Cortical Bone Formation Research Articles

Effects of Type 2 Diabetes Mellitus on Osteoclast Differentiation, Activity, and Cortical Bone Formation in POSTmenopausal MRONJ Patients.

Osteoporosis is a common metabolic bone disease in patients with diabetes, which can develop simultaneously with type 2 diabetes (T2D) in postmenopausal women. Bisphosphonate (BP) is administered to patients with both conditions and may cause medication-related osteonecrosis of the jaw (MRONJ). It affects the differentiation and function of osteoclasts as well as the thickness of the cortical bone through bone mineralization. Therefore, this study aimed to investigate the effects of T2D on osteoclast differentiation and activity as well as cortical bone formation in postmenopausal patients with MRONJ. Tissue samples were collected from 10 patients diagnosed with T2D and stage III MRONJ in the experimental group and from 10 patients without T2D in the control group. A histological examination was conducted, and the expression of dendritic cell-specific transmembrane protein (DC-STAMP) and tartrate-resistant acid phosphatase (TRAP) was assessed. Cortical bone formation was analyzed using CBCT images. The number of TRAP-positive osteoclasts and DC-STAMP-positive mononuclear cells was significantly less in the experimental group (p < 0.05). Furthermore, the thickness and ratio of cortical bone were significantly greater in the experimental group (p < 0.05). In conclusion, T2D decreased the differentiation and function of osteoclasts and increased cortical bone formation in postmenopausal patients with MRONJ.

Understanding the Role of Surface Modification of Randomized Trabecular Titanium Structures in Bone Tissue Regeneration: An Experimental Study.

Background and Objectives: Three-dimensional (3D) metallic trabecular structures made by additive manufacturing (AM) technologies promote new bone formation and osteointegration. Surface modifications by chemical treatments can improve the osteoconductive properties of metallic structures. An in vivo study in sheep was conducted to assess the bone response to randomized trabecular titanium structures that underwent a surface modification by chemical treatment compared to the bone response to the untreated specimens. Material and Methods: Sixteen specimens with a randomized trabecular titanium structure were implanted in the spongious bone of the distal femur and proximal tibia and the cortical bone of the tibial diaphysis of two sheep. Of them, eight implants had undergone a chemical treatment (treated) and were compared to eight implants with the same structure but native surfaces (native). The sheep were sacrificed at 6 weeks. Surface features of the lattice structures (native and treated) were analyzed using a 3D non-contact profilometer. Compression tests of 18 lattice cubes were performed to investigate the mechanical properties of the two structures. Excellent biocompatibility for the trabecular structures was demonstrated in vitro using a cell mouse fibroblast culture. Histomorphometric analysis was performed to evaluate bone implant contact and bone ingrowth. Results: A compression test of lattice cubic specimens revealed a comparable maximum compressive strength value between the two tested groups (5099 N for native surfaces; 5558 N for treated surfaces; p > 0.05). Compared to native surfaces, a homogenous formation of micropores was observed on the surface of most trabeculae that increased the surface roughness of the treated specimens (4.3 versus 3.2 µm). The cellular viability of cells seeded on three-dimensional structure surfaces increased over time compared to that on plastic surfaces. The histomorphometric data revealed a similar behavior and response in spongious and cortical bone formation. The percentage of the implant surface in direct contact with the regenerated bone matrix (BIC) was not significantly different between the two groups either in the spongious bone (BIC: 27% for treated specimens versus 30% for native samples) or in the cortical bone (BIC: 75% for treated specimens versus 77% for native samples). Conclusions: The results of this study reveal rapid osseointegration and excellent biocompatibility for the trabecular structure regardless of surface treatment using AM technologies. The application of implant surfaces can be optimized to achieve a strong press-fit and stability, overcoming the demand for additional chemical surface treatments.

PDGFR\u03b1 reporter activity identifies periosteal progenitor cells critical for bone formation and fracture repair

The outer coverings of the skeleton, which is also known as the periosteum, are arranged in concentric layers and act as a reservoir for tissue-specific bone progenitors. The cellular heterogeneity within this tissue depot is being increasingly recognized. Here, inducible PDGFRα reporter animals were found to mark a population of cells within the periosteum that act as a stem cell reservoir for periosteal appositional bone formation and fracture repair. During these processes, PDGFRα reporter+ progenitors give rise to Nestin+ periosteal cells before becoming osteoblasts and osteocytes. The diphtheria toxin-mediated ablation of PDGFRα reporter+ cells led to deficits in cortical bone formation during homeostasis and a diminutive hard callus during fracture repair. After ossicle transplantation, both mouse PDGFRα reporter+ periosteal cells and human Pdgfrα+ periosteal progenitors expand, ossify, and recruit marrow to a greater extent than their counterpart periosteal cells, whereas PDGFRα reporter− periosteal cells exhibit a predisposition to chondrogenesis in vitro. Total RNA sequencing identified enrichment of the secreted factors Fermt3 and Ptpn6 within PDGFRα reporter+ periosteal cells, which partly underlie the osteoblastogenic features of this cell population.

Sweep imaging with Fourier transform as a tool with MRI for evaluating the effect of teriparatide on cortical bone formation in an ovariectomized rat model

BackgroundTeriparatide (TPTD) is a drug for osteoporosis that promotes bone formation and improves bone quality. However, the effects of TPTD on cortical bone are not well understood. Sweep imaging with Fourier transform (SWIFT) has been reported as a useful tool for evaluating bound water of cortical bone, but it has yet to be used to investigate the effects of TPTD on cortical bone. This study aimed to evaluate the consequences of the effect of TPTD on cortical bone formation using SWIFT.MethodsTwelve-week-old female Sprague-Dawley rats (n = 36) were reared after ovariectomy to create a postmenopausal osteoporosis model. They were divided into two groups: the TPTD and non-TPTD groups. Rats were euthanized at 4, 12, and 24 weeks after initiating TPTD treatment. Tibial bones were evaluated using magnetic resonance imaging (MRI) and bone histomorphometry. In MRI, proton density-weighted imaging (PDWI) and SWIFT imaging were performed. The signal-to-noise ratio (SNR) was calculated for each method. The same area evaluated by MRI was then used to calculate the bone formation rate by bone histomorphometry. Measurements were compared using the Mann-Whitney U-test, and a P-value of < 0.05 was considered significant.ResultsPDWI-SNR was not significantly different between the two groups at any time point (P = 0.589, 0.394, and 0.394 at 4, 12, and 24 weeks, respectively). Contrarily, SWIFT-SNR was significantly higher in the TPTD group than in the non-TPTD group at 4 weeks after initiating treatment, but it was not significantly different at 12 and 24 weeks (P = 0.009, 0.937, and 0.818 at 4, 12, and 24 weeks, respectively). The bone formation rate assessed by histomorphometry was significantly higher in the TPTD group than in the non-TPTD group at all timepoints (P < 0.05, all weeks). In particular, at 4 weeks, the bone formation rate was markedly higher in the TPTD group than in the non-TPTD group (P = 0.028, 1.98 ± 0.33 vs. 0.09 ± 0.05 μm3/μm2/day).ConclusionsSWIFT could detect increased signals of bound water, reflecting the effect of TPTD on the cortical bone. The signal detected by SWIFT reflects a marked increase in the cortical bone formation rate.

Periosteal CD68+ F4/80+ Macrophages Are Mechanosensitive for Cortical Bone Formation by Secretion and Activation of TGF-β1.

Mechanical force regulates bone density, modeling, and homeostasis. Substantial periosteal bone formation is generated by external mechanical stimuli, yet its mechanism is poorly understood. Here, it is shown that myeloid‐lineage cells differentiate into subgroups and regulate periosteal bone formation in response to mechanical loading. Mechanical loading on tibiae significantly increases the number of periosteal myeloid‐lineage cells and the levels of active transforming growth factor β (TGF‐β), resulting in cortical bone formation. Knockout of Tgfb1 in myeloid‐lineage cells attenuates mechanical loading‐induced periosteal bone formation in mice. Moreover, CD68+F4/80+ macrophages, a subtype of myeloid‐lineage cells, express and activate TGF‐β1 for recruitment of osteoprogenitors. Particularly, mechanical loading induces the differentiation of periosteal CD68+F4/80− myeloid‐lineage cells to the CD68+F4/80+ macrophages via signaling of piezo‐type mechanosensitive ion channel component 1 (Piezo1) for TGF‐β1 secretion. Importantly, CD68+F4/80+ macrophages activate TGF‐β1 by expression and secretion of thrombospondin‐1 (Thbs1). Administration of Thbs1 inhibitor significantly impairs loading‐induced TGF‐β activation and recruitment of osteoprogenitors in the periosteum. The results suggest that periosteal myeloid‐lineage cells respond to mechanical forces and consequently produce and activate TGF‐β1 for periosteal bone formation.

Differential Effects of Neurectomy and Botox-induced Muscle Paralysis on Bone Phenotype and Titanium Implant Osseointegration.

Metabolic bone is highly innervated by both sensory and sympathetic nerves. In addition to skeletal development, neural regulation participates in local bone remodeling, which is important for successful osseointegration of titanium implants. Neurectomy is a model used to investigate the lack of neural function on bone homeostasis, but the relative impacts of direct denervation to bone or denervation-induced muscle paralysis are less well defined. To investigate this difference, we used two nerve intervention models, sciatic and femoral neurectomy (SFN) v. botox-induced muscle paralysis (BTX) and assessed the resulting femoral bone phenotype and Ti implant osseointegration. Male Sprague Dawley rats (19) were randomly divided into three groups: implant control (n=5), SFN (n=7), and BTX (n=7). Ti implants (microrough/hydrophilic [modSLA], Institut Straumann AG) were placed in the distal metaphysis of each femur on day 24 post-SFN or BTX. Bone and muscle were examined on day 28 after implant insertion. Both nerve intervention models impaired osseointegration. MicroCT and histology indicated that both models had reduced trabecular bone formation. Only BTX reduced cortical bone formation and increased cortical bone porosity. BTX resulted in more bone loss characterized by the least trabecular and cortical bone, as well as osseointegration. Osteoblasts isolated from the tibia exhibited a model-specific phenotype when they were grown on Ti substrates in vitro. Neurectomy caused more severe muscle atrophy than botox injection. These results indicate that neural regulation directly modulates bone formation and osseointegration. Muscle paralysis modulated the effects of loss of neural inputs into bone, supporting the hypothesis that mechanical loading of bone is a factor in achieving successful osseointegration. The different effects of botox and neurectomy on bone phenotype indicated that the sensory and sympathetic nerves had a role in the osseointegration process.

Decreased Trabecular Bone Mass in Col22a1-Deficient Mice.

The bone matrix is constantly remodeled by the coordinated activities of bone-forming osteoblasts and bone-resorbing osteoclasts. Whereas type I collagen is the most abundant bone matrix protein, there are several other proteins present, some of them specifically produced by osteoblasts. In a genome-wide expression screening for osteoblast differentiation markers we have previously identified two collagen-encoding genes with unknown function in bone remodeling. Here we show that one of them, Col22a1, is predominantly expressed in bone, cultured osteoblasts, but not in osteoclasts. Based on this specific expression pattern we generated a Col22a1-deficient mouse model, which was analyzed for skeletal defects by µCT, undecalcified histology and bone-specific histomorphometry. We observed that Col22a1-deficient mice display trabecular osteopenia, accompanied by significantly increased osteoclast numbers per bone surface. In contrast, cortical bone parameters, osteoblastogenesis or bone formation were unaffected by the absence of Col22a1. Likewise, primary osteoblasts from Col22a1-deficient mice did not display a cell-autonomous defect, and they did not show altered expression of Rankl or Opg, two key regulators of osteoclastogenesis. Taken together, we provide the first evidence for a physiological function of Col22a1 in bone remodeling, although the molecular mechanisms explaining the indirect influence of Col22a1 deficiency on osteoclasts remain to be identified.

Feeding intervention potentiates the effect of mechanical loading to induce new bone formation in mice.

The benefits of increased human lifespan depend upon duration of healthy, independent living; the healthspan. Bone-wasting disorders contribute significantly to loss of independence, frailty, and morbidity in older people. Therefore, there is an unmet need globally for lifestyle interventions to reduce the likelihood of bone fractures with age. Although many mechanisms are involved in disorders of bone loss, there is no single regulatory pathway and, therefore, there is no single treatment available to prevent their occurrence. Our aim in these studies was to determine whether fasting/feeding interventions alter the effect of mechanical loading on bone anabolic activities and increase bone mass. In young 17-week-old mice, 16-hour fasting period followed by reintroduction of food for 2hours increased markedly the potency of mechanical loading, that mimics the effect of exercise, to induce new cortical bone formation. Consistent with this finding, fasting and re-feeding increased the response of bone to a loading stimulus that, alone, does not stimulate new bone formation in ad-lib fed mice. Older mice (20months) experienced no potentiation of loading-induced bone formation with the same timing of feeding interventions. Interestingly, the pre-, prandial, and postprandial endocrine responses in older mice were different from those in young animals. The hormones that change in response to timing of feeding have osteogenic effects that interact with loading-mediated effects. Our findings indicate associations between timing of food ingestion and bone adaptation to loading. If translated to humans, such non-pharmacological lifestyle interventions may benefit skeletal health of humans throughout life-course and in older age.

Early mature bone formation using a bioactive membrane in dogs

The purpose of study was to compare the amounts of mature bone formation between a new bioactive collagen membrane (BCM) and a conventional collagen membrane (CCM) when used in a canine model of dental guided bone and tissue regeneration (GBR). GBR surgery was performed in 36 skeletally mature (9-14kg / 20-30lb) Beagle dogs using a split mouth design. The implant site was prepared with a titanium implant in the tooth socket and remaining void filled with a granulated bone substitute. The collagen membranes were trimmed and placed over the implant, and the gingiva closed around the treatment site. Animals were sacrificed (n=6 each group) at 4, 8, and 12 weeks after implant placement. Bone formation and tissue reactions were measured by micro-CT and histomorphometry. The study was approved by the institutional Animal Ethics committee. Bone volume in sites treated with both collagen membranes was significantly greater than controls at 12 weeks. However, the BCM displayed comparatively more bone formation at the early time point of 4 weeks than the CCM. Further, histological assessment of sites treated with the bioactive membrane revealed that mature cortical bone formation occurred as early as 8 weeks, which was likely due to the minimal inflammatory reaction observed and retention of the bioactive barrier structure. The new bioactive collagen membrane induces early mature bone formation in a canine model of dental GBR.

Anti-RANKL monoclonal antibody and bortezomib prevent mechanical unloading-induced bone loss.

Bone loss is a major health concern for astronauts during long-term spaceflight and for patients during prolonged bed rest or paralysis. It is essential to develop therapeutic strategies to combat the bone loss occurring in people afflicted with disuse atrophy on earth as well as in astronauts in space, especially during prolonged missions. Although several drugs have been demonstrated for treating postmenopausal osteoporosis or bone-related diseases, their effects on microgravity-induced bone loss are still unclear. Here, we employed the hindlimb-unloading (HLU) tail suspension model and compared the preventive efficiencies of five agents including alendronate (ALN), raloxifene (Rox), teriparatide (TPTD), anti-murine RANKL monoclonal antibody (anti-RANKL) and proteasome inhibitor bortezomib (Bzb) on mechanical unloading-induced bone loss. Bone mineral density (BMD) was measured by quantitative computed tomography. The osteoblastic and osteoclastic activity were measured by serum ELISA, histology analysis, and histomorphometric analysis. Compared to the control, ALN and anti-RANKL antibody could restore bone mass close to sham levels by inhibiting bone resorption. Bzb could increase the whole bone mass and strength by inhibiting bone resorption and promoting bone formation simultaneously. Meanwhile, Rox did not affect bone loss caused by HLU. TPTD stimulated cortical bone formation but the total bone mass was not increased significantly. We demonstrated for the first time that anti-RANKL antibody and Bzb had a positive effect on preventing mechanical unloading-induced bone loss. This finding puts forward the potential use of anti-RANKL and Bzb on bone loss therapies or prophylaxis of astronauts in spaceflight.

Targeted Remodeling of the Tumor Microenvironment Via Mechanical Stimulation Defines a Novel Therapeutic Strategy in Myeloma Bone Disease

Multiple myeloma (MM) patients develop devastating osteolytic bone disease causing non-healing bone fractures and pain. MM cells in the bone marrow microenvironment stimulate osteoclast activity and inhibit osteoblast and osteocyte function leading to massive bone resorption and therefore destruction. Bones have the capacity to locally adapt to changing loads and for self-repair and remodeling as dramatically seen in athletes during training. In this study we determined if mechanical loading can counterbalance osteolytic bone disease in the presence of established MM. We hypothesized that mechanical stimulation increases bone formation and decreases bone resorption by activating known bone (re)modeling pathways, which are deleteriously altered by MM cells in bone. We performed in vivo cyclic axial compressive tibial loading (Fig. 1) to MOPC315.BM BALB/c injected mice (n=19) with established MM bone disease (Fig. 2, bioluminescence imaging). The bone (re)modeling response to mechanical loading (5d/wk, over 3 wks) was investigated by longitudinal in vivo microCT imaging at days 14, 18, 23, 28 and 33 (Fig. 3, 4). Loading was administered to the left tibia starting 14 days after i.t. injection of left limbs of MOPC315.BM (n=10) and PBS (n=7) mice (left limb: εmax=2000µε determined by strain gauging, right limb was nonloaded). 15 mice served as nonloaded control mice (Fig. 3). We also analyzed a group of loaded, but not injected mice (n=10). MicroCT data (Fig. 3), such as cortical thickness (Ct.Th), cortical bone area (Ct.Ar) of loaded left limbs demonstrate that metaphyseal cortical bone formation was significantly greater and cortical porosity (Ct.Po) was lower compared to nonloaded control limbs. Our data provide for the first time an indication that mechanical loading counteracts MM induced bone loss over time. To further study mechanisms of mechanotransduction we used a small scale cell culture bioreactor system to analyze mechanical stimulation of human mesenchymal stem cells (hMSCs) and the translation of mechanical forces into biochemical signals. The bioreactor system allows for analysis of molecular mechanisms of mechanotransduction using a transcription factor activator protein (AP1) luciferase reporter gene construct. Cyclic stretching of telomerase immortalized hMSCs (hMSC-TERT cells) which were stably transfected by an AP1 construct resulted in stimulation of AP1 activity as measured by luciferase gene expression. We analyzed how MM cells interfere with mechanotransduction when co-cultured with hMSC-TERT cells. Our data show that in presence of MOPC315.BM cells and 2 (MM1S, INA-6) out of 4 human MM cell lines hMSCs can be mechanically stimulated and have osteogenic activity. In contrast the human MM cell lines Amo-1 and U266 inhibit the hMSC-TERT response to mechanical stimulation under these conditions. Biochemical and molecular genetic studies are underway to determine how MM cells in bone alter known bone (re)modeling pathways and which yet unanticipated factors regulate the anabolic response to mechanical loading in the MOPC315.BM model. Our study provides a first fundamental understanding of the mechano-biological mechanisms of anabolic bone adaption during MM bone disease in response to mechanical stimuli. We conclude that mechanical loading in the form of exercise has the potential to be a potent novel anabolic strategy for primary and secondary prevention of MM bone disease. Moreover dissection of the underlying molecular pathways will identify new targets for innovative antimyeloma strategies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cortical Thickness Adaptive Response to Mechanical Loading Depends on Periosteal Position and Varies Linearly With Loading Magnitude.

The aim of the current study was to quantify the local effect of mechanical loading on cortical bone formation response at the periosteal surface using previously obtained μCT data from a mouse tibia mechanical loading study. A novel image analysis algorithm was developed to quantify local cortical thickness changes (ΔCt.Th) along the periosteal surface due to different peak loads (0N ≤ F ≤ 12N) applied to right-neurectomised mature female C57BL/6 mice. Furthermore, beam analysis was performed to analyse the local strain distribution including regions of tensile, compressive, and low strain magnitudes. Student’s paired t-test showed that ΔCt.Th in the proximal (25%), proximal/middle (37%), and middle (50%) cross-sections (along the z-axis of tibia) is strongly associated with the peak applied loads. These changes are significant in a majority of periosteal positions, in particular those experiencing high compressive or tensile strains. No association between F and ΔCt.Th was found in regions around the neutral axis. For the most distal cross-section (75%), the association of loading magnitude and ΔCt.Th was not as pronounced as the more proximal cross-sections. Also, bone formation responses along the periosteum did not occur in regions of highest compressive and tensile strains predicted by beam theory. This could be due to complex experimental loading conditions which were not explicitly accounted for in the mechanical analysis. Our results show that the bone formation response depends on the load magnitude and the periosteal position. Bone resorption due to the neurectomy of the loaded tibia occurs throughout the entire cross-sectional region for all investigated cortical sections 25, 37, 50, and 75%. For peak applied loads higher than 4 N, compressive and tensile regions show bone formation; however, regions around the neutral axis show constant resorption. The 50% cross-section showed the most regular ΔCt.Th response with increased loading when compared to 25 and 37% cross-sections. Relative thickness gains of approximately 70, 60, and 55% were observed for F = 12 N in the 25, 37, and 50% cross-sections. ΔCt.Th at selected points of the periosteum follow a linear response with increased peak load; no lazy zone was observed at these positions.

Heterotopic Ossifications of the Forearm: A Cause of Post-Traumatic Loss of Pronation-Supination

HO is defined by the development of ectopic mature bone within nonosseous tissues. It is a well-described phenomenon that complicates forearm fractures, especially when there is an open fracture, a significant soft tissue injury, and associated neural axis or thermal injury. HO mainly forms near metal hardware and may lead to the formation of radio-ulnar synostosis. CT is superior to plain radiographs, as it identifies the ectopic bone earlier, defines its exact localization, and helps planning the surgical intervention. Radiologic features are variable; in the early stage, CT shows a low-attenuation mass with indistinct surroundings. As the ossification process progresses, zones of mineralization are visible before leading to the formation of mature cortical bone at the periphery (Figure 1 and 2: arrows). Hastings classification describes 5 classes according to how HO affects the forearm range of motion.

Osseointegration Improvement of Co-Cr-Mo Alloy Produced by Additive Manufacturing.

Cobalt-base alloys (Co-Cr-Mo) are widely employed in dentistry and orthopedic implants due to their biocompatibility, high mechanical strength and wear resistance. The osseointegration of implants can be improved by surface modification techniques. However, complex geometries obtained by additive manufacturing (AM) limits the efficiency of mechanical-based surface modification techniques. Therefore, plasma immersion ion implantation (PIII) is the best alternative, creating nanotopography even in complex structures. In the present study, we report the osseointegration results in three conditions of the additively manufactured Co-Cr-Mo alloy: (i) as-built, (ii) after PIII, and (iii) coated with titanium (Ti) followed by PIII. The metallic samples were designed with a solid half and a porous half to observe the bone ingrowth in different surfaces. Our results revealed that all conditions presented cortical bone formation. The titanium-coated sample exhibited the best biomechanical results, which was attributed to the higher bone ingrowth percentage with almost all medullary canals filled with neoformed bone and the pores of the implant filled and surrounded by bone ingrowth. It was concluded that the metal alloys produced for AM are biocompatible and stimulate bone neoformation, especially when the Co-28Cr-6Mo alloy with a Ti-coated surface, nanostructured and anodized by PIII is used, whose technology has been shown to increase the osseointegration capacity of this implant.

Pain related behaviors and disease related outcomes in an immunocompetent mouse model of prostate cancer induced bone pain

The purpose of this study is to characterize a syngeneic mouse model of prostate cancer induced bone pain. We stably transfected a prostate cancer cell line (RM-1) with green fluorescent protein (GFP) or red fluorescent protein (RFP) and luciferase reporters in order to visualize tumor growth longitudinally in vivo and to assess the relationship between sensory neurons and tumor cells within the bone microenvironment. Following intra-femoral injection of the RM-1 prostate cancer cell line or Hank's buffered saline into male C57Bl6 mice, we observed a progressive increase in spontaneous guarding of the inoculated limb between 12-21 days post inoculation. Daily running wheel performance was assessed as a measure of functional impairment and movement evoked pain. We observed a progressive reduction in the distance traveled and percentage of time at an optimal velocity between 12-21 days post inoculation. We utilized histological, radiographic and μCT analysis to examine tumor induced bone remodeling and observed osteolytic lesions within the distal femur as well as extra-periosteal aberrant cortical bone formation in the tumor bearing femur similar to clinical findings in patients with mixed bone metastatic prostate cancer. Tumor bearing mice displayed significant increases in markers of central sensitization (pERK, pDyn), astrocyte activation (GFAP) but not microglial activation (IBA1) in the ipsilateral spinal cord compared to sham operated mice by three weeks post inoculation. We observed reorganization of blood vessels (endomucin) and alterations in nerve fibers (PGP 9.5) in the intramedullary space of the tumor bearing femur. This immunocompetent mouse model will be useful when combined with transgenic Cre/Flpo driver mice to examine tumor, immune cell and sensory neuron interactions in the bone microenvironment and their role in pain and disease progression associated with bone cancer. Supported by DOD grants W81XWH-17-1-0542 to CMP and W81XWH-17-1-0541 o YS.

Possible Roles of Vitamin D in Bone Grafting.

Bone grafting is one of the most commonly used options to treat large bone defects. Evidence has shown that vitamin D may affect osseointegration, a major component for successful bone grafting. In vitro studies have proved that implants coated with activated vitamin D stimulate bone production and reduce bone resorption around implants. Animal studies have noticed that oral administration of vitamin D may stimulate bone formation as well as strengthen and support the interaction between bone and implants. Vitamin D insufficiency may affect negatively the cortical peri-implant bone formation, suggesting a negative effect in graft incorporation. Few clinical studies have observed that vitamin D administration enhanced graft incorporation and bone formation, while severe vitamin D deficiency is associated with failed implant osseointegration. Even though there are encouraging results of vitamin D supplementation on graft incorporation in animal studies, the use of vitamin D as an adjuvant in bone grafting procedures cannot be fully supported at the moment. However, there is theoretical support in the use of vitamin D after surgery and the use of bone grafts to support the bone structure, relieve pain and increase graft absorption. Further experimental and clinical studies are required to support the administration of vitamin D and its analogues in such cases.

Formation Dominates Resorption With Increasing Mineralized Density and Time Postfracture in Cortical but Not Trabecular Bone: A Longitudinal HRpQCT Imaging Study in the Distal Radius.

ABSTRACTClinical evaluation of fracture healing is often limited to an assessment of fracture bridging from radiographic images, without consideration for other aspects of bone quality. However, recent advances in HRpQCT offer methods to accurately monitor microstructural bone remodeling throughout the healing process. In this study, local bone formation and resorption were investigated during the first year post fracture in both the fractured (n = 22) and contralateral (n = 19) radii of 34 conservatively treated patients (24 female, 10 male) who presented with a unilateral radius fracture at the Innsbruck University Hospital, Austria. HRpQCT images and clinical metrics were acquired at six time points for each patient. The standard HRpQCT image acquisition was captured for all radii, with additional distal and proximal image acquisitions for the fractured radii. Measured radial bone densities were isolated with a voxel‐based mask and images were rigidly registered to images from the previous imaging session using a pyramid‐based approach. From the registered images, bone formation and resorption volume fractions were quantified for multiple density‐based thresholds and compared between the fractured and contralateral radius and relative to demographics, bone morphometrics, and fracture metrics using regression. Compared with the contralateral radius, both bone formation and resorption were significantly increased in the fractured radius throughout the study for nearly all evaluated thresholds. Higher density cortical bone formation continually increased throughout the duration of the study and was significantly greater than resorption during late‐stage healing in both the fractured and intact regions of the radius. With the small and diverse study population, only weak relationships between fracture remodeling and patient‐specific parameters were unveiled. However this study provides methods for the analysis of local bone remodeling during fracture healing and highlights relevant considerations for future studies, specifically that remodeling postfracture is likely to continue beyond 12‐months postfracture. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

3D-printed, bioactive ceramic scaffold with rhBMP-2 in treating critical femoral bone defects in rabbits using the induced membrane technique.

Although autogenous bone grafts are an optimal filling material for the induced membrane technique, limited availability and complications at the harvest site have created a need for alternative graft materials. We aimed to investigate the effect of an rhBMP-2-coated, 3D-printed, macro/microporous CaO-SiO2 -P2 O5 -B2 O3 bioactive ceramic scaffold in the treatment of critical femoral bone defects in rabbits using the induced membrane technique. A 15-mm segmental bone defect was made in the metadiaphyseal area of the distal femur of 14 rabbits. The defect was filled with polymethylmethacrylatecement and stabilized with a 2.0 mm locking plate. After the membrane matured for 4 weeks, the scaffold was implanted in two randomized groups: Group A (3D-printed bioceramic scaffold) and Group B (3D-printed, bioceramic scaffold with rhBMP-2). Eight weeks after implantation, the radiographic assessment showed that the healing rate of the defect was significantly higher in Group B (7/7, 100%) than in Group A (2/7, 29%). The mean volume of new bone formation around and inside the scaffold doubled in Group B compared to that in Group A. The mean static and dynamic stiffness were significantly higher in Group B. Histological examination revealed newly formed bone in both groups. Extensive cortical bone formation along the scaffold was found in Group B. Successful bone reconstruction in critical-sized bone defects could be obtained using rhBMP-2-coated, 3D-printed, macro/microporous bioactive ceramic scaffolds. This grafting material demonstrated potential as an alternative graft material in the induced membrane technique for reconstructing critical-sized bone defects.

Low-Dose Tamoxifen Induces Significant Bone Formation in Mice.

ABSTRACTUse of the selective estrogen receptor modulator Tamoxifen (TAM) is a mainstay to induce conditional expression of Cre recombinase in transgenic laboratory mice. To excise β‐cateninfl/fl in 28‐day‐old male and female Prrx1‐CreER/β‐cateninfl/fl mice (C57BL/6), we utilized TAM at 150 mg/kg; despite β‐catenin knockout in MSC, we found a significant increase in trabecular and cortical bone volume in all genders. Because TAM was similarly anabolic in KO and control mice, we investigated a dose effect on bone formation by treating wild‐type mice (WT C57BL/6, 4 weeks) with TAM (total dose 0, 20, 40, 200 mg/kg via four injections). TAM increased bone in a dose‐dependent manner analyzed by micro–computed tomography (μCT), which showed that, compared to control, 20 mg/kg TAM increased femoral bone volume fraction (bone volume/total volume [BV/TV]) (21.6% ± 1.5% to 33% ± 2.5%; 153%, p < 0.005). With TAM 40 mg/kg and 200 mg/kg, BV/TV increased to 48.1% ± 4.4% (223%, p < 0.0005) and 58% ± 3.8% (269%, p < 0.0001) respectively, compared to control. Osteoblast markers increased with 200 mg/kg TAM: Dlx5 (224%, p < 0.0001), Alp (166%, p < 0.0001), Bglap (223%, p < 0.0001), and Sp7 (228%, p < 0.0001). Osteoclasts per bone surface (Oc#/BS) nearly doubled at the lowest TAM dose (20 mg/kg), but decreased to <20% control with 200 mg/kg TAM. Our data establish that use of TAM at even very low doses to excise a floxed target in postnatal mice has profound effects on trabecular and cortical bone formation. As such, TAM treatment is a major confounder in the interpretation of bone phenotypes in conditional gene knockout mouse models. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Copper-Containing Alloy as Immunoregulatory Material in Bone Regeneration via Mitochondrial Oxidative Stress.

In the mammalian skeletal system, osteogenesis and angiogenesis are closely linked by type H vessels during bone regeneration and repair. Our previous studies confirmed the promotion of these processes by copper-containing metal (CCM) in vitro and in vivo. However, whether and how the coupling of angiogenesis and osteogenesis participates in the promotion of bone regeneration by CCM in vivo is unknown. In this study, M2a macrophages but not M2c macrophages were shown to be immunoregulated by CCM. A CCM, 316L−5Cu, was applied to drilling hole injuries of the tibia of C57/6 mice for comparison. We observed advanced formation of cortical bone and type H vessels beneath the new bone in the 316L−5Cu group 14 and 21 days postinjury. Moreover, the recruitment of CD206-positive M2a macrophages, which are regarded as the primary source of platelet-derived growth factor type BB (PDGF-BB), was significantly promoted at the injury site at days 14 and 21. Under the stimulation of CCM, mitochondria-derived reactive oxygen species were also found to be upregulated in CD206hi M2a macrophages in vitro, and this upregulation was correlated with the expression of PDGF-BB. In conclusion, our results indicate that CCM promotes the evolution of callus through the generation of type H vessels during the process of bone repair by upregulating the expression of PDGF-BB derived from M2a macrophages.