Increased Bone Volume Research Articles

VEGF with AMD3100 endogenously mobilizes mesenchymal stem cells and improves fracture healing.

ABSTRACTA significant number of fractures develop non‐union. Mesenchymal stem cell (MSC) therapy may be beneficial, however, this requires cell acquisition, culture and delivery. Endogenous mobilization of stem cells offers a non‐invasive alternative. The hypothesis was administration of VEGF and the CXCR4 antagonist AMD3100 would increase the circulating pool of available MSCs and improve fracture healing. Ex‐breeder female wistar rats received VEGF followed by AMD3100, or sham PBS. Blood prepared for culture and colonies were counted. P3 cells were analyzed by flow cytometry, bi‐differentiation. The effect of mobilization on fracture healing was evaluated with 1.5 mm femoral osteotomy stabilized with an external fixator in 12–14 week old female Wistars. The mobilized group had significantly greater number of cfus/ml compared to controls, p = 0.029. The isolated cells expressed 1.8% CD34, 35% CD45, 61% CD29, 78% CD90, and differentiated into osteoblasts but not into adipocytes. The fracture gap in animals treated with VEGF and AMD3100 showed increased bone volume; 5.22 ± 1.7 µm3 and trabecular thickness 0.05 ± 0.01 µm compared with control animals (4.3 ± 3.1 µm3, 0.04 ± 0.01 µm, respectively). Radiographic scores quantifying fracture healing (RUST) showed that the animals in the mobilization group had a higher healing score compared to controls (9.6 vs. 7.7). Histologically, mobilization resulted in significantly lower group variability in bone formation (p = 0.032) and greater amounts of bone and less fibrous tissue than the control group. Clinical significance: This pre‐clinical study demonstrates a beneficial effect of endogenous MSC mobilization on fracture healing, which may have translation potential to prevent or treat clinical fractures at risk of delayed or non‐union fractures. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:1294–1302, 2019.

Effects of methods of hydration of a biphasic ceramic graft on bone regeneration of extraction socket defects.

Alveolar volumetric changes are inherent to the healing process after tooth extraction and filling of the extraction site with biomaterials can reduce these volumetric changes and provide a more favorable environment for the future prosthetic restoration with implants. Among the biomaterials available, biphasic ceramic compounds have shown promissing clinical results, however, histologic evaluation of bone tissue response to different hydration and incorporation methods are limited. The objective of this study was to comparatively evaluate in vivo the osteoconductive potential of the biphasic ceramic bone substitute (SBC) composed of beta-tricalcium phosphate and hydroxyapatite after different hydration methodologies in rat extraction sockets. Wistar rats (n=20) were randomly distributed in two subgroups (G1, G2) according to the hydration methods used. After extraction of the right upper central incisor the alveoli were filled with SBC hydrated by two different methods. Group 1: biomaterial hydrated in blood; Group 2: biomaterial hydrated in physiologic saline. The animals were euthanized after 1 and 6weeks for removal of the bone blocks containing the biomaterial and were submitted to histologic processing. Five-μm-thick demineralized sections of the samples were stained with hematoxylin and eosin (HE) and subjected to histomorphometric analysis. Bone formation was limited 7 days after the extraction procedure and increased in both groups between 7 and 42 days from surgery, demonstrating a time dependent increase of bone volume throughtout the experimental period (p<0.05). The hydration of SBC with saline significantly increased new bone formation and reduced connective tissue volume after 42 days demonstrating that hydration method may significantly influence bone healing in such defects, and, thus should be carefully performed.

Evaluation of Custom made Xenogenic Bone grafts in Mandibular Alveolar Ridge Augmentation versus Particulate bone graft with titanium mesh

Aim: This study was to evaluate clinically and radiographically the volume changes of alveolar ridge grafting using customized xenogenic bone graft.Materials and Methods: A total of 12 patients with mandibular horizontal and vertical alveolar ridge defects ≥ 3 mm were selected. They were divided into 2 groups: Group I (Test Group) included 6 patients in which mandibular alveolar ridges were reconstructed with customized Xenogenic bone graft Smartbone (IBI S.A., Switzerland). Group II (Control Group) included 6 patients in which mandibular alveolar ridges were reconstructed with particulate Xenogenic bone (Smart bone, IBI S.A., Switzerland) grafting to posterior mandibular ridge with titanium mesh was performed. Volume analysis of the changes in alveolar ridge in both Groups were obtained before and four months after the procedure using CBCT. Densitometric analysis of the Postoperative bone formed and compared with native bone. .Results: Four months postoperatively. Measurements made on cone-beam computerized tomograms, four months postoperative showed significance increase in bone volume by 40 % in the area of newly formed bone in Group I (Customized bone) compared with 23% in Control Group. Statistical significant changes was found in the density of newly formed bone four months post-operatively in both Groups, however there was no significant difference in bone density postoperatively between Group I (customized Bone) and Group II (Control).Conclusion: According to the results, the treatment of defective alveolar ridge augmentation of the mandibular ridge with customized xenogenic bone graft Smartbone (IBI S.A., Switzerland) is successful and produces results consistent with the control Group.

IL6 receptor blockade preserves articular cartilage and increases bone volume following ischemic osteonecrosis in immature mice

Juvenile ischemic osteonecrosis (JIO) of the femoral head is one of the most serious hip disorders causing a permanent deformity of the femoral head in childhood. We recently reported that interleukin 6 (IL6) is predominantly increased in the hip synovial fluid of patients with JIO and that articular chondrocytes are primary source of IL6. This study investigated whether an inhibition of IL6 receptor improves cartilage preservation and bone healing in JIO. A small animal model (i.e., 6-week-old mouse) of JIO was treated with either saline or tocilizumab, an IL6 receptor blocker, for 6 weeks. TUNEL-positive chondrocytes in the articular cartilage were reduced by the tocilizumab treatment, concomitant with the increase in cartilage matrix. The levels of a cartilage anabolic marker Sox9 was significantly increased in the articular cartilage of mice treated with tocilizumab. Micro-CT assessment showed tocilizumab treatment significantly increased trabecular epiphyseal bone volume (P=0.001, n=10), thickness (P=0.007) and number (P=0.014) and decreased bone separation (P=0.002) and its deformity (P=0.003). A bone formation marker, BMP2, and an angiogenic marker, vascular endothelial growth factor (VEGF), were both significantly increased by tocilizumab treatment under hypoxia using human chondrocytes while the bone resorption marker, RANKL/OPG ratio, was reduced. Tocilizumab treatment following ischemic osteonecrosis has cartilage anabolic effect and increases bone volume in JIO mouse model. The findings lead to a possible application of tocilizumab for preclinical study using a large animal model of JIO and a clinical trial to validate this treatment.

Lentivirus‑mediated microRNA‑26a overexpression in bone mesenchymal stem cells facilitates bone regeneration in bone defects of calvaria in mice

Repair of bone defects presents a serious clinical challenge as it is difficult to restore bone function and regenerate bone loss. In the present study, the effects of lentivirus-mediated transfection of bone marrow mesenchymal stem cells (BMSCs) with microRNA (miR)-26a on bone regeneration were investigated in a mouse bone defect repair model. Marker of proliferation Ki67 (Ki67) staining was employed to detect the cell proliferation capacity and alkaline phosphatase (ALP) staining was used to investigate osteogenic differentiation. A mouse model of cranial bone defects was established. β-tricalcium phosphate biomaterials co-cultured with the transfected BMSCs were implanted into the defect areas of mouse models. Micro-computed tomography, and hematoxylin and eosin and toluidine blue staining, were used to detect bone regeneration in the defect areas and the degradation of scaffolds. miR-26a expression, and the mRNA and protein expression of osteogenesis-associated cytokines, were detected using reverse transcription-quantitative polymerase chain reaction and western blot analysis. Separated and cultured BMSCs highly expressed CD29 and CD105, but not CD34 and CD45, as determined by flow cytometry. miR-26a expression and the positive cell rate of Ki67 and ALP staining in BMSCs transfected with pLVTHM-miR-26a were increased. The BMSC and negative control-transfected BMSC groups exhibited increased bone regeneration in the defect areas, increased bone volume of newly formed bones, and elevated mRNA and protein expression of runt-related transcription factor 2 (Runx2) and osteocalcin (OC), compared with the blank group. However, the miR-26a-transfected BMSC group exhibited further increases in bone regeneration and the volume of newly formed bones, and further elevations of the mRNA and protein expression levels of Runx2 and OC. The present findings demonstrated that lentivirus-mediated modification of BMSCs enhanced bone regeneration during the repair of cranial bone defects in mice.

A12839 Angiotensin 1-7 functionally recovers aging-induced locomotive dysfunction, but is not associated with accelerated aging phenotypes in angiotensin-converting-enzyme 2 knockout mice

Objectives: The angiotensin-converting enzyme 2 (ACE2)-angitotensin 1-7 (A1-7)-A1-7 receptor (Mas) axis has been shown to have a protective role in the renin-angiotensin system (RAS). We recently found that ACE2 knockout (KO) mice exhibit earlier aging-associated muscle weakness, and A1-7 restores muscle weakness in aging mice. As ACE2 has a multifunctional role outside the RAS, we investigated the dependence on the A1-7-Mas pathway in the effect of ACE2 on physiological aging including locomotive dysfunction with muscle weakness and osteoporosis in mice. Methods: Wildtype (WT), ACE2KO, and MasKO mice were subjected to periodical grip strength measurement, followed by administration of A1–7 (100 ng/kg/min) or saline for 4 weeks with an osmotic pump in 24-month-old mice. Results: ACE2KO mice exhibited decreased grip strength compared with WT mice after 6 months of age, while MasKO mice had grip strength similar to WT mice during aging. A1-7 infusion at 24 months old improved grip strength with increased muscle size in WT and ACE2KO mice but not in MasKO mice. A1–7 significantly increased bone volume in WT and ACE2KO mice, but not in MasKO mice. Histological and gene expression analysis revealed that tissue aging including skin and adipose tissue was accelerated in aging ACE2KO mice, but not in MASKO mice, compared to WT mice. Conclusion: A1-7 improves muscle weakness and osteoporosis in aged mice via a Mas-dependent pathway. In contrast, the different aging phenotypes between ACEKO and MasKO mice suggest the A1-7-independent effect of ACE2 on senescence.

Sinus lift without graft material – stability of the increased bone volume (4–8 years follow up study)

Background Implant-supported prosthetic restorations in the severely atrophic posterior maxilla have been successfully performed for the last 2 decades with various sinus augmentation techniques. The use of grafting material is widespread practice (Jensen et al. 1996) but studies (Lundgren 2004, Thor 2007, Cricchio 2011, Volpe 2011 Riben 2012, Fermergard 2012, Si 2013) have demonstrated that the elevation of the Schneiderian membrane with simultaneous implants placement result in bone formation. Aim/Hypothesis The aim of the study is to evaluate the quantity and the stability of the increased bone volume around implants inserted in a void space created by the elevation of the sinus membrane without adding any bone grafting material. Material and Methods Sixty-eight implants with a residual bone height requiring sinus lift were placed from January 2009 to January 2014. The sinus mucosal lining was elevated implants were installed in the residual subantral bone without adding any bone grafting material. Radiographs were performed (at T0, T0, 6 m, and every years as soon as is possible) to evaluate bone formation and the stability of bone volume around implants. The length of implants was used as a reference point for measurements. Results All implants were functional after 6 months. The survival rate at 8 years is equal to or greater than that reported when using biomaterials. These results are in agreement with the recent studies. Radiographic examination demonstrated new bone formation in 98.2% of the cases after 6 months. Average bone gain in the sinus was 5.81 ± 2.22 mm after a minimum of 6 months. The results are comparable with those of the literature. The bone gain is sometime lower because the implants used are on average shorter than in the reference studies. At T0, 92.86% of the implants were radiographically covered with less than 8 mm of bone. At T0, 6 months, only 12.50% had a bone level <8 mm. Bone volume remains stable from one to 8 years (98.4%). In only one of the implant is a clear resorption giving a pyramidal structure very likely compatible with a long-term survival of the implant. Studies denouncing resorption are animal studies with no loading implant. Conclusions and Clinical Implications Our investigation shows that sinus membrane elevation and simultaneous implant placement without the use of additional bone grafting material is a predictable technique for Sinus lift (Riben 2016, Stefanski 2017). According to the study, we assume that bone gain volume remains stable. Progression and clinical appearance of peri-implantitis in this new living bone seems more predictable and allow earlier care. Further human long-term studies on the stability of the bone volume are necessary .

Alendronate improves bone density and type I collagen accumulation but increases the amount of pentosidine in the healing dental alveolus of ovariectomized rabbits.

It has been shown that the oral aminobisphosphonate sodium alendronate (ALN) therapy reduces the risk of main fractures in osteoporotic women, but its effect on the jaw bones is poorly known. Here, we hypothesized that ALN affects the newly formed alveolar bone, particularly the quality of the type I collagen cross-linking. Osteoporosis was induced by ovariectomy (OVX) in 6-month old rabbits. Six weeks following surgery, eight animals were treated by oral gavage with ALN (OVX + ALN) and ten received placebo (OVX + Pbo). Another six rabbits which were sham operated also received placebo (SHAM + Pbo). One month following the beginning of treatment, the upper and lower left first premolars were removed. Six weeks later, the upper and the lower right first premolars were also extracted. One month after the second extraction, biopsies were collected from the maxillary extraction sites and collagen crosslinks were analyzed in the newly formed bone tissue by HPLC. Also, at this time, mandibular bone segments were subjected to μCT. Animals treated with ALN achieved a roughly 2-time greater bone volume fraction value at a late healing period than animals in the other groups (p < 0.05). Collagen mean results were 2- to 4-times superior in the OVX + ALN group than in the control groups (p < 0.05). ALN-treated animals presented higher amounts of the non-enzymatic collagen cross-link pentosidine (PEN) than the sham-operated rabbits (p < 0.05), whereas the OVX + Pbo group presented the highest amount of PEN (p < 0.05). Alendronate increases bone volume and collagen accumulation, but does not fully rescue the non-osteoporotic alveolar tissue quality as is evident from the increased quantity of pentosidine.

The Cocktail Effect of BMP-2 and TGF-β1 Loaded in Visible Light-Cured Glycol Chitosan Hydrogels for the Enhancement of Bone Formation in a Rat Tibial Defect Model.

Bone tissue engineering scaffolds offer the merits of minimal invasion as well as localized and controlled biomolecule release to targeted sites. In this study, we prepared injectable hydrogel systems based on visible light-cured glycol chitosan (GC) hydrogels containing bone morphogenetic protein-2 (BMP-2) and/or transforming growth factor-beta1 (TGF-β1) as scaffolds for bone formation in vitro and in vivo. The hydrogels were characterized by storage modulus, scanning electron microscopy (SEM) and swelling ratio analyses. The developed hydrogel systems showed controlled releases of growth factors in a sustained manner for 30 days. In vitro and in vivo studies revealed that growth factor-loaded GC hydrogels have no cytotoxicity against MC3T3-E1 osteoblast cell line, improved mRNA expressions of alkaline phosphatase (ALP), type I collagen (COL 1) and osteocalcin (OCN), and increased bone volume (BV) and bone mineral density (BMD) in tibia defect sites. Moreover, GC hydrogel containing BMP-2 (10 ng) and TGF-β1 (10 ng) (GC/BMP-2/TGF-β1-10 ng) showed greater bone formation abilities than that containing BMP-2 (5 ng) and TGF-β1 (5 ng) (GC/BMP-2/TGF-β1-5 ng) in vitro and in vivo. Consequently, the injectable GC/BMP-2/TGF-β1-10 ng hydrogel may have clinical potential for dental or orthopedic applications.

Zygomatic implants in our daily practice. Part II: Prosthetic rehabilitation and effect on quality of life

Effect of prosthesis and dental implants on quality of life related to the patient’s oral health can guide the clinician in providing best services. The zygomatic implant (ZI) is a partial or complete alternative technique to so-called traditional rehabilitation techniques (aiming to increase bone volume) of edentulous atrophic maxillae. The objective of this study was to evaluate the impact of fixed prosthetic rehabilitation by ZI on patients’ quality of life. This study highlighted the level of satisfaction of nine patients treated with ZI via their pre-therapeutic and post-therapeutic scores on the Oral Health Impact Profile (OHIP)-14 scale. Effect of ZI on quality of life in reference literature was also discussed. The mean pre-therapeutic and post-therapeutic OHIP scores were 29.1 and 5.8, respectively. Results about the quality of life of our patients was very satisfying. In accordance with literature, fixed prosthetic rehabilitation on ZI brought satisfaction in both its functional and aesthetic dimensions. This option of therapeutic arsenal for advanced atrophy of the edentulous maxillary is becoming more and more interesting in our daily practice.

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.

Remodelling of human bone on the chorioallantoic membrane of the chicken egg: De novo bone formation and resorption.

Traditionally used as an angiogenic assay, the chorioallantoic membrane (CAM) assay of the chick embryo offers significant potential as an in vivo model for xenograft organ culture. Viable human bone can be cultivated on the CAM and increases in bone volume are evident; however, it remains unclear by what mechanism this change occurs and whether this reflects the physiological process of bone remodelling. In this study we tested the hypothesis that CAM-induced bone remodelling is a consequence of host and graft mediated processes. Bone cylinders harvested from femoral heads post surgery were placed on the CAM of green fluorescent protein (GFP)-chick embryos for 9days, followed by micro computed tomography (μCT) and histological analysis. Three-dimensional registration of consecutive μCT-scans showed newly mineralised tissue in CAM-implanted bone cylinders, as well as new osteoid deposition histologically. Immunohistochemistry demonstrated the presence of bone resorption and formation markers (Cathepsin K, SOX9 and RUNX2) co-localising with GFP staining, expressed by avian cells only. To investigate the role of the human cells in the process of bone formation, decellularised bone cylinders were implanted on the CAM and comparable increases in bone volume were observed, indicating that avian cells were responsible for the bone mineralisation process. Finally, CAM-implantation of acellular collagen sponges, containing bone morphogenetic protein 2, resulted in the deposition of extracellular matrix and tissue mineralisation. These studies indicate that the CAM can respond to osteogenic stimuli and support formation or resorption of implanted human bone, providing a humanised CAM model for regenerative medicine research and a novel short-term in vivo model for tissue engineering and biomaterial testing.

(−)-Epigallocatechin-3-gallate (EGCG) enhances healing of femoral bone defect

BackgroundPreviously, we found that (−)-epigallocatechin-3-gallate (EGCG) enhanced osteogenic differentiation of murine bone marrow mesenchymal stem cells by increasing the mRNA expression of osteogenesis-related genes, alkaline phosphatase activity and eventually mineralization. We further found EGCG supplementation preserved bone mass and microarchitecture in female rats during estrogen deficiency in the proximal tibia and lumbar spine at least in part by increasing bone morphogenetic protein-2 (BMP2). BMP2 can enhance de novo bone formation. PurposeIn this study, we evaluate the effect of local EGCG application in de novo bone formation in bone defect healing. MethodsTwenty-four rats aged 4 months were weight-matched and randomly allocated to 2 groups: defect control with vehicle treatment (control) and defect with 10 µM EGCG treatment (EGCG). Daily vehicle and EGCG were applied locally by percutaneous local injection 2 days after defect creation for 2 weeks. Four weeks after treatment, animals were sacrificed for micro-computed tomography (μ-CT) and biomechanical analysis. ResultsLocal EGCG at femoral defect can enhance de novo bone formation by increasing bone volume and subsequently improve mechanical properties including max load, break point, stiffness, area under the max load curve, area under the break point curve and ultimate stress. ConclusionsLocal EGCG may enhance bone defect healing via at least partly by the de novo bone formation of BMP-2.

Zygomatic implants in our daily practice. Part I: Treatment Plan and Surgical Technique

Prosthetic rehabilitation of the atrophic edentulous maxilla is a challenge for which zygomatic implants (ZI) stand out from traditional techniques (aiming to increase bone volume) with reduced treatment duration and immediate loading. The aim of this study was to review the ZI treatment plan and its surgical technique based on a series of nine edentulous atrophic maxillary cases. Implant survival rate and postoperative follow-up were reported as well as those described in the literature. The advantages and disadvantages of ZI versus traditional methods of rehabilitation in our daily work were also discussed. Implant survival rate was 100% over follow-up periods varying from 5 to 47 months. During postoperative follow-up, two patients presented with slight palate inflammation. The results obtained with zygomatic implants are satisfactory in terms of reproducibility and speed of rehabilitation of the maxillary. When the patient wishes a fixed prosthetic rehabilitation, the solution provided by the ZI becomes more common in the daily practice of the clinician.

Abstract 2116: Tumor-specific effects of estrogen drive a murine model of human estrogen receptor-positive breast cancer bone metastasis independent of bone microenvironment changes

Abstract Estrogen receptor α-positive (ER+) breast cancers (BrCAs) have the greatest predilection for forming bone metastases (BMETs). To begin to query ERα's role in osteolytic BrCA BMET progression, tumoral vs bone microenvironment effects of 17β-estradiol (E2) were determined in a human xenograft murine model of ER+ BrCA BMET. For these studies, naïve or 17β-estradiol (E2)-treated female athymic mice aged 4 weeks (young) or 15 weeks (mature) were inoculated with 1x105 human ER+ MCF-7 BrCA cells via the left cardiac ventricle 2 days post-placement of 60-day release E2 pellets (0.05, 0.10, 0.18, 0.36, &amp; 0.72 mg E2). For studies examining E2 effects on bone, MCF-7 cells were not inoculated. Osteolytic lesion formation was assessed by hind limb radiographs, and E2 effects on bone were assessed by DXA (Faxitron), μCT (Scanco), and biochemical markers of bone formation or resorption (P1NP and TRAcP, respectively; Immunodiagnostic Systems). BMET incidence and lesion size in young mice were E2 dose-dependent, achieving 100% incidence at the highest dose tested, with smaller lesions and lower incidence occurring in response to lower doses, and no lesions forming in the absence of E2 even after 8 months. In contrast, E2 effects on the bone microenvironment were not dose dependent, and resulted in identical increases in bone mineral density (BMD) and bone volume (BV/TV), primarily via an increase in bone formation. To further query the importance of tumoral vs bone effects of E2 in driving ER+ BrCA progression, the tumoral effects of an identical E2 dose (0.72 mg) were compared in the skeletally immature young mice, which accrued increased bone (BV/TV) due to increases in bone formation in response to E2, vs skeletally mature mice, where E2-induced increases in bone were less marked and primarily driven by decreases in bone resorption. In these two very different bone milieus, progression of E2-dependent BMET was identical. While the relative roles of E2-induced tumor cell proliferation vs osteolytic factor secretion cannot be distinguished here, the development of osteolytic lesions similar in size to those occurring in ER- models driven by tumoral secretion of osteolytic factors, despite a marked increase in bone volume, suggests that tumoral effects of E2 may drive osteolytic ER+ BrCA BMET progression in a bone-specific fashion. Citation Format: Julia N. Cheng, Jennifer B. Frye, Susan A. Whitman, Andrew G. Kunihiro, Madison M. Egan, Julia A. Brickey, Janet L. Funk. Tumor-specific effects of estrogen drive a murine model of human estrogen receptor-positive breast cancer bone metastasis independent of bone microenvironment changes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2116.

Low power laser irradiation and human adipose-derived stem cell treatments promote bone regeneration of critical-sized calvarial defects in rats

The treatments of large bone defects are often accompanied by various limitations and complications. Stem cell therapy and physical treatments have both been shown to be beneficial in accelerating bone healing. However, the efficacy of combined treatment with stem cells and physical stimuli on large bone defects remains uncertain. The aim of this study was to evaluate the bone regeneration effects of low power laser irradiation (LPLI) and human ADSC treatments during fracture repair using a comparative rat calvarial defect model. The viability of human ADSCs was cultured on a porous PLGA scaffold using an MTS assay. The critical-sized (7 mm) calvarial bone defect Sprague–Dawley (SD) rats were divided into 4 groups: a control group, an LPLI group, an ADSC group, and an ADSC + LPLI group. Bone formation was evaluated using micro-CT at 4, 8, 12, and 16 weeks after surgery. New bone formation areas and osteogenic factor expression levels were then examined by histomorphological analysis and immunohistochemical staining. PLGA had no cytotoxic effect on human ADSCs. Micro-CT analyses showed that both the LPLI and ADSC groups improved calvarial bone defect healing compared to the control group. In addition, the ADSC + LPLI group showed significantly increased bone volume at 16 weeks after surgery. The area of new bone formation ranked as follows: control group < LPLI group < ADSC group < ADSC + LPLI group. There were significant differences between the groups. The ADSC and ADSC + LPLI groups showed strong signals of vWF expression in week 16. ADSC and LPLI treatments improve fracture repair in critical-sized calvarial defects in rats. Importantly, the combined treatment of ADSCs and further enhances the bone healing process.

Therapeutic Effect of Strontium Ranelate on Bone in Chemotherapy-Induced Osteopenic Rats via Increased Bone Volume and Reduced Bone Loss.

The purpose of this study was to determine whether treatment with strontium ranelate (SrR) can restore bone mass and strength at a skeletal site with established osteopenia in chemotherapy-induced rats. Forty-five Sprague-Dawley male rats were randomly assigned to three study groups (n = 15 rats per group): normal rats (control group), osteopenic rats (chemo group), and osteopenic rats with SrR (chemo-SrR group). We chose a male rat model that mimicked a clinical setting by weekly intravenous injection of cyclophosphamide at 20mg/kg. The control and chemo groups were maintained without treatment, and the chemo-SrR group was intragastrically administered strontium ranelate at a dosage of 900mg/kg/day. All animals were fed a standard laboratory diet, and blood samples were collected for biochemical analysis. After 12weeks of treatment, micro-CT, biomechanical testing, and histomorphometry were examined. In addition, bone samples were obtained to evaluate the content of mineral substances in bones. SrR treatment of chemo rats significantly increased tibial trabecular bone volume, trabecular thickness, and BV/TV. Serum levels of the bone formation marker alkaline phosphatase (ALP) in the SrR group were significantly higher than those in the chemo animals, which was accompanied by an increase in the bone mineral content, bone calcium and phosphate, as well as reduced serum Ca and P concentrations. The serum level of tartrate-resistant acid phosphatase (TRAP) in the SrR treatment group showed no obvious changes. Histomorphological analyses revealed that chemotherapy resulted in decreased osteoclast number, which may be due to the inhibition of bone turnover. However, SrR treatment enhanced the number of osteoblasts while restoring bone mass and improving bone strength in chemo rats. Therefore, the results of this study indicate that SrR treatment has a positive effect on bone in chemotherapy-induced osteoporosis.

PD053: Sinus lift without graft material: stability of the increased bone volume (1–7 years follow up study)

PD053: Sinus lift without graft material: stability of the increased bone volume (1–7 years follow up study)

ONO-1301 Enhances in vitro Osteoblast Differentiation and in vivo Bone Formation Induced by Bone Morphogenetic Protein.

In vitro and in vivo assessment of osteogenic effect by prostacyclin agonist (ONO-1301). The aim of this study was to investigate the effects of ONO-1301 on in vitro osteoblastic differentiation and in vivo bone formation induced by bone morphogenetic protein (BMP). Among prostaglandins (PGs), PGE2 is the most abundant in bone tissue and its effects on bone formation have been well studied. PGI2 (prostacyclin) is the second most abundant PG in bone tissue and plays important roles in hemodynamics. However, the effects of PGI2 on osteoblast differentiation and bone regeneration have not been elucidated. The effects of PGI2 agonist (ONO-1301), with and without recombinant human (rh) BMP-2, on osteoblastic differentiation and cell proliferation were investigated in vitro using alkaline phosphatase (ALP) and WST-1 assays. Murine primary osteoblasts and cell lines (ST2, MC3T3-E1, C2C12, and CH310T1/2) were used for the study. The effects of ONO-1301 on rhBMP-2 induced bone formation were investigated in a mouse model of muscle pouch transplantation (ectopic model) and in a rat model of spinal fusion (orthotopic model). ONO-1301 significantly increased ALP activity in the primary osteoblasts and ST2 cells. In addition, cotreatment with ONO-1301 and rhBMP-2 significantly increased ALP activity in the primary osteoblasts, as well as in ST2 and MC3T3-E1 cells. Cell proliferation was not affected by both ONO-1301 and ONO-1301 as well as rhBMP-2. In the ectopic model, ONO-1301 significantly increased the volume of ectopic bone whose formation was induced by BMP. In addition, in the orthotopic model, ONO-1301 significantly increased bone volume and fusion rate. This study has demonstrated that the PG IP agonist ONO-1301 improves in vitro BMP-2 induced osteoblast differentiation and in vivo ectopic and orthotopic bone formation. The results suggest that ONO-1301 has a potential clinical application as an enhancer of BMP-induced bone formation. N/A.

Dkk1 KO Mice Treated with Sclerostin Antibody Have Additional Increases in Bone Volume.

Dickkopf-1 (DKK1) and sclerostin are antagonists of the Wnt/β-catenin pathway and decreased expression of either results in increased bone formation and mass. As both affect the same signaling pathway, we aimed to elucidate the redundancy and/or compensation of sclerostin and DKK1. Weekly sclerostin antibody (Scl-Ab) was used to treat 9-week-old female Dkk1 KO (Dkk1-/-:Wnt3+/-) mice and compared to Scl-Ab-treated wild-type mice as well as vehicle-treated Dkk1 KO and wild-type animals. While Wnt3 heterozygote (Wnt3+/-) mice show no bone phenotype, Scl-Ab and vehicle-treated control groups of this genotype were included. Specimens were harvested after 3 weeks for microCT, bone histomorphometry, anti-sclerostin immunohistochemistry, and biomechanical testing. Scl-Ab enhanced bone anabolism in all treatment groups, but with synergistic enhancement seen in the cancellous compartment of Dkk1 KO mice (bone volume + 55% Dkk1 KO p < 0.01; + 22% wild type p < 0.05). Scl-Ab treatment produced less marked increases in cortical bone of the tibiae, with anabolic effects similar across genotypes. Mechanical testing confirmed that Scl-Ab improved strength across all genotypes; however, no enhancement was seen within Dkk1 KO mice. Dynamic bone labeling showed that Scl-Ab treatment was associated with increased bone formation, regardless of genotype. Immunohistochemical staining for sclerostin protein indicated no differences in the Dkk1 KO mice, indicating that the increased Wnt signaling associated with DKK1 deficiency was not compensated by upregulation of sclerostin protein. These data suggest complex interactions between Wnt signaling factors in bone, but critically illustrate synergy between DKK1 deficiency and Scl-Ab treatment. These data support the application of dual-targeted therapeutics in the modulation of bone anabolism.