding the effects of AEDs on growth in epileptic children is limited. The aim of this study was to investigate the potential effects of valproate (VPA) and/or oxcarbazepine (OXC) therapy on growth velocity and bone metabolism.MethodsSeventy-three ambulatory children (40 boys and 33 girls) with epilepsy, aged between 1 and 18 years (mean age 9.8 ± 4.1 years), were evaluated for growth velocity before and for 1 year after VPA and/or OXC treatment. The bone resorption marker serum tartrate-resistant acid phosphatase 5b (TRAcP5b) and the bone formation marker serum bone-specific alkaline phosphatase (BAP) were measured post-AEDs therapy for 1 year.ResultsThe difference in growth velocity (ΔHt) and body weight change (ΔWt) between pre- and post-AEDs treatment were -1.0 ± 2.8 cm/year (P < 0.05) and 0.1 ± 3.9 kg/year (P = 0.84), respectively. The study population had serum TRAcP5b-SDS of -1.6 ± 1.2 and BAP-SDS of 1.7 ± 3.7 compared with sex- and age-matched healthy children. Significant correlation between serum TRAcP 5b and BAP activities was noted (r = 0.60, p < 0.001). There was a positive correlation between growth velocity and serum TRAcP 5b activity after AED treatment (r = 0.42, p < 0.01). No correlation was found between ΔHt, ΔWt, serum TRAcP 5b, BAP activity and types of AEDs.ConclusionGrowth velocity was significantly decreased in epileptic children after 1 year of VPA and/or OXC treatment. The effect of VPA and/or OXC therapy on dysregulation of bone metabolism might play a crucial role in physical growth.Electronic supplementary materialThe online version of this article (doi:10.1186/s12887-016-0597-7) contains supplementary material, which is available to authorized users.

High Uric Acid (UA) Negatively Affects Serum Tartrate-Resistant Acid Phosphatase 5b (TRACP 5b) Immunoassay.

BackgroundBone metastases often occur in the majority of patients with advanced cancer, such as prostate cancer, lung cancer and breast cancer. Serum tartrate-resistant acid phosphatase 5b (TRACP 5b), a novel bone resorption marker, has been used gradually in the clinics as a specific and sensitive marker of bone resorption for the early diagnosis of cancer patients with bone metastasis. Here, we reported that high concentrations of uric acid (UA) lead to decrease of TRACP 5b levels and determined whether TRACP 5b level was associated with UA in interference experiment.MethodsA total of 77 patients with high concentrations of UA and 77 healthy subjects were tested to evaluate the differences in their TRACP 5b levels. Serial dilutions of UA were respectively spiked with a known concentration of TRACP 5b standard sample, then Serum TRACP 5b was detected by using bone TRAP® Assay. A correction equation was set to eliminate UA-derived TRACP 5b false-decrease. The effect of this correction was evaluated in high-UA individuals.ResultsThe average TRACP level of the high-UA individuals (1.47± 0.62 U/L) was significantly lower than that of the healthy subjects (2.62 ± 0.63 U/L) (t-test, p<0.0001). The UA correction equation derived: ΔTRACP 5b = -1.9751lgΔUA + 3.7365 with an R2 = 0.98899. Application of the UA correction equation resulted in a statistically non-significant difference in TRACP 5b values between the healthy subjects and high-UA individuals (p = 0.24).ConclusionsHigh UA concentrations can falsely decrease TRACP 5b levels due to a method-related systematic error. To avoid misdiagnoses or inappropriate therapeutic decisions, increased attention should be paid to UA interference, when TRACP 5b is used for early diagnosis of cancer patients with bone metastasis, evaluation of the aggressiveness of osteosarcoma or prediction of survival in prostate cancer and breast cancer with bone metastases.