Expression Of Bone Formation Markers Research Articles

Microbiome dysbiosis by antibiotics protects cartilage degradation in OAOP mice.

The role of this study was to evaluate the impact of gut microbiota depletion on the progression of osteoarthritis (OA) and osteoporosis (OP). We conducted an experimental mouse model of OA and OP over an 8-week period. The model involved destabilization of the medial meniscus and bilateral ovariectomy (OVX). To deplete the gut microbiota, we administered a course of antibiotics for 8 weeks. The severity of OA was assessed through micro-CT scanning, X-rays, and immunohistochemical staining. Microbiome analysis was performed using PCR of 16S DNA on fecal samples, and the levels of serum lipopolysaccharide, interleukin 6, tumor necrosis factor-α (TNF-α), osteocalcin, and estrogen were measured using enzyme-linked immunosorbent assay. We found that in comparison to the OVX+OA group, the OVX+OA+ABT group exhibited increased bone mineral density (P < 0.0001), bone volume fraction (P = 0.0051), and trabecular number (P = 0.0023) in the metaphyseal bone. Additionally, cartilage injury and levels of matrix metalloproteinase 13 were reduced in the OVX+OA+ABT group compared to the OVX+OA group. Moreover, the OVX+OA+ABT group demonstrated decreased relative abundance of Bacteroidetes, serum lipopolysaccharide (P = 0.0005), TNF-α (P < 0.0001), CTX-1 (P = 0.0002), and increased expression of bone formation markers. These findings were further supported by correlation network analyses. Depletion of gut microbiota was shown to protect against bone loss and cartilage degradation by modulating the composition of the gut microbiota in osteoporosis and osteoarthritis.

Combination effect of Chinese kidney-tonifying granules and platelet-rich plasma gels on enhancing bone healing in rat models with femur defects

BackgroundThe traditional Chinese kidney-tonifying granules, known as Bushen Zhongyao Keli (BSZYKL), have been found to stimulate calcium salt deposition, enhance bone formation, and foster bone growth within the bone matrix at sites of bone defects. On the other hand, platelet-rich plasma (PRP) is enriched with various growth factors capable of facilitating the repair of bone defects and enhancing bone strength following fractures. This study is dedicated to investigating the combined efficacy of BSZYKL and PRP gel (PRP-G) in the treatment of bone defects.MethodsWe established a femur defect model in male Sprague-Dawley (SD) rats and filled the defect areas with autologous coccygeal bone and PRP-G. For 8 consecutive weeks, those rats were given with intragastric administration of BSZYKL. Biomechanical characteristics of the femur were assessed 28 days after intramuscular administration. On day 56, bone formation was examined using X-ray, micro-CT, and transmission electron microscopy. Additionally, we analyzed the expression of bone formation markers, Runx2 and Osterix, in femur tissues through qPCR, Western blotting, and immunohistochemistry.ResultsRats receiving the combined treatment of BSZYKL and PRP-G exhibited drastically enhanced femoral peak torsion, failure angle, energy absorption capacity, and torsional stiffness as compared to control group. This combination therapy also led to marked improvements in bone volume, mass, and microarchitecture, accompanied by elevated expressions of Runx2 and Osterix when compared to control group. Notably, the synergistic effects of BSZYKL and PRP-G in treating bone defects surpassed the effects of either treatment alone.ConclusionsThese findings revealed the potential of BSZYKL in combination with PRP-G in improving bone defects.

Comparison of two non-union models with damaged periosteum in mice: Segmental defect and pin-clip fixation versus transverse fracture and K-wire stabilization.

Despite growing knowledge about the mechanisms of fracture healing, non-union formation still represents a major complication in trauma and orthopedic surgery. Non-union models in mice gain increasing interest, because they allow investigating the molecular and cellular mechanisms of failed fracture healing. These models often use segmental defects to achieve non-union formation. Alternatively, failed fracture healing can be induced by transverse fractures with additional periosteal injury. The present study systematically compared the reliability of these two approaches to serve as non-union model. A 0.6mm K-wire was inserted into the femora of CD-1 mice in a retrograde fashion and a closed transverse femoral fracture was created. Subsequently, the fracture site was exposed and the periosteum was cauterized. This approach was compared with a well-established non-union model involving the pin-clip fixation of a 1.8mm segmental defect. The callus tissue was analyzed by means of radiography, biomechanics, histology and Western blotting. At 10weeks after surgery 10 out of 12 femora (83.3%) of the K-wire group showed a non-union formation. The pin-clip model resulted in 100% non-union formation. The K-wire group showed increased bone formation, osteoclast activity and bending stiffness when compared to the group with pin-clip fixation. This was associated with a higher expression of bone formation markers. However, the number of CD31-positive microvessels was reduced in the K-wire group, indicating an impaired angiogenic capacity after periosteal cauterization. These findings suggest that the pin-clip model is more reliable for the study of non-union formation in mice. The K-wire model including periosteal injury by cauterization however, may be particularly applied in preclinical studies which explore the effects of damaged periosteum and reduced angiogenic capacity to trauma-induced fractures.

Establishment of a Reliable Model to Study the Failure of Fracture Healing in Aged Mice.

The failure of fracture healing represents a substantial clinical problem. Moreover, aged patients demonstrate an elevated risk for failed bone healing. However, murine models to study the failure of fracture healing are established only in young adult animals. Therefore, the aim of this study was to develop a reliable model to study failed fracture healing in aged mice. After creation of a 1.8-mm segmental defect and periosteal resection, femora of aged mice (18-20 months) and young adult control mice (3-4 months) were stabilized by pin-clip fixation. Segmental defects were analyzed by means of biomechanics, x-ray, and micro-computed tomography, as well as histomorphometric, immunohistochemical, and Western blot analysis. After 10 weeks, all animals showed a complete lack of osseous bridging, resulting in fracture healing failure. Segmental defects in aged mice revealed a reduced bone formation and vascularization when compared to young adult mice. This was associated with a decreased expression of bone formation markers. In addition, we detected a reduced number of tartrate-resistant acid phosphatase-positive osteoclasts and an elevated osteoprotegerin/receptor activator of NF-ĸB ligand ratio in aged animals, indicating a reduced osteoclast activity. Moreover, aged animals showed also an enhanced inflammatory response, characterized by an increased infiltration of macrophages within the callus tissue. Taken together, we herein report for the first time a reliable model to study fracture healing failure in aged mice. In the future, the use of this model enables us to study novel therapeutic strategies and molecular mechanics of failed fracture healing during aging.

Osthole ameliorates simulated microgravity-induced bone loss through down-regulation of miR-34c-5p

Bone loss severely threatens the heath of astronauts in long-term spaceflight and the effects of the developed countermeasures are limited. Emerging studies have revealed the positive role of osthole (OST) in induction of osteogenic differentiation and bone formation. In this study, the effects of OST on bone metabolism were investigated in osteoblasts and rats treated with random positioning machine and hindlimb unloading, respectively. The results showed that OST treatment promoted the cellular proliferation and elevated the expression levels of RUNX2 and BMP2 in osteoblasts under simulated microgravity. In addition, hindlimb unloading rats administered with OST (5 mg kg/d, i.g.) exhibited improved bone mass, bone strength, expression of bone formation markers (BALP and OCN), and decreased expression of bone resorption markers (CTX-1 and TRACP-5b) in serum compared with rats treated with placebo (0.9% saline). Notably, we found that miR-34c-5p expression level was significantly up-regulated when exposed to simulated microgravity both in vitro and in vivo, and suppression of miR-34c-5p by antagomiR in rats obviously rescued the damages of BMD and trabecular bone microstructures caused by simulated microgravity. Importantly, OST treatment reversed miR-34c-5p expression level in hindlimb unloading rats, and ectopic expression of miR-34c-5p by transfection of agomiR in osteoblasts treated with OST decreased the expression levels of bone formation genes COL1α1, RUNX2, and BMP2. Overall, these data suggest an undisclosed role for OST in regulating microgravity-induced bone loss via control of miR-34c-5p expression, which may help to countermeasure development to overcome bone loss in spaceflight.

Semaphorin3B Promotes Proliferation and Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in a High-Glucose Microenvironment.

Bone marrow mesenchymal stem cells (BMSCs) play an essential role in osteogenesis and bone metabolism and have already been recognized as one of the most popular seed cells for bone tissue engineering for bone diseases. However, high-glucose (HG) conditions in type 2 diabetes mellitus (T2DM) exert deleterious effects on BMSC proliferation and osteogenic differentiation. Semaphorin 3B (Sema3B) increases osteoblast differentiation in bone metabolism. Here, we determined the role of Sema3B in the proliferation and osteogenic differentiation of BMSCs in the HG microenvironment. The HG microenvironment decreased Sema3B expression in BMSCs. Moreover, HG inhibited BMSC proliferation. Furthermore, HG inhibited osteogenic differentiation in BMSCs by decreasing the expression of bone formation markers, alkaline phosphatase (ALP) activity, and mineralization. However, the administration of recombinant Sema3B reversed all of these effects. Moreover, our study found that Sema3B could activate the Akt pathway in BMSCs. Sema3B rescues defects in BMSC proliferation and osteogenic differentiation in the HG microenvironment by activating the Akt pathway. These effects were significantly reduced by treatment with an Akt inhibitor. Together, these findings demonstrate that Sema3B promotes the proliferation and osteogenic differentiation of BMSCs via the Akt pathway under HG conditions. Our study provides new insights into the potential ability of Sema3B to ameliorate BMSC proliferation and osteogenic differentiation in an HG microenvironment.

Telmisartan Prevents Alveolar Bone Loss by Decreasing the Expression of Osteoclasts Markers in Hypertensive Rats With Periodontal Disease.

Periodontal disease (PD) is a prevalent inflammatory disease with the most severe consequence being the loss of the alveolar bone and teeth. We therefore aimed to evaluate the effects of telmisartan (TELM), an angiotensin II type 1 receptor (Agtr1) antagonist, on the PD-induced alveolar bone loss, in Wistar (W) and Spontaneous Hypertensive Rats (SHRs). PD was induced by ligating the lower first molars with silk, and 10 mg/kg TELM was concomitantly administered for 15 days. The hemimandibles were subjected to microtomography, ELISA was used for detecting tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), CXCL3, and CCL2, while qRT-PCR was used for analyzing expression of components of renin-angiotensin system (RAS) (Agt, Ace, Agt1r, Agt2r, Ace2, and Masr), and bone markers (Runx2, Osx, Catnb, Alp, Col1a1, Opn, Ocn, Bsp, Bmp2, Trap, Rank, Rankl, CtsK, Mmp-2, Mmp-9, and osteoclast-associated receptor (Oscar)). The SHR + PD group showed greater alveolar bone loss than the W + PD group, what was significantly inhibited by treatment with TELM, especially in the SHR group. Additionally, TELM reduced the production of TNF-α, IL-1β, and CXCL3 in the SHR group. The expression of Agt increased in the groups with PD, while Agtr2 reduced, and TELM reduced the expression of Agtr1 and increased the expression of Agtr2, in W and SHRs. PD did not induce major changes in the expression of bone formation markers, except for the expression of Alp, which decreased in the PD groups. The bone resorption markers expression, Mmp9, Ctsk, and Vtn, was higher in the SHR + PD group, compared to the respective control and W + PD group. However, TELM attenuated these changes and increased the expression of Runx2 and Alp. Our study suggested that TELM has a protective effect on the progression of PD, especially in hypertensive animals, as evaluated by the resorption of the lower alveolar bone. This can be partly explained by the modulation in the expression of Angiotensin II receptors (AT1R and AT2R), reduced production of inflammatory mediators, the reduced expression of resorption markers, and the increased expression of the bone formation markers.

3D Bioprinted Osteogenic Tissue Models for In Vitro Drug Screening.

Metabolic bone disease affects hundreds of millions of people worldwide, and as a result, in vitro models of bone tissue have become essential tools to help analyze bone pathogenesis, develop drug screening, and test potential therapeutic strategies. Drugs that either promote or impair bone formation are in high demand for the treatment of metabolic bone diseases. These drugs work by targeting numerous signaling pathways responsible for regulating osteogenesis such as Hedgehog, Wnt/β-catenin, and PI3K-AKT. In this study, differentiated bone marrow-derived mesenchymal stem cell (BM-MSC) scaffold-free 3D bioprinted constructs and 2D monolayer cultures were utilized to screen four drugs predicted to either promote (Icariin and Purmorphamine) or impair osteogenesis (PD98059 and U0126). Osteogenic differentiation capacity was analyzed over a four week culture period by evaluating mineralization, alkaline phosphatase (ALP) activity, and osteogenesis related gene expression. Responses to drug treatment were observed in both 3D differentiated constructs and 2D monolayer cultures. After four weeks in culture, 3D differentiated constructs and 2D monolayer cultures treated with Icariin or Purmorphamine showed increased mineralization, ALP activity, and the gene expression of bone formation markers (BGLAP, SSP1, and COL1A1), signaling molecules (MAPK1, WNT1, and AKT1), and transcription factors (RUNX2 and GLI1) that regulate osteogenic differentiation relative to untreated. 3D differentiated constructs and 2D monolayer cultures treated with PD98059 or U0126 showed decreased mineralization, ALP activity, and the expression of the aforementioned genes BGLAP, SPP1, COL1A1, MAPK1, AKT1, RUNX2, and GLI1 relative to untreated. Differences in ALP activity and osteogenesis related gene expression relative to untreated cells cultured in a 2D monolayer were greater in 3D constructs compared to 2D monolayer cultures. These findings suggest that our bioprinted bone model system offers a more sensitive, biologically relevant drug screening platform than traditional 2D monolayer in vitro testing platforms.

Preventive Effects of Chrysanthemum coronarium L. Extract on Bone Metabolism In Vitro and In Vivo.

Osteoporosis is characterized by decreased bone mass and bone microarchitectural failure, leading to an enhanced risk of bone fractures. Chrysanthemum coronarium L. (CC) is a natural plant with powerful antioxidant activity. This study investigated the antiosteoporotic effects of CC extracts in in vitro cell cultures and in vivo bone loss animal models. CC stimulated osteoblast differentiation and mineralized bone formation by osteoblasts by increasing the expression of bone formation markers (alkaline phosphatase, osteoprotegerin, and osteoprotegerin/receptor activator nuclear factor-κB ligand ratio) in the murine preosteoblastic cell line MC3T3-E1. Additionally, CC was found to inhibit osteoclast differentiation by downregulating bone resorption markers (tartrate-resistant acid phosphatase, cathepsin K, dendritic cell-specific transmembrane protein, and calcitonin receptor) in the murine macrophage-like cell line RAW264.7. CC prevented ovariectomy-induced bone loss, preserved trabecular microarchitecture, and improved serum bone turnover markers in an osteoporotic mouse model. These findings suggest that CC extract may be considered as a natural therapeutic or preventive agent for osteoporotic bone loss.

Abstract P031: Effect of Exercise on Function and Differentiation of Adipose Tissue Derived Mesenchymal Stromal Cells (MSCs)

Approximately 422 million people have diabetes worldwide (2014) and it is predicted that diabetes will rise by nearly 54% by 2030. Aerobic exercise is known to show positive effect on health of diabetic and pre diabetic patients. The effect of exercise has been studied extensively using plasma biochemistry but cellular data is scares. Previously, we have shown endothelial progenitor cells (EPCs) can act as a strong cellular biomarker of endothelial function following aerobic exercise as an intervention. In this study, we are examining the effect of aerobic exercise on adipocyte derived MSCs to study stromal cell differentiation and as a cellular surrogate of fat metabolism. Methods: Overweight and obese subjects (n=5) were enrolled in a 12 week exercise intervention study. The biweekly exercise sessions were supervised by a trained exercise physiologist and consisted of a 1 hour sessions that included warm-up and cool-down and 30 min of combined aerobic and resistance training at an exercise intensity of 50-80% of heart rate reserve. Physical and biochemical parameters were tested pre and post exercise. Subcutaneous abdominal fat biopsies were obtained and fat derived stromal cells were cultured in vitro for 2-3 weeks. MSCs were analyzed for mRNA gene expression (qRT-PCR) and cellular oxygen consumption rate (OCR), pre and post 12 week exercise. Results: With exercise, A1C reduced significantly. An increase of METs was also noticed post exercise. Both basal and maximal respiration increased significantly post exercise when compared with commercially obtained MSCs. Simultaneously, mitochondrial genes COX4 and ATP5B (p= 0.01, 1.4 fold, 0.02, 1.5 fold respectively), Glucose transporter, GLUT1 (p=0.04, 1.8 fold), antioxidants GPX3 and CAT (p= 0.01, 3.2 fold and p=0.04, 1.5 fold respectively) upregulated whereas pro-inflammatory cyclo-oxygenase-2 (p=0.04, 2.5 fold) gene reduced significantly, post exercise. Regarding differentiation potential of multipotent MSCs, post exercise, we noted enhanced expression of bone markers such Alkaline Phosphatase (p= 0.03, 2.7 fold) BGLAP and RUNX2 (1.3 and 1.2 fold) and also for collagen marker COL2 (2.4 fold) expression. For adipogenic differentiation potential PPARG mRNA expression was reduced. Interestingly, serum value of osteocalcin increased significantly from 15.0 (5.5) to 16.3(6.1) ng/ml (9% increase, p=0.03) with 1% increase in bone alkaline phosphatase level, post exercise. Conclusion: We conclude that exercise augments cellular glucose transporters (GLUT1), anti-oxidants and reduce MSC inflammation and up-regulates mitochondrial function and gene expression profile of MSCs. Increased serum value of osteocalcin complement increased gene expression of bone formation markers indicating a cross talk between fat derived MSCs and bone formation, post exercise.

Effects of blood flow restriction exercises on bone metabolism: a systematic review.

This study analysed the effect of low-intensity (LI) exercises with blood flow restriction (BFR) on bone metabolism compared with high-intensity (HI) exercises without BFR. The following databases were searched using the keywords therapeutic occlusion training OR BFR training OR vascular occlusion training OR KAATSU training OR ischaemia training AND osteogenesis OR bone biomarkers OR bone metabolic marker OR bone mass OR bone turnover OR osteoporosis OR osteopenia: PubMed, Web of Science, SPORTDiscus, CINAHL, Science Direct, Cochrane and Google Scholar. Two researchers, independently and blindly, selected the studies based on established inclusion and exclusion criteria. Electronic and manual searches located 170 articles published in English; after screening, only four studies showed that BFR training increases the expression of bone formation markers (e.g. bone-specific alkaline phosphatase) and decreases bone resorption markers (e.g. the amino-terminal telopeptides of type I collagen) after both aerobic and anaerobic exercise across several populations. The results of this study show that few studies have confirmed the positive effect of exercise with BFR on bone metabolism, formation and resorption. Furthermore, no methodological standardization of the samples, exercise type, intervention frequency or duration was observed.

Titanium implants coated with UV‐irradiated vitamin D precursor and vitamin E: in vivo performance and coating stability

This study aimed at evaluating the biological response of titanium implants coated with UV-irradiated 7-dehydrocholesterol (7-DHC) and vitamin E (VitE) invivo and analyzing the effects of aging on their stability and bioactivity invitro. Titanium surfaces were coated with 7-DHC and VitE, UV-irradiated and incubated for 48h at 23°C to allow cholecalciferol synthesis. The invivo biological response was tested using a rabbit tibia model after 8weeks of healing by analyzing the wound fluid and the mRNA levels of several markers at the bone-implant interface (N=8). The stability of the coating after storage up to 12weeks was determined using HPLC analysis, and the bioactivity of the stored modified implants was studied by an invitro study with MC3T3-E1 cells (N=6). A significant increase in gene expression levels of osteocalcin was found in the bone tissue attached to implants coated with the low dose of 7-DHC and VitE, together with a higher ALP activity in the wound fluid. Implants treated with the high dose of 7-DHC and VitE showed increased tissue necrosis and inflammation. Regarding the aging effects, coated implants were stable and bioactive up to 12weeks when stored at 4°C and avoiding oxygen, light and moisture. This study demonstrates that Ti implants coated with UV-irradiated 7-DHC and VitE promote invivo gene expression of bone formation markers and ALP activity, while they keep their osteopromotive potential invitro and composition when stored up to 12weeks at 4°C.

Pharmaceutical inhibition of glycogen synthetase kinase 3 beta suppresses wear debris-induced osteolysis

Aseptic loosening is associated with the development of wear debris-induced peri-implant osteolytic bone disease caused by an increased osteoclastic bone resorption and decreased osteoblastic bone formation. However, no effective measures for the prevention and treatment of peri-implant osteolysis currently exist. The aim of this study was to determine whether lithium chloride (LiCl), a selective inhibitor of glycogen synthetase kinase 3 beta (GSK-3β), mitigates wear debris-induced osteolysis in a murine calvarial model of osteolysis. GSK-3β is activated by titanium (Ti) particles, and implantation of Ti particles on the calvarial surface in C57BL/6 mice resulted in osteolysis caused by an increase in the number of osteoclasts and a decrease in the number of osteoblasts. Mice implanted with Ti particles were gavage-fed LiCl (50 or 200 mg kg−1d−1), 6 days per week for 2 weeks. The LiCl treatment significantly inhibited GSK-3β activity and increased β-catenin and axin-2 expression in a dose-dependent manner, dramatically mitigating the Ti particle-induced suppression of osteoblast numbers and the expression of bone formation markers. Finally, we demonstrated that inhibition of GSK-3β suppresses osteoclast differentiation and reduces the severity of Ti particle-induced osteolysis. The results of this study indicate that Ti particle-induced osteolysis is partly dependent on GSK-3β and, therefore, the canonical Wnt signaling pathway. This suggests that selective inhibitors of GSK-3β such as LiCl may help prevent and treat wear debris-induced osteolysis.

Skeletal development of mice lacking bone sialoprotein (BSP)--impairment of long bone growth and progressive establishment of high trabecular bone mass.

Adult Ibsp-knockout mice (BSP−/−) display shorter stature, lower bone turnover and higher trabecular bone mass than wild type, the latter resulting from impaired bone resorption. Unexpectedly, BSP knockout also affects reproductive behavior, as female mice do not construct a proper "nest" for their offsprings. Multiple crossing experiments nonetheless indicated that the shorter stature and lower weight of BSP−/− mice, since birth and throughout life, as well as their shorter femur and tibia bones are independent of the genotype of the mothers, and thus reflect genetic inheritance. In BSP−/− newborns, µCT analysis revealed a delay in membranous primary ossification, with wider cranial sutures, as well as thinner femoral cortical bone and lower tissue mineral density, reflected in lower expression of bone formation markers. However, trabecular bone volume and osteoclast parameters of long bones do not differ between genotypes. Three weeks after birth, osteoclast number and surface drop in the mutants, concomitant with trabecular bone accumulation. The growth plates present a thinner hypertrophic zone in newborns with lower whole bone expression of IGF-1 and higher IHH in 6 days old BSP−/− mice. At 3 weeks the proliferating zone is thinner and the hypertrophic zone thicker in BSP−/− than in BSP+/+ mice of either sex, maybe reflecting a combination of lower chondrocyte proliferation and impaired cartilage resorption. Six days old BSP−/− mice display lower osteoblast marker expression but higher MEPE and higher osteopontin(Opn)/Runx2 ratio. Serum Opn is higher in mutants at day 6 and in adults. Thus, lack of BSP alters long bone growth and membranous/cortical primary bone formation and mineralization. Endochondral development is however normal in mutant mice and the accumulation of trabecular bone observed in adults develops progressively in the weeks following birth. Compensatory high Opn may allow normal endochondral development in BSP−/− mice, while impairing primary mineralization.

The effect of dose on rhBMP-2 signaling, delivered via collagen sponge, on osteoclast activation and in vivo bone resorption

While recombinant human bone morphogenetic protein (rhBMP)-2-based bone therapy presents potential osteoinductivity, it also leads concern due to transient osteoclast activation during early healing periods, ultimately limiting its clinical use. Therefore, we investigated in vivo and in vitro rhBMP-2 signaling which mediates early bone resorbing effect, depending on the dose, and attempted to inhibit this resorption phenomenon using NFAT inhibitor as a target molecule. High-dose of rhBMP-2 (20 μg/defect) enhanced osteoclast activation and the expression of bone resorption markers, compared to low dose (5 μg/defect) at one week after surgery in collagen sponge-delivered rat calvarial defect models. Interestingly, this trend was also observed in the expression of bone formation markers. In particular, rhBMP-2 upregulated RANKL expression, while it downregulated osteoprotegerin (OPG) expression, resulting in a dose-dependent increase in the ratio of RANKL to OPG. NFAT inhibitor (150 μm) treatment in vivo suppressed the high-dose effect of rhBMP-2 on both resorption and formation. In vitro results of rhBMP-2 signaling and NFAT inhibitor effects in rat mesenchymal stem cells showed similar trends as in vivo results. Microcomputer tomography-based evaluation at 4 weeks showed that combined treatment of NFAT inhibitor with 20 μg rhBMP-2 in vivo increased bone volume (BV) more than 20 μg rhBMP-2 alone, which showed little difference in BV compared to 5 μg of rhBMP-2. These results demonstrated that rhBMP-2 implantation concurrently signalized into enhanced osteoclastogenesis and osteoblastogenesis in vivo, dose-dependently. Ratio of RANKL/OPG might be an index for early bone resorbing activity of implanted rhBMP-2. A local cocktail treatment of NFAT inhibitor and high-dose rhBMP-2 might be an alternative to overcome early bone resorbing effects, thereby accelerating bone formation.

Preclinical Efficacy of the Investigational Orally Bioavailable Proteasome Inhibitor MLN9708 in Myeloma Bone Disease

AbstractAbstract 4014 Introduction:Bone destruction, a hallmark of multiple myeloma (MM), arises as a consequence of the interactions between MM cells and the bone marrow microenvironment, which lead to an increase in the bone-resorptive activity and number of osteoclasts (OC) and a reduction of the bone-forming activity and differentiation of osteoblasts (OB).MLN9708, which hydrolyzes to pharmacologically active MLN2238 in aqueous solution, is an investigational proteasome inhibitor (PI) with demonstrated preclinical anti-myeloma activity. However, it is currently not known whether MLN9708, may have a beneficial effect on myeloma-associated bone disease. Here, we have conducted in vitro and in vivo studies to evaluate its ability to promote osteogenic differentiation and to inhibit OC formation and function in the myeloma setting. Patient samples, material and methods:The human MM cell lines RPMI-8226 and MM.1S (or RPMI-8226-luc and MM.1S-luc) together with the mesenchymal stem hMSC-TERT cell line were employed. Also, MSCs from BM samples of healthy donors and MM patients were used in OB differentiation studies, whereas PBMCs from healthy volunteers were used to generate OCs. NOD.SCID.IL2Rγ−/− mice were used in the in vivo model of disseminated human MM. MLN2238 and bortezomib (Velcade) were provided by Millennium Pharmaceuticals, Inc.OB differentiation from MSCs and OB function were investigated by measurement of ALP activity, quantitative mineralization, luciferase reporter assays, siRNA gene silencing and real time RT-PCR. The effect of the new PI on OC formation was assessed by enumeration of multinucleated (≥3) TRAP-positive cells. Measurement of resorbed area, immunofluorescence and flow cytometry were used to further investigate the effect of MLN2238 on OC function. In our in vivo model, bioluminescence imaging, micro-CT analysis and serum levels of Igλ and bone markers were determined. Results:Physiologic concentrations of MLN2238 were able to stimulate the osteogenic differentiation of MSCs from both myeloma patients and healthy donors in vitro to an extent comparable to bortezomib; this was assessed by increased levels of ALP activity, higher expression of bone formation markers (Runx2, osterix, osteopontin and osteocalcin) and augmented matrix mineralization. The enhanced OB formation and function induced by MLN2238 was at least partly due to induction of T-cell factor 4 (TCF4) transcriptional activity, as well as to activation of the unfolded protein response. A similar range of MLN2238 doses also markedly inhibited OC formation and resorption from human progenitors. Similarly to that described with bortezomib, MLN2238 treatment of human pre-OCs prevented RANKL-induced NF-κB activation, disrupted the integrity of the F-actin ring and also reduced the expression of the αVβ3 integrin, thus contributing to inhibition of OC function. MLN2238 was also able to overcome the growth advantage conferred to MM.1S-luc cells by co-culture with MSCs or OCs.Oral administration of MLN2238 in a mouse model of disseminated human MM decreased human RPMI-8226-luc tumor burden as assessed by diminished bioluminescence signal and decreased serum levels of Igλ secreted by RPMI-8226-luc cells. In addition, MLN2238 prevented tumor-associated bone loss with significant increases in femoral trabecular bone parameters as compared to vehicle control animals. Serum markers of bone turnover showed that MLN2238 inhibited bone resorption (decreased levels of CTX) while enhancing bone formation (increased levels of P1NP). Conclusion:MLN2238 in vitro was capable of promoting osteoblastogenesis and OB activity as well as of inhibiting OC formation and function to an extent similar to bortezomib. In a disseminated human MM mouse model, orally administered MLN2238 showed anti-resorptive and bone-anabolic effects in addition to its anti-tumor properties. Given the thus far available data on the preclinical safety and favorable pharmacologic properties of MLN2238, it is conceivable that MLN9708, the clinical formulation of this proteasome inhibitor, may also achieve bone benefits in myeloma patients. Disclosures:Berger:Millennium Pharmaceuticals, Inc.: Employment. San-Miguel:Millennium Pharmaceuticals, Inc.: Consultancy.

Pantoprazole, a Proton Pump Inhibitor, Delays Fracture Healing in Mice

Proton pump inhibitors (PPIs), which are widely used in the treatment of dyspeptic problems, have been shown to reduce osteoclast activity. There is no information, however, on whether PPIs affect fracture healing. We therefore studied the effect of the PPI pantoprazole on callus formation and biomechanics during fracture repair. Bone healing was analyzed in a murine fracture model using radiological, biomechanical, histomorphometric, and protein biochemical analyses at 2 and 5 weeks after fracture. Twenty-one mice received 100 mg/kg body weight pantoprazole i.p. daily. Controls (n = 21) received equivalent amounts of vehicle. In pantoprazole-treated animals biomechanical analysis revealed a significantly reduced bending stiffness at 5 weeks after fracture compared to controls. This was associated with a significantly lower amount of bony tissue within the callus and higher amounts of cartilaginous and fibrous tissue. Western blot analysis showed reduced expression of the bone formation markers bone morphogenetic protein (BMP)-2, BMP-4, and cysteine-rich protein (CYR61). In addition, significantly lower expression of proliferating cell nuclear antigen indicated reduced cell proliferation after pantoprazole treatment. Of interest, the reduced expression of bone formation markers was associated with a significantly diminished expression of RANKL, indicating osteoclast inhibition. Pantoprazole delays fracture healing by affecting both bone formation and bone remodeling.

Maternal Vitamin D Supplementation Results in Higher Expression of Bone Formation Markers at the Growth Plate and Site Specific Effects on Bone Strength in Male Offspring

Lower maternal serum 25‐hydroxyvitamin D or ultraviolet B exposure is associated with lower bone mineral in offspring during childhood. The effect of higher maternal vitamin D status on offspring bone health at adulthood is less clear. Using the CD‐1 mouse model, this study determined if dams fed supplemental (5,000 IU/kg diet) versus low (25 IU/kg diet) levels of vitamin D during pregnancy and lactation affected bone health of male offspring. At weaning, expression of osterix, osteoprotegrin, RANKL and vitamin D receptor (VDR) in the tibia growth plate was measured by immunohistochemical analyses in a subset of male mice (n=8/group). Some offspring were fed their mothers diet or the opposite diet (n=14/group) until age 3 months when bone strength, i.e. peak load, at lumbar vertebra and femur neck and midpoint was measured. At weaning, mice exposed to supplemental vitamin D had higher (p&lt;0.05) expression of osteoprotegrin and osterix and lower (p&lt;0.05) expression of VDR with no difference in expression of RANKL in proliferating or hypertrophic chondrocytes in the tibia growth plate. At 3 months of age, offspring of mothers fed supplemental vitamin D had lower (p&lt;0.05) femur neck peak load with no difference in peak load at other skeletal sites regardless of post‐weaning diet. Further study is required to understand the structural changes that contribute to the lower femur neck peak load.

Expression of RANKL/OPG during bone remodeling in vivo

The interaction between receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) plays a dominant role in osteoclastogenesis. As both proteins are produced by osteoblast lineage cells, they are considered to represent a key link between bone formation and resorption. In this study, we investigated the expression of RANKL and OPG during bone remodeling in vivo to determine the relationship between osteoclastogenic stimulation and osteoblastic differentiation.Total RNA was prepared from rat femurs after marrow ablation on days 0, 3, 6, and 9. The temporal activation patterns of osteoblast-related genes (procollagen α1 (I), alkaline phosphatase, osteopontin, and osteocalcin) were examined by Northern blot analysis. An appreciable increase in the expression of these osteoblast markers was observed on day 3. The peak increase in gene expression was observed on day 6 followed by a slight reduction by day 9. Real-time PCR analysis showed that the OPG mRNA expression was markedly upregulated on day 6 and slightly decreased on day 9. In contrast, RANKL mRNA expression was increased by more than 20-fold on day 9. The RANKL/OPG ratio, an index of osteoclastogenic stimulation, peaked on day 9. Histological analysis showed that RANKL and OPG immunoreactivity were predominantly associated with bone marrow cells. The expression of bone formation markers was activated in the bone formation phase, followed by the stimulation of RANKL/OPG expression in the bone resorption phase, which confirmed that these molecules are key factors linking bone formation to resorption during bone remodeling.

In vivo performance of titanium implants functionalized with eicosapentaenoic acid and UV irradiation

The aim of this study was to design implant surfaces that attach less to bone but at the same time improve osseous healing for use as temporary bone fracture plates. The strategy was to combine the nonadhesive properties of smooth titanium (Ti) surfaces with the differentiative and anti-inflammatory properties of eicosapentaenoic acid (EPA). Machined Ti implant surfaces coated with a layer of EPA, with or without UV irradiation, were characterized by X-ray photoelectron spectroscopy, and their in vivo performance was evaluated in New Zealand White rabbits. The performance of the functionalised implants was analyzed after 10 weeks of healing by mechanical pull-out testing, molecular biology, and histological and microcomputed tomography analysis. The results indicate that surface functionalization with UV light can reduce bone attachment and volumetric bone mineral density in the peri-implant bone tissue. The presence of EPA on the surfaces enhanced this effect further. Gene expression of bone formation markers showed a trend toward higher mRNA levels in all EPA treated groups. The histological analyses demonstrated lower inflammation in the UV-irradiated group and immature bone formation in all the groups. In conclusion, surface functionalization of Ti implants with UV light and EPA could be a biocompatible coating for reduced bone bonding ability of Ti while promoting bone formation.