Increased Bone Volume Fraction Research Articles

Calorie restriction in mice impairs cortical but not trabecular peak bone mass by suppressing bone remodeling.

Calorie restriction (CR) can lead to weight loss and decreased substrate availability for bone cells. Ultimately, this can lead to impaired peak bone acquisition in children and adolescence and bone loss in adults. But the mechanisms that drive diet-induced bone loss in humans are not well characterized. To explore those in greater detail, we examined the impact of 30% CR for 4 and 8wk in both male and female 8-wk-old C57BL/6J mice. Body composition, areal bone mineral density (aBMD), skeletal microarchitecture by micro-CT, histomorphometric parameters, and in vitro trajectories of osteoblast and adipocyte differentiation were examined. After 8wk, CR mice lost weight and exhibited lower femoral and whole-body aBMD vs ad libitum (AL) mice. By micro-CT, CR mice had lower cortical bone area fraction vs AL mice, but males had preserved trabecular bone parameters and females showed increased bone volume fraction compared to AL mice. Histomorphometric analysis revealed that CR mice had a profound suppression in trabecular as well as endocortical and periosteal bone formation in addition to reduced bone resorption compared to AL mice. Bone marrow adipose tissue was significantly increased in CR mice. In vitro, the pace of adipogenesis in bone marrow stem cells was greatly accelerated with higher markers of adipocyte differentiation and more oil red O staining, whereas osteogenic differentiation was reduced. qRT-PCR and western blotting suggested that the expression of Wnt16 and the canonical β-catenin pathway was compromised during CR. In sum, CR causes impaired peak cortical bone mass due to a profound suppression in bone remodeling. The increase in marrow adipocytes in vitro and in vivo is related to both progenitor recruitment and adipogenesis in the face of nutrient insufficiency. Long-term CR may lead to lower bone mass principally in the cortical envelope, possibly due to impaired Wnt signaling.

Clodronate disodium does not produce measurable effects on bone metabolism in an exercising, juvenile, large animal model.

Bisphosphonates are commonly used to treat and prevent bone loss, but their effects in active, juvenile populations are unknown. This study examined the effects of intramuscular clodronate disodium (CLO) on bone turnover, serum bone biomarkers (SBB), bone mineral density (BMD), bone microstructure, biomechanical testing (BT), and cartilage glycosaminoglycan content (GAG) over 165 days. Forty juvenile sheep (253 ± 6 days of age) were divided into four groups: Control (saline), T0 (0.6 mg/kg CLO on day 0), T84 (0.6 mg/kg CLO on day 84), and T0+84 (0.6 mg/kg CLO on days 0 and 84). Sheep were exercised 4 days/week and underwent physical and lameness examinations every 14 days. Blood samples were collected for SBB every 28 days. Microstructure and BMD were calculated from tuber coxae (TC) biopsies (days 84 and 165) and bone healing was assessed by examining the prior biopsy site. BT and GAG were evaluated postmortem. Data, except lameness data, were analyzed using a mixed-effects model; lameness data were analyzed as ordinal data using a cumulative logistic model. CLO did not have any measurable effects on the skeleton of sheep. SBB showed changes over time (p ≤ 0.03), with increases in bone formation and decreases in some bone resorption markers. TC biopsies showed increasing bone volume fraction, trabecular spacing and thickness, and reduced trabecular number on day 165 versus day 84 (p ≤ 0.04). These changes may be attributed to exercise or growth. The absence of a treatment effect may be explained by the lower CLO dose used in large animals compared to humans. Further research is needed to examine whether low doses of bisphosphonates may be used in active juvenile populations for analgesia without evidence of bone changes.

Enlarged colony housing promotes linear progression of subchondral bone remodeling in joint instability rat models.

Objective: Osteoarthritis (OA) is a disease with high prevalence and an unmet medical need for disease modifying treatments. In rat models, OA-like subchondral bone and cartilage changes can be induced by instability surgery with different severity levels. Factors which determine structural changes additionally comprise the study duration and activity-impacted joint loading. Methods: A medial meniscal tear (MMT) or anterior cruciate ligament transection with partial meniscectomy (ACLT+pMx) was induced unilaterally in rats housed in a rat colony cage (RCC), allowing high activity levels including jumping and stair climbing. In parallel, ACLT+pMx rats were housed in Type IV cages. The time course of OA-related changes was investigated at 4, 8, 12, and 16weeks after surgery by micro-CT, gait analysis and joint diameter measurements. Results: Gait disturbance was observed after 2weeks and to a similar extend in all models. The increase in ipsilateral joint diameters peaked after 2weeks and were more pronounced after ACLT+pMx compared to MMT-surgery, but independent of housing. Micro-CT analysis revealed that increases in osseous tibial width were most distinct after ACLT+pMx in RCC and progressed continuously until week sixteen. In contrast, osseous tibial width of ipsilateral joints in MMT RCC and ACLT+pMx Type IV groups did not increase further after week twelve. In contralateral joints, this parameter was not affected, regardless of the model or caging. However, a significant increase in bone volume fraction and trabecular thickness was observed over time in the femur and tibia of both ipsilateral and contralateral knees. Here, the medial tibial compartment of the operated joint was most affected and linear changes were most pronounced in the ACLT+pMx RCC group. Conclusion: Increased movement of animals in colony cages leads to robust structural changes in subchondral bone after surgically induced joint instability over time, while in regular Type IV housing maximal changes are reached in week twelve. The new insights into the differentiation of the models, particularly with respect to the linear progression of bone changes in ACLT+pMx in the RCC, may be useful for the design of chronic OA-studies within a longer lifespan and therefore supporting the development of novel therapies.

Thermal control of photothermal implants inspired by polar bear skin for the treatment of infected bone defects.

Photothermal therapy (PTT) encounters challenges in addressing deep tissue infections, characterized by limited penetration or potential hyperthermal damage to surrounding tissues, initiating undesirable inflammatory cascades. Inspired by polar bear thermal regulation, we present a "bio-based endogenic thermal-adaptive booster" implant coating. This coating integrates a photothermal poly(tannic acid) (pTA) layer, mimicking the "polar bear dark skin", securely linked with anti-inflammatory dexamethasone (Dex), resembling the "secretion", and a red blood cell membrane (RBCM) layer, forming the insulating "transparent fur". The RBCM "fur" demonstrates unexpectedly superior local heat storage, amplifying the photothermal effect of the pTA "skin" by 1.30 times and boosting localized photothermal antibacterial efficiency by 1.30-fold (approximately 99%) compared to those without RBCM. Furthermore, RBCM sustains Dex release and offers additional protection against thermal inflammation, releasing Dex 1.90 times more under NIR irradiation than under non-photothermal conditions. In a rat infectious bone model, the photothermal-boosting implant coating provides a favorable biological interface and achieves a 99.97% photothermal antibacterial ratio, enhancing osseointegration without evident tissue harm, evidenced by a 2.47-fold increase in bone volume fraction and a 2.24-fold reduction in pro-inflammatory cytokines compared to those lacking a RBCM. Insights derived from cell membrane-based thermal-adaptive coatings herald a paradigm shift in efficient and safe PTT.

CAN ACUTE MUSCLE DAMAGE LEAD TO OSTEOARTHRITIS?

Abstract Skeletal muscles are crucial for maintaining musculoskeletal homeostasis, as they provide mechanical support, joint stability, and biochemical functions via myokine secretion. We investigated whether acute muscle damage or weakness could contribute to the development of osteoarthritis (OA). Using a chemical damage model, we induced muscle damage by injecting 1.2% barium chloride into the tibialis anterior muscle, resulting in an immune response characterized by a significant increase in the concentration of Prostaglandin E2 (PGE2) and other lipid mediators derived from the EPA and DHA signaling pathways, as quantified using our innovative lipidomics methods. The muscle damage was confirmed and visualized by muscle histomorphometry. The damage resulted in muscle weakness, detected by a significant decrease in grip strength at 4-days post-injury, but without changes in mice activity measured with force-plate actimeter. After 1-month post-injury, grip strength and muscle histology returned to normal, but histological changes were observed in the articulating cartilage, indicating surface erosion and cartilage matrix loss (OARSI grade 4). We used Raman Spectroscopy and micro-CT to confirm the joint alterations observed in the changes in subchondral bone and increased bone volume fraction in the cortical shell of the subchondral bone, respectively, features characteristic of OA in rodents and humans. These findings suggest that muscle weakness or imbalance due to injury can contribute to joint degeneration. This study highlights the significance of muscles in preventing OA and provides a novel approach for exploring the relationship between muscle damage and OA development. This new model could prove valuable for aging research.

The bone-protective benefits of amino-conjugated calcium in an ovariectomized (OVX) rat model

Low bone density, fragility, and microarchitectural disintegration are the symptoms of osteoporosis. An imbalance between bone growth and resorption can lead to osteoporosis. This study evaluated the effects of amino-calcium (AC) on bone protection in ovariectomized control group (NC) rats. Amino-calcium (AC) was characterized using Fourier-transform infrared spectroscopy (FT-IR), energy-dispersive X-ray spectroscopy (EDS), and nuclear magnetic resonance spectroscopy analyses (NMR). After determining the biocompatibility of amino-calcium (AC) with MC3T3-E1 cells, alkaline phosphatase staining revealed significant changes on day 7. Three of the four groups underwent ovariectomy, whereas one group received a placebo. On micro-computed tomography, in vivo, data showed increased bone volume fraction in the femoral head and shaft areas in the amino-calcium (AC) group. Hematoxylin and eosin staining showed a bone mass and architectural protection in the amino-calcium (AC) group compared with the calcium carbonate and OVX control group. RNA sequencing analysis revealed high expression of osteogenesis-related genes in MC3T3-E1 cells. RNA sequencing revealed a significant fold change in the expression of integrin-binding sialoprotein (IBSP), bone gamma-carboxyglutamate proteins 1 and 2(BGLAP1 and BGLAP2), and periostin (POSTN). The study concluded that supplementing the OVX rats with calcium enhanced bone protection.

How does alendronate affect orthodontic tooth movement in osteogenesis imperfecta: an in vivo study on a mice model.

The purpose of this study was to evaluate the effects of alendronate on orthodontic tooth movement (OTM) and bone modelling/remodelling in an osteogenesis imperfecta (OI) mice model. Ten-week-old male and female OI mice (Col1a2oim, n = 32) were divided into four groups: 1. Alendronate male (AM, n = 8), 2. Alendronate female (AF, n = 8), 3. saline male (SM, n = 8), and 4. saline female (SF, n = 8). The mice in all four groups received either Alendronate (0.05 mg/kg) or vehicle (saline 0.05 mg/kg) subcutaneously for 2 weeks prior to the placement of orthodontic spring. A nickel-titanium spring applying 3-5 cN of force was used to perform the OTM for 1 week. After 7 days of OTM, the OI mice were euthanized with CO2 inhalation and microfocus computed tomography and histological analyses were performed. AM and AF mice showed a significant decrease (P < 0.05) in the rate of OTM compared with SM and SF mice, respectively. In addition, AM and AF mice showed a significant increase (P < 0.05) in the bone volume fraction (BVF) and tissue density (TD) compared with SM and SF mice. Histological analysis of haematoxylin-eosin staining revealed a hyalinization zone in AM and AF mice compared with SM and SF mice. Furthermore, tartrate-resistant acid phosphatase staining indicated decreased number of osteoclasts in AM and AF mice compared with SM and SF mice. Picrosirius red staining showed, Alendronate treatment led to thick uniform and smooth morphology of collagen fibres as compared with saline group. Similarly, second harmony generation images also revealed thicker collagen fibres at the periodontal ligament (PDL)-cementum entheses and PDL-alveolar bone entheses in AM and AF mice compared with SM and SF mice. Alendronate led to a decrease in the rate of OTM, increase in BVF and TD, decrease in the number of osteoclasts, and smooth and thick collagen fibres compared with saline in both male and female OI mice.

Bone Sialoprotein Is Critical for Alveolar Bone Healing in Mice

Bone sialoprotein (BSP) is an extracellular matrix (ECM) protein associated with mineralized tissues, particularly bone and cementum. BSP includes functional domains implicated in collagen binding, hydroxyapatite nucleation, and cell signaling, although its function(s) in osteoblast and osteoclast differentiation and function remain incompletely understood. Genetic ablation of BSP in Ibsp knockout (Ibsp–/–) mice results in developmental bone mineralization and remodeling defects, with alveolar bone more severely affected than the femurs and tibias of the postcranial skeleton. The role of BSP in alveolar bone healing has not been studied. We hypothesized that BSP ablation would cause defective alveolar bone healing. We employed a maxillary first molar extraction socket healing model in 42-d postnatalIbsp–/– and wild-type (WT) control mice. Tissues were collected at 0, 7, 14, 21, and 56 d postprocedure (dpp) for analysis by micro–computed tomography (microCT), histology, in situ hybridization (ISH), immunohistochemistry (IHC), and quantitative polymerase chain reaction (qPCR) array. As expected, alveolar bone healing progressed in WT mice with increasing bone volume fraction (BV/TV), bone mineral density (BMD), and tissue mineral density (TMD), transitioning from woven to mature bone from 7 to 56 dpp. Ibsp messenger RNA (mRNA) and BSP protein were strongly expressed during alveolar bone healing in parallel with other osteogenic markers. Compared to WT, Ibsp–/– mice exhibited 50% to 70% reduced BV/TV and BMD at all time points, 7% reduced TMD at 21 dpp, abnormally increased Col1a1 and Alpl mRNA expression, and persistent presence of woven bone and increased bone marrow in healing sockets. qPCR revealed substantially dysregulated gene expression in alveolar bone of Ibsp–/– versus WT mice, with significantly disrupted expression of 45% of tested genes in functional groups, including markers for osteoblasts, osteoclasts, mineralization, ECM, cell signaling, and inflammation. We conclude that BSP is a critical and nonredundant factor for alveolar bone healing, and its absence disrupts multiple major pathways involved in appropriate healing.

A multi-model approach to predict efficacious clinical dose for an anti-TGF-β antibody (GC2008) in the treatment of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a heterogeneous group of inherited bone dysplasias characterized by reduced skeletal mass and bone fragility. Although the primary manifestation of the disease involves the skeleton, OI is a generalized connective tissue disorder that requires a multidisciplinary treatment approach. Recent studies indicate that application of a transforming growth factor beta (TGF-β) neutralizing antibody increased bone volume fraction (BVF) and strength in an OI mouse model and improved bone mineral density (BMD) in a small cohort of patients with OI. In this work, we have developed a multitiered quantitative pharmacology approach to predict human efficacious dose of a new anti-TGF-β antibody drug candidate (GC2008). This method aims to translate GC2008 pharmacokinetic/pharmacodynamic (PK/PD) relationship in patients, using a number of appropriate mathematical models and available preclinical and clinical data. Compartmental PK linked with an indirect PD effect model was used to characterize both pre-clinical and clinical PK/PD data and predict a GC2008 dose that would significantly increase BMD or BVF in patients with OI. Furthermore, a physiologically-based pharmacokinetic model incorporating GC2008 and the body's physiological properties was developed and used to predict a GC2008 dose that would decrease the TGF-β level in bone to that of healthy individuals. By using multiple models, we aim to reveal information for different aspects of OI disease that will ultimately lead to a more informed dose projection of GC2008 in humans. The different modeling efforts predicted a similar range of pharmacologically relevant doses in patients with OI providing an informed approach for an early clinical dose setting.

Do Knee-Straining Activities Influence the Subchondral Bone Microarchitecture and Accelerate Knee Osteoarthritis Progression?

Knee overload was detrimental to knee osteoarthritis subjects. We aim to analyze the association between the subchondral bone microarchitecture sclerosis and typical knee-straining activities. The frequency of experiencing squatting, kneeling, lifting, and climbing of 481 knee osteoarthritis subjects was investigated. Subchondral bone microarchitecture (bone volume fraction, trabecular thickness, trabecular separation, and trabecular number) was measured by the fast imaging with steady-state free precession magnetic resonance imaging applying trabecular sequencing, at baseline and 12-mo follow-up. Logistic regression was conducted to investigate the relationship between microarchitecture change and each knee-straining activity, adjusted by age, sex, weight, and Kellgren-Lawrence grade. All data were from the Osteoarthritis Initiative. Long kneeling was associated with increased bone volume fraction (adjusted odds ratio = 1.16, confidence interval = 1.01-1.33) and trabecular number (adjusted odds ratio = 1.16, confidence interval = 1.02-1.33). Long squatting was associated with a lower risk of increased trabecular separation (adjusted odds ratio = 0.84, confidence interval = 0.71-0.98) and a higher risk of increased trabecular thickness (adjusted odds ratio = 1.29, confidence interval = 1.06-1.55). Long squatting also increased the medial compartment joint width narrowing (-0.21 in squatting group vs -0.03 in no squatting group, P < 0.05). Knee osteoarthritis patients should avoid long squatting and kneeling, and subchondral bone microarchitecture possesses excellent potential as a monitoring indicator in subjects who kneel or squat for long.

CCL2 is a critical mechano-responsive mediator in crosstalk between osteoblasts and bone mesenchymal stromal cells.

It has been known that moderate mechanical loading, like that caused by exercise, promotes bone formation. However, its underlying mechanisms remain elusive. Here we showed that moderate running dramatically improved trabecular bone in mice tibias with an increase in bone volume fraction and trabecular number and a decrease in trabecular pattern factor. Results of immunohistochemical and histochemical staining revealed that moderate running mainly increased the number of osteoblasts but had no effect on osteoclasts. In addition, we observed a dramatic increase in the number of colony forming unit-fibroblast in endosteal bone marrow and the percentage of CD45- Leptin receptor+ (CD45- LepR+ ) endosteal mesenchymal progenitors. Bioinformatics analysis of the transcriptional data from gene expression omnibus (GEO) database identified chemokine c-c-motif ligands (CCL2) as a critical candidate induced by mechanical loading. Interestingly, we found that CCL2 was up-regulated mainly in osteoblastic cells in the tibia of mice after moderate running. Further, we found that mechanical loading up-regulated the expression of CCL2 by activating ERK1/2 pathway, thereby stimulating migration of endosteal progenitors. Finally, neutralizing CCL2 abolished the recruitment of endosteal progenitors and the increased bone formation in mice after 4weeks running. These results therefore uncover an unknown connection between osteoblasts and endosteal progenitors recruited in the increased bone formation induced by mechanical loading.

Effects of Active Vitamin D or FGF23 Antibody on Hyp Mice Dentoalveolar Tissues

Mutations in the PHEX gene lead to X-linked hypophosphatemia (XLH), a form of inherited rickets featuring elevated fibroblast growth factor 23 (FGF23), reduced 1,25-dihydroxyvitamin D (1,25D), and hypophosphatemia. Hyp mutant mice replicate the XLH phenotype, including dentin, alveolar bone, and cementum defects. We aimed to compare effects of 1,25D versus FGF23-neutralizing antibody (FGF23Ab) monotherapies on Hyp mouse dentoalveolar mineralization. Male Hyp mice, either injected subcutaneously with daily 1,25D or thrice weekly with FGF23 blocking antibody from 2 to 35 d postnatal, were compared to wild-type (WT) controls and untreated Hyp mice. Mandibles were analyzed by high-resolution micro–computed tomography (micro-CT), histology, and immunohistochemistry. Both interventions maintained normocalcemia, increased serum phosphate levels, and improved dentoalveolar mineralization in treated versus untreated Hyp mice. 1,25D increased crown dentin volume and thickness and root dentin/cementum volume, whereas FGF23Ab effects were limited to crown dentin volume. 1,25D increased bone volume fraction, bone mineral density, and tissue mineral density in Hyp mice, whereas FGF23Ab failed to significantly affect these alveolar bone parameters. Neither treatment fully attenuated dentin and bone defects to WT levels, and pulp volumes remained elevated regardless of treatment. Both treatments reduced predentin thickness and improved periodontal ligament organization, while 1,25D promoted a more profound improvement in acellular cementum thickness. Altered cell densities and lacunocanalicular properties of alveolar and mandibular bone osteocytes and cementocytes in Hyp mice were partially corrected by either treatment. Neither treatment normalized the altered distributions of bone sialoprotein and osteopontin in Hyp mouse alveolar bone. Moderate improvements from both 1,25D and FGF23Ab treatment regimens support further studies and collection of oral health data from subjects receiving a newly approved anti-FGF23 therapy. The inability of either treatment to fully correct Hyp mouse dentin and bone prompts further experiments into underlying pathological mechanisms to identify new therapeutic approaches.

The Dual Effects of Reactive Oxygen Species on the Mandibular Alveolar Bone Formation in SOD1 Knockout Mice: Promotion or Inhibition.

The status of reactive oxygen species (ROS) correlates closely with the normal development of the oral and maxillofacial tissues. Oxidative stress caused by ROS accumulation not only affects the development of enamel and dentin but also causes pathological changes in periodontal tissues (periodontal ligament and alveolar bone) that surround the root of the tooth. Although previous studies have shown that ROS accumulation plays a pathologic role in some oral and maxillofacial tissues, the effects of ROS on alveolar bone development remain unclear. In this study, we focused on mandibular alveolar bone development of mice deficient in superoxide dismutase1 (SOD1). Analyses were performed using microcomputerized tomography (micro-CT), TRAP staining, immunohistochemical (IHC) staining, and enzyme-linked immunosorbent assay (ELISA). We found for the first time that slightly higher ROS in mandibular alveolar bone of SOD1(-/-) mice at early ages (2-4 months) caused a distinct enlargement in bone size and increased bone volume fraction (BV/TV), trabecular thickness (Tb.Th), and expression of alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx2), and osteopontin (OPN). With ROS accumulation to oxidative stress level, increased trabecular bone separation (Tb.Sp) and decreased expression of ALP, Runx2, and OPN were found in SOD1(-/-) mice at 6 months. Additionally, dosing with N-acetylcysteine (NAC) effectively mitigated bone loss and normalized expression of ALP, Runx2, and OPN. These results indicate that redox imbalance caused by SOD1 deficiency has dual effects (promotion or inhibition) on mandibular alveolar bone development, which is closely related to the concentration of ROS and the stage of growth. We present a valuable model here for investigating the effects of ROS on mandibular alveolar bone formation and highlight important roles of ROS in regulating tissue development and pathological states, illustrating the complexity of the redox signal.

Preparation and osteogenic effect study of small intestinal submucosa sponge

To prepare and evaluate the basic properties in vitro of a novel small intestinal submucosa (SIS) sponge, and to describe the bone formation ability of the SIS sponge in vivo. The SIS sponge was prepared by freeze-drying method. To evaluate the physicochemical properties of the sponge, electron microscope observation, porosity test, water absorption ability and mechanical property were conducted in vitro. The cytotoxicity of the SIS sponge was performed by cell counting kit-8 method. In vivo experiments, eighteen extraction sockets of premolar of three Beagle dogs were randomly divided into three groups: SIS sponge group (SIS sponge), positive control group (Bio-Oss granules and Bio-Gide membrane) and control group(no treatment). The animals were sacrificed 4 weeks and 12 weeks after operation, and micro computed tomography (Micro-CT) was applied to measure the bone volume fraction (BV/TV) and bone mineralized density (BMD). The data were analyzed with one-way ANOVA. The average pore diameter of the SIS sponge was (194.90±30.39) μm, the porosity was 92.31%±0.24%, the water absorption rate was 771.50%±40.90%, and the compressive elastic modulus was (2.20±0.19) kPa. There was no significant difference in cell proliferation ability between SIS sponge and control group (P>0.05). Micro-CT quantitative results showed that BV/TV of SIS sponge group (52.81%±3.21%) and positive control group (58.30%±9.36%) were significantly higher than that of control group (38.65%±4.80%) 4 weeks after operation (P < 0.05). The BMD of SIS sponge group [(887.09±61.02) mg/cm3], positive control group [(952.05±132.78) mg/cm3] and control group [(879.29±74.27) mg/cm3] showed no statistical difference 4 weeks after operation (P>0.05). The BV/TV of positive control group (60.57%± 6.56%) was significantly higher than that of SIS sponge group (47.89%±3.59%) and control group (42.99%±2.54%) 12 weeks after operation (P < 0.05). BMD of SIS sponge group [(1047±89.95) mg/cm3] and positive control group [(1101.37±98.85) mg/cm3] were significantly higher than that of control group [(890.36±79.79) mg/cm3] 12 weeks after operation (P < 0.05). The SIS sponge has satisfying physicochemical properties and biocompatibility. The SIS sponge significantly increased bone volume fraction in the early stage of bone formation (4 weeks) and bone mineralized density in the late stage of bone formation (12 weeks).

Alendronate enhances osseointegration in a murine implant model.

Administration of bisphosphonates following total joint arthroplasty might be beneficial to reduce aseptic loosening. However, their effects on peri-implant bone formation and bone-implant interface strength have not been investigated yet. We used a physiologically loaded mouse implant model to investigate the short-term effects of postoperative systemic alendronate on osseointegration. A titanium implant with a rough surface was inserted in the proximal tibiae of 17-week-old female C57BL/6 mice (n = 44). Postimplantation mice were given alendronate (73 μg/kg/days, n = 22) or vehicle (n = 22) 5 days/week. At 7- and 14-daypostimplantation, histology and histomorphometry were conducted. At 28 days,microcomputed tomography and biomechanical testing were performed (n = 10/group). Postoperative alendronate treatment enhanced osseointegration, increasing maximum pullout load by 45% (p < .001) from 19.1 ± 4.5 N in the control mice to 27.6 ± 4.9 N in the treated mice, at day 28 postimplantation. Alendronate treatment increased the bone volume fraction by 139% (p < .001) in the region distal to the implant and 60% (p < .05) in the peri-implant region. At 14-daypostimplantation, alendronate treatment decreased the number of osteoclasts per bone perimeter (p < .05) and increased bone volume fraction (p < .01) when compared with the control group. Postimplantation, short-term alendronate treatment enhanced osseointegration as demonstrated by increased bone mass, trabecular bone thickness, and maximum pullout load. Alendronate decreased peri-implant osteoclasts while preserving peri-implant osteoblasts and endothelial cells, in turn, increasing bone volume fraction. This data supports the postoperative clinical use of bisphosphonates, especially in patients with high risks of aseptic loosening.

Comparative Analysis of Femoral Macro- and Micromorphology in Males and Females With and Without Hyperostosis Frontalis Interna: A Cross-Sectional Cadaveric Study.

We hypothesized that subjects with hyperostosis frontalis interna (HFI), which represents local, endocranial thickening of the frontal bone, would express extra-calvarial manifestations of this condition. Therefore, we compared femoral bone mineral density, geometry, and microarchitecture of males and females with HFI to those without this condition as well as between males and females with HFI. The sample was taken from human donor cadavers, 38 males (19 with and 19 without HFI) and 34 females (17 with and 17 without HFI) that were age-matched within the same sex. The specimens of femoral bones were scanned using microcomputed tomography and dual-energy X-ray absorptiometry (DXA). Parameters of hip structure analysis (HSA) were calculated from data derived from DXA scans. Females with HFI had increased cortical bone volume fraction and their cortical bone was less porous compared to females without HFI. Males with HFI showed microarchitectural differences only with the trabecular bone. They had increased bone volume fraction and decreased trabecular separation compared to males without HFI, although with borderline significance. These microarchitectural changes did not have significant impact on femoral geometry and bone mineral density. The same, still unknown etiological factor behind HFI might be inducing changes at the level of bone microarchitecture at a remote skeletal site (femoral bone), in both sexes. These alterations still do not have the magnitude to induce obvious, straightforward overall increase of bone mineral density measured by DXA. HFI could be a systemic phenomenon that affects both males and females in a similar manner.

Varying development of femoral and tibial subchondral bone tissue and their interaction with articular cartilage during progressing osteoarthritis.

Differences between tibial and femoral joint surfaces and knee compartments concerning coupled bone and cartilage turnover or bone-cartilage cross talk have not been previously examined, although the mechanical and biological interaction of the mineralized subchondral tissues with articular cartilage is of great importance for advancing osteoarthritis. Therefore, with the help of immunohistochemistry and real-time polymerase chain reaction (RT-PCR), human knee joint cartilage tissue was investigated for expression of key molecules of the extracellular matrix and cartilage composition (collagen type I and II, aggrecan) plus proteoglycan content (colorimetric analysis). Furthermore, we correlated the results with 3D microcomputed tomography of the underlying subchondral bone (high-resolution micro-CT system). Measurements were performed in dependence of the anatomical site (femoral vs. tibial, medial and lateral each) to identify regional differences during the osteoarthritic process. From an enduring series of 108 patients undergoing implantation of TKA, 34 osteochondral samples with lesions macroscopically classified as ICRS grade 1b (group A) and 34 samples with ICRS grade 3a/3b lesions (group B) were compared with 21 healthy controls. Concerning 3D analysis, the medial femoral condyle and tibia showed the most significant increase in bone volume fraction and a decrease in the trabecular number in group B frequently accompanied by subchondral bone resorption pits and enchondral ossification. Under physiological conditions, tibia plateaus show lower bone volume fraction than the corresponding femoral site and this difference enlarges with advancing OA. Partially even contradictory behavior was observed such as trabecular separation at the lateral tibial and medial plateau in osteochondral OA samples of the same patients. Collagen type II expression levels show faster and varying changes than type I during the OA process, leading to a lower positive or negative correlation with bone microstructural analysis, especially on the tibia plateau. Structural bone and cartilage parameter changes showed varying developments and correlations among each other in the different compartments of the knee. As a clinical conclusion, therapies to postpone or prevent cartilage degeneration by influencing the loss of mineralized bone could be site dependent.

The Paradox of Fragile but Dense Bones in Type 2 Diabetes

Epidemiologic evidence from the past decade has demonstrated that increased bone fracture risk is one of the myriad clinical complications of type 2 diabetes mellitus (T2DM). Counterintuitively, the increased fracture risk in T2DM occurs despite greater bone mineral density, and persists after accounting for potential confounders like body mass index, neuropathies, and falls. Therefore, T2DM may alter aspects of bone’s intrinsic resistance to fracture beyond bone mineral density, such as material properties or microarchitecture, thereby increasing bone fragility independently of bone mass. Although the mechanisms that underlie bone fragility in T2DM are not yet well established, a multiplicity of factors, including hyperglycemia, altered bone remodeling, and non‐enzymatic collagen crosslinking in the bone matrix are implicated. In this work we elucidate the factors that influence fragility in T2DM by characterizing the biochemical, material, microarchitectural, and mechanical properties of bone from a clinical population of men with and without T2DM. The cancellous bone from patients with T2DM had greater concentrations of the advanced glycation endproduct pentosidine and sugars bound to the collagen matrix, had greater mineral content, and trended toward a greater bone volume fraction than the non‐DM specimens. Surprisingly, the T2DM specimens had similar strength and toughness as those from non‐DM controls. Regression modeling of the mechanical outcomes revealed competing effects of T2DM on bone mechanical behavior. The trend toward greater bone volume fraction and greater mineral content of the T2DM specimens increased strength, while the greater concentrations of pentosidine in the T2DM group increased brittleness. These data suggest that high concentrations of AGEs can increase fragility by reducing the ability of bone to absorb energy prior to failure, especially for the subset of T2DM patients with low bone volume fraction. Further, our model shows that an increase in bone volume fraction offers a protective effect for T2DM patients, even with the inferior intrinsic quality of the T2DM bone, whereas loss of bone volume fraction substantially increases risk of bone fragility for T2DM patients. The primary clinical implications of these findings are that preservation of bone volume fraction is critical in patients with T2DM.Support or Funding InformationSupported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases (K01 AR064314)

Subchondral and Trabecular Bone Respond Differently to Exercise in Juvenile Sheep

Evaluating the responses of subchondral and underlying trabecular bone properties to exercise interventions have implications for understanding normal developmental processes as well as the etiology of diseases affecting these tissues. We hypothesized that exercise would result in (1) increased subchondral and trabecular thickness and density, and increased bone volume fraction, (2) the distribution of increased bone properties would reflect experimentally induced postural differences, and (3) shallower trabecular bone would show a greater response to exercise than deeper trabecular bone. To evaluate these hypotheses we randomly assigned thirty juvenile sheep to flat exercise (n=11), inclined exercise (15% grade; n=11) which used more flexed knee postures, and non‐exercised control (n=8) groups. Exercise was conducted on motorized treadmills twice daily for 20 min at 1.12m/s for 60 days. Distal femora were micro‐CT‐scanned at 50μm resolution. Trabecular thickness, apparent density, bone volume fraction, anisotropy, as well as subchondral thickness and apparent density were measured using custom algorithms in AMIRA, ImageJ and MATLAB. Subchondral thickness showed significant and predictable response to exercise and with approximately 20% thicker bone in both exercise groups compared to controls, and postural changes with increased thickness toward the posterior side of the condyle in the incline group. Subchondral density did not increase substantially with exercise. In contrast trabecular density was increased slightly with exercise (~5%) but the response did not correspond to incline‐exercised and control groups. These results suggest a greater responsiveness to exercise in subchondral than trabecular bone, and imply that increases in stiffness in these tissues may be achieved through modulation of different tissue properties.Support or Funding InformationNational Science Foundation 1638756

Administration of allogeneic mesenchymal stem cells in lengthening phase accelerates early bone consolidation in rat distraction osteogenesis model

BackgroundDistraction osteogenesis (DO) is a surgical technique to promote bone regeneration which may require long duration for bone consolidation. Bone marrow-derived mesenchymal stem cells (MSCs) have been applied to accelerate bone formation in DO. However, the optimal time point for cell therapy in DO remains unknown. This study sought to determine the optimal time point of cell administration to achieve early bone consolidation in DO. We hypothesized that the ratio of circulating MSCs to peripheral mononuclear cells and the level of cytokines in serum might be indicators for cell administration in DO.MethodsUnilateral tibial osteotomy with an external fixator was performed in adult Sprague Dawley rats. Three days after osteotomy, the tibia was lengthened at 0.5 mm/12 h for 5 days. At first, 5 rats were used to analyze the blood components at 6 different time points (3 days before lengthening, on the day lengthening began, or 3, 6, 10, or 14 days after lengthening began) by sorting circulating MSCs and measuring serum levels of stromal cell-derived factor 1 (SDF-1) and interleukin 1β. Then, 40 rats were used for cell therapy study. A single dose of 5 × 105 allogeneic MSCs was locally injected at the lengthening site on day 3, 6, or 10 after lengthening began, or 3 doses of MSCs were injected at the three time points. Sequential X-ray radiographs were taken weekly. Endpoint examinations included micro-computed tomography analysis, mechanical testing, histomorphometry, and histology.ResultsThe number of circulating MSCs and serum level of SDF-1 were significantly increased during lengthening, and then decreased afterwards. Single injection of MSCs during lengthening phase (on day 3, but not day 6 or 10) significantly increased bone volume fraction, mechanical maximum loading, and bone mineralization of the regenerate. Triple injections of MSCs at three time points also significantly increased bone volume and maximum loading of the regenerates.ConclusionThis study demonstrated that bone consolidation could be accelerated by a single injection of MSCs during lengthening when the ratio of peripheral MSCs to mononuclear cells and the serum SDF-1 presented at peak levels concurrently, suggesting that day 3 after lengthening began may be the optimal time point for cell therapy to promote early bone consolidation.