Irisin Modulates Inflammatory, Angiogenic, and Osteogenic Factors during Fracture Healing.

Bone fractures are a widespread clinical event due to accidental falls and trauma or bone fragility; they also occur in association with various diseases and are common with aging. In the search for new therapeutic strategies, a crucial link between irisin and bone fractures has recently emerged. To explore this issue, we subjected 8-week-old C57BL/6 male mice to tibial fracture, and then we treated them with intra-peritoneal injection of r-Irisin (100 µg/kg/weekly) or vehicle as control. At day 10 post fracture, histological analysis showed a significant reduced expression of inflammatory cytokines as tumor necrosis factor-alpha (TNFα) (p = 0.004) and macrophage inflammatory protein-alpha (MIP-1α) (p = 0.015) in the cartilaginous callus of irisin-treated mice compared to controls, supporting irisin's anti-inflammatory role. We also found increased expressions of the pro-angiogenic molecule vascular endothelial growth factor (VEGF) (p = 0.002) and the metalloproteinase MMP-13 (p = 0.0006) in the irisin-treated mice compared to the vehicle ones, suggesting a myokine involvement in angiogenesis and cartilage matrix degradation processes. Moreover, the bone morphogenetic protein (BMP2) expression was also upregulated (p = 0.002). Taken together, our findings suggest that irisin can contribute to fracture repair by reducing inflammation and promoting vessel invasion, matrix degradation, and bone formation, supporting its possible role as a novel molecule for fracture treatment.

Matrix/mineral ratio and domain size variation with bone tissue age: A photothermal infrared study.

Atomic force microscopy-infrared spectroscopy (AFM-IR) and optical photothermal infrared spectroscopy (O-PTIR), which feature spectroscopic imaging spatial resolution down to âˆ¼ 50 nm and âˆ¼ 500 nm, respectively, were employed to characterize the nano- to microscale chemical compositional changes in bone. Since these changes are known to be age dependent, fluorescently labelled bone samples were employed. The average matrix/mineral ratio values decrease as the bone tissue matures as measured by both AFM-IR and O-PTIR, which agrees with previously published FTIR and Raman spectroscopy results. IR ratio maps obtained by AFM-IR reveal variation in matrix/mineral ratio-generating micron-scale bands running parallel to the bone surface as well as smaller domains within these bands ranging from âˆ¼ 50 to 700 nm in size, which is consistent with the previously published length scale of nanomechanical heterogeneity. The matrix/mineral changes do not exhibit a smooth gradient with tissue age. Rather, the matrix/mineral transition occurs sharply within the length scale of 100-200 nm. O-PTIR also reveals matrix/mineral band domains running parallel to the bone surface, resulting in waves of matrix/mineral ratios progressing from the youngest to most mature tissue. Both AFM-IR and O-PTIR show a greater variation in matrix/mineral ratio value for younger tissue as compared to older tissue. Together, this data confirms O-PTIR and AFM-IR as techniques that visualize bulk spectroscopic data consistent with higher-order imaging techniques such as Raman and FTIR, while revealing novel insight into how mineralization patterns vary as bone tissue ages.

Tibial nerve stimulation increases vaginal blood perfusion and bone mineral density and yield load in ovariectomized rat menopause model.

INTRODUCTION AND HYPOTHESIS: Human menopause transition and post-menopausal syndrome, driven by reduced ovarian activity and estrogen levels, are associated with an increased risk for symptoms including but not limited to sexual dysfunction, metabolic disease, and osteoporosis. Current treatments are limited in efficacy and may have adverse consequences, so investigation for additional treatment options is necessary. Previous studies have demonstrated that percutaneous tibial nerve stimulation (PTNS) and electro-acupuncture near the tibial nerve are minimally invasive treatments that increase vaginal blood perfusion or serum estrogen in the rat model. We hypothesized that PTNS would protect against harmful reproductive and systemic changes associated with menopause. METHODS: We examined the effects of twice-weekly PTNS (0.2 ms pulse width, 20 Hz, 2× motor threshold) under ketamine-xylazine anesthesia in ovariectomized (OVX) female Sprague-Dawley rats on menopause-associated physiological parameters including serum estradiol, body weight, blood glucose, bone health, and vaginal blood perfusion. Rats were split into three groups (n = 10 per group): (1) intact control (no stimulation), (2) OVX control (no stimulation), and (3) OVX stimulation (treatment group). RESULTS: PTNS did not affect serum estradiol levels, body weight, or blood glucose. PTNS transiently increased vaginal blood perfusion during stimulation for up to 5 weeks after OVX and increased areal bone mineral density and yield load of the right femur (side of stimulation) compared to the unstimulated OVX control. CONCLUSIONS: PTNS may ameliorate some symptoms associated with menopause. Additional studies to elucidate the full potential of PTNS on menopause-associated symptoms under different experimental conditions are warranted.

Position-Specific Physical Workload Intensities in American Collegiate Football Training.

ABSTRACT: MamonJr, MA, Olthof, SBH, Burns, GT, Lepley, AS, Kozloff, KM, and Zernicke, RF. Position-specific physical workload intensities in American collegiate football training. J Strength Cond Res 36(2): 420-426, 2022-Quantifying player training loads allows football coaching staff to make informed adjustments to the volume and intensity of training. Physical workload intensity in American football practices have not been extensively quantified. The current study examined physical workload intensities across positions in American collegiate football during training. Data from player tracking technology (Catapult Vector) were collected from 72 American football players (National Collegiate Athletic Association Division I) during in-season practices. Players were involved in individualized skill (indy), team playbook (team), and special team (ST) drills during practice and analyzed for their specialist offensive or defensive role (e.g., linebacker or wide receiver). Player running (i.e., high-speed running and sprint) and accelerations (i.e., high-intensity PlayerLoad and high-intensity inertial movement analysis) per minute were of interest. Drill type and practice day had significant effects on all workload intensity metrics (p < 0.01), but not position. Greater running intensities were seen in ST drills compared with other drill types. Tuesday practice sessions had greater overall intensities compared with other days. Interaction effect of position and drill type was significant (p < 0.001) for all intensity metrics, indicating that position groups exhibited unique workload responses to the drill types. Drill type and practice day interaction effect was significant for all intensity metrics (p < 0.01). The findings may be informative for coaches to tailor physical workloads of practice drills for positional roles in preparation for games and practices. Player tracking technology can add value for strength and conditioning coaches to adjust training programs based on position-specific on-field demands of players.

Systemic Administration of Recombinant Irisin Accelerates Fracture Healing in Mice.

To date, pharmacological strategies designed to accelerate bone fracture healing are lacking. We subjected 8-week-old C57BL/6 male mice to closed, transverse, mid-diaphyseal tibial fractures and treated them with intraperitoneal injection of a vehicle or r-irisin (100 µg/kg/weekly) immediately following fracture for 10 days or 28 days. Histological analysis of the cartilaginous callus at 10 days showed a threefold increase in Collagen Type X (p = 0.0012) and a reduced content of proteoglycans (40%; p = 0.0018). Osteoclast count within the callus showed a 2.4-fold increase compared with untreated mice (p = 0.026), indicating a more advanced stage of endochondral ossification of the callus during the early stage of fracture repair. Further evidence that irisin induced the transition of cartilage callus into bony callus was provided by a twofold reduction in the expression of SOX9 (p = 0.0058) and a 2.2-fold increase in RUNX2 (p = 0.0137). Twenty-eight days post-fracture, microCT analyses showed that total callus volume and bone volume were increased by 68% (p = 0.0003) and 67% (p = 0.0093), respectively, and bone mineral content was 74% higher (p = 0.0012) in irisin-treated mice than in controls. Our findings suggest that irisin promotes bone formation in the bony callus and accelerates the fracture repair process, suggesting a possible use as a novel pharmacologic modulator of fracture healing.

LINE-1 Mediated Insertion into Poc1a (Protein of Centriole 1 A) Causes Growth Insufficiency and Male Infertility in Mice.

Skeletal dysplasias are a common, genetically heterogeneous cause of short stature that can result from disruptions in many cellular processes. We report the identification of the lesion responsible for skeletal dysplasia and male infertility in the spontaneous, recessive mouse mutant chagun. We determined that Poc1a, encoding protein of the centriole 1a, is disrupted by the insertion of a processed Cenpw cDNA, which is flanked by target site duplications, suggestive of a LINE-1 retrotransposon-mediated event. Mutant fibroblasts have impaired cilia formation and multipolar spindles. Male infertility is caused by defective spermatogenesis early in meiosis and progressive germ cell loss. Spermatogonial stem cell transplantation studies revealed that Poc1a is essential for normal function of both Sertoli cells and germ cells. The proliferative zone of the growth plate is small and disorganized because chondrocytes fail to re-align after cell division and undergo increased apoptosis. Poc1a and several other genes associated with centrosome function can affect the skeleton and lead to skeletal dysplasias and primordial dwarfisms. This mouse mutant reveals how centrosome dysfunction contributes to defects in skeletal growth and male infertility.

Altered cytoskeletal organization characterized lethal but not surviving Brtl+/- mice: insight on phenotypic variability in osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a heritable bone disease with dominant and recessive transmission. It is characterized by a wide spectrum of clinical outcomes ranging from very mild to lethal in the perinatal period. The intra- and inter-familiar OI phenotypic variability in the presence of an identical molecular defect is still puzzling to the research field. We used the OI murine model Brtl(+/-) to investigate the molecular basis of OI phenotypic variability. Brtl(+/-) resembles classical dominant OI and shows either a moderately severe or a lethal outcome associated with the same Gly349Cys substitution in the α1 chain of type I collagen. A systems biology approach was used. We took advantage of proteomic pathway analysis to functionally link proteins differentially expressed in bone and skin of Brtl(+/-) mice with different outcomes to define possible phenotype modulators. The skin/bone and bone/skin hybrid networks highlighted three focal proteins: vimentin, stathmin and cofilin-1, belonging to or involved in cytoskeletal organization. Abnormal cytoskeleton was indeed demonstrated by immunohistochemistry to occur only in tissues from Brtl(+/-) lethal mice. The aberrant cytoskeleton affected osteoblast proliferation, collagen deposition, integrin and TGF-ß signaling with impairment of bone structural properties. Finally, aberrant cytoskeletal assembly was detected in fibroblasts obtained from lethal, but not from non-lethal, OI patients carrying an identical glycine substitution. Our data demonstrated that compromised cytoskeletal assembly impaired both cell signaling and cellular trafficking in mutant lethal mice, altering bone properties. These results point to the cytoskeleton as a phenotypic modulator and potential novel target for OI treatment.

Abnormal type I collagen post-translational modification and crosslinking in a cyclophilin B KO mouse model of recessive osteogenesis imperfecta.

Cyclophilin B (CyPB), encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib-/- mice that recapitulate the OI phenotype. Knock-out (KO) mice are small, with reduced femoral areal bone mineral density (aBMD), bone volume per total volume (BV/TV) and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2-11%) collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity. Ppib-/- fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS) analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered crosslink pattern was associated with decreased collagen deposition into matrix in culture, altered fibril structure in tissue, and reduced bone strength. These studies demonstrate novel consequences of the indirect regulatory effect of CyPB on collagen hydroxylation, impacting collagen glycosylation, crosslinking and fibrillogenesis, which contribute to maintaining bone mechanical properties.

A novel loss-of-function mutation in Npr2 clarifies primary role in female reproduction and reveals a potential therapy for acromesomelic dysplasia, Maroteaux type.

We discovered a new spontaneous mutant allele of Npr2 named peewee (pwe) that exhibits severe disproportionate dwarfism and female infertility. The pwe phenotype is caused by a four base-pair deletion in exon 3 that generates a premature stop codon at codon 313 (L313X). The Npr2(pwe/pwe) mouse is a model for the human skeletal dysplasia acromesomelic dysplasia, Maroteaux type (AMDM). We conducted a thorough analysis of the female reproductive tract and report that the primary cause of Npr2(pwe/pwe) female infertility is premature oocyte meiotic resumption, while the pituitary and uterus appear to be normal. Npr2 is expressed in chondrocytes and osteoblasts. We determined that the loss of Npr2 causes a reduction in the hypertrophic and proliferative zones of the growth plate, but mineralization of skeletal elements is normal. Mutant tibiae have increased levels of the activated form of ERK1/2, consistent with the idea that natriuretic peptide receptor type 2 (NPR2) signaling inhibits the activation of the MEK/ERK mitogen activated protein kinase pathway. Treatment of fetal tibiae explants with mitogen activated protein kinase 1 and 2 inhibitors U0126 and PD325901 rescues the Npr2(pwe/pwe) growth defect, providing a promising foundation for skeletal dysplasia therapeutics.

Dissection of complex adult traits in a mouse synthetic population.

Finding the causative genetic variations that underlie complex adult traits is a significant experimental challenge. The unbiased search strategy of genome-wide association (GWAS) has been used extensively in recent human population studies. These efforts, however, typically find only a minor fraction of the genetic loci that are predicted to affect variation. As an experimental model for the analysis of adult polygenic traits, we measured a mouse population for multiple phenotypes and conducted a genome-wide search for effector loci. Complex adult phenotypes, related to body size and bone structure, were measured as component phenotypes, and each subphenotype was associated with a genomic spectrum of candidate effector loci. The strategy successfully detected several loci for the phenotypes, at genome-wide significance, using a single, modest-sized population (N = 505). The effector loci each explain 2%-10% of the measured trait variation and, taken together, the loci can account for over 25% of a trait's total population variation. A replicate population (N = 378) was used to confirm initially observed loci for one trait (femur length), and, when the two groups were merged, the combined population demonstrated increased power to detect loci. In contrast to human population studies, our mouse genome-wide searches find loci that individually explain a larger fraction of the observed variation. Also, the additive effects of our detected mouse loci more closely match the predicted genetic component of variation. The genetic loci discovered are logical candidates for components of the genetic networks having evolutionary conservation with human biology.

Characterization of bone microstructure using photoacoustic spectrum analysis.

Osteoporosis is a progressive bone disease that is characterized by a decrease in bone mass and the deterioration in bone microarchitecture. This study investigates the feasibility of characterizing bone microstructure by analyzing the frequency spectrum of the photoacoustic (PA) signal from the bone. Modeling and numerical simulation of PA signal were performed on trabecular bone simulations and CT scans with different trabecular thicknesses. The resulting quasi-linear photoacoustic spectra were fittted by linear regression, from which the spectral parameter slope was quantified. The simulation based on two different models both demonstrate that bone specimens with thinner trabecular thicknesses have higher slope. Experiment on osteoporotic rat femoral heads with different mineral content was conducted. The finding from the experiment was in good agreement with the simulation, demonstrating that the frequency-domain analysis of PA signals can provide an objective assessment of bone microstructure and deterioration. Considering that PA measurement is non-ionizing, non-invasive, and has sufficient penetration in both calcified and non-calcified tissues, this new bone evaluation method based on photoacoustic spectral analysis holds potential for clinical management of osteoporosis and other bone diseases.

Bone assessment via thermal photo-acoustic measurements.

The feasibility of an innovative biomedical diagnostic technique, thermal photo-acoustic (TPA) measurement, for non-ionizing and non-invasive assessment of bone health is investigated. Unlike conventional photo-acoustic PA methods that are mostly focused on the measurement of absolute signal intensity, TPA targets the change in PA signal intensity as a function of the sample temperature, i.e., the temperature-dependent Grueneisen parameter that is closely relevant to the chemical and molecular properties in the sample. Based on the differentiation measurement, the results from TPA technique are less susceptible to the variations associated with sample and system, and could be quantified with improved accurately. Due to the fact that the PA signal intensity from organic components such as blood changes faster than that from non-organic mineral under the same modulation of temperature, TPA measurement is able to objectively evaluate bone mineral density (BMD) and its loss as a result of osteoporosis. In an experiment on well-established rat models of bone loss and preservation, PA measurements of rat tibia bones were conducted over a temperature range from 37°C to 44°C. The slope of PA signal intensity verses temperature was quantified for each specimen. The comparison among three groups of specimens with different BMD shows that bones with lower BMD have higher slopes, demonstrating the potential of the proposed TPA technique in future clinical management of osteoporosis.

Measure bone volume and density changes of sites grafted with bone allografts and a titanium mesh on cone-beam computed tomography: a technique.

PURPOSE: The aim of this study was to introduce a novel method to evaluate volumetric and density changes of the augmented sites. MATERIALS AND METHODS: A ridge augmentation procedure, using particulate bone allografts and a titanium mesh, was performed on the posterior edentulous mandible of 3 participants. Cone-beam computed tomography was taken preoperatively (1st scan), immediately (2nd scan), and 5 months (3rd scan) after the surgery. The grafted area was segmented on the 2nd and 3rd scans, using the 1st scan as the reference. The volume of the grafted area and the newly formed bone-graft complex that is defined by a preselected threshold was then determined. The bone mineral density (BMD) of the grafted area was determined by comparing gray-scale histograms of the grafted area for the 2nd and 3rd scans using the cortical bone adjacent to the grafted area as the reference. RESULTS: The mean volumetric shrinkage was 13.5%. The BMD increase was 4.65%. The mean error in determining cortical bone density between the scans was 1.69%. CONCLUSIONS: A novel technique to measure bone volume and density changes after bone augmentation was described. The low measurement/scanning error suggested that this technique is reliable and reproducible.

Development of a semi-anthropomorphic photoacoustic calcaneus phantom based on nano computed tomography and stereolithography 3D printing.

Osteoporosis is a major public health threat with significant physical, psychosocial, and financial consequences. The calcaneus bone has been used as a measurement site for risk prediction of osteoporosis by noninvasive quantitative ultrasound (QUS). By adding optical contrast to QUS, our previous studies indicate that a combination of photoacoustic (PA) and QUS, that is, PAQUS, provides a novel opportunity to assess the health of human calcaneus. Calibration of the PAQUS system is crucial to realize quantitative and repeatable measurements of the calcaneus. Therefore, a phantom which simulates the optical, ultrasound, and architectural properties of the human calcaneus, for PAQUS system calibration, is required. Additionally, a controllable phantom offers researchers a versatile framework for developing versatile structures, allowing more controlled assessment of how varying bone structures cause defined alterations in PA and QUS signals. In this work, we present the first semi-anthropomorphic calcaneus phantom for PAQUS. The phantom was developed based on nano computed-tomography (nano-CT) and stereolithography 3D printing, aiming to maximize accuracy in the approximation of both trabecular and cortical bone microstructures. Compared with the original digital input calcaneus model from a human cadaveric donor, the printed model achieved accuracies of 71.15% in total structure and 87.21% in bone volume fraction. Inorganic materials including synthetic blood, mineral oil, intralipid, and agar gel were used to model the substitutes of bone marrow and soft tissue, filling and covering the calcaneus phantom. The ultrasound and optical properties of this phantom were measured, and the results were consistent with those measured by a commercialized device and from previous in vivo studies. In addition, a short-term stability test was conducted for this phantom, demonstrating that the optical and ultrasound properties of the phantom were stable without significant variation over 1 month. This semi-anthropomorphic calcaneus phantom shows structural, ultrasound, and optical properties similar to those from a human calcaneus in vivo and, thereby, can serve as an effective source for equipment calibration and the comprehensive study of human patients.

Ultrasound-based jawbone surface quality evaluation after alveolar ridge preservation.

BACKGROUND: Bone readiness for implant placement is typically evaluated by bone quality/density on 2-dimensional radiographs and cone beam computed tomography at an arbitrary time between 3 and 6 months after tooth extraction and alveolar ridge preservation (ARP). The aim of this study is to investigate if high-frequency ultrasound (US) can classify bone readiness in humans, using micro-CT as a reference standard to obtain bone mineral density (BMD) and bone volume fraction (BVTV) of healed sockets receiving ARP in humans. METHODS: A total of 27 bone cores were harvested during the implant surgery from 24 patients who received prior extraction with ARP. US images were taken immediately before the implant surgery at a site co-registered with the tissue biopsy collection location, made possible with a specially designed guide, and then classified into 3 tiers using B-mode image criteria (1) favorable, (2) questionable, and (3) unfavorable. Bone mineral density (hydroxyapatite) and BVTV were obtained from micro-CT as the gold standard. RESULTS: Hydroxyapatite and BVTV were evaluated within the projected US slice plane and thresholded to favorable (>2200 mg/cm3; >0.45 mm3/mm3), questionable (1500-2200 mg/cm3; 0.4-0.45 mm3/mm3), and unfavorable (<1500 mg/cm3; <0.4 mm3/mm3). The present US B-mode classification inversely scales with BMD. Regression analysis showed a significant relation between US classification and BMD as well as BVTV. T-test analysis demonstrated a significant correlation between US reader scores and the gold standard. When comparing Tier 1 with the combination of Tier 2 and 3, US achieved a significant group differentiation relative to mean BMD (p = 0.004, true positive 66.7%, false positive 0%, true negative 100%, false negative 33.3%, specificity 100%, sensitivity 66.7%, receiver operating characteristics area under the curve 0.86). Similar results were found between US-derived tiers and BVTV. CONCLUSION: Preliminary data suggest US could classify jawbone surface quality that correlates with BMD/BVTV and serve as the basis for future development of US-based socket healing evaluation after ARP.

Collagen mutation and age contribute to differential craniofacial phenotypes in mouse models of osteogenesis imperfecta.

Craniofacial and dentoalveolar abnormalities are present in all types of osteogenesis imperfecta (OI). Mouse models of the disorder are critical to understand these abnormalities and underlying OI pathogenesis. Previous studies on severely affected OI mice report a broad spectrum of craniofacial phenotypes, exhibiting some similarities to the human disorder. The Brtl/+ and G610c/+ are moderately severe and mild-type IV OI, respectively. Little is known about the aging effects on the craniofacial bones of these models and their homology to human OI. This study aimed to analyze the Brtl/+ and G610c/+ craniofacial morphometries during aging to establish suitability for further OI craniofacial bone intervention studies. We performed morphological measurements on the micro-CT-scanned heads of 3-wk-old, 3-mo-old, and 6-mo-old female Brtl/+ and G610c/+ mice. We observed that Brtl/+ skulls are shorter in length than WT (P < .05), whereas G610c/+ skulls are similar in length to their WT counterparts. The Brtl/+ mice exhibit alveolar bone with a porotic-like appearance that is not observed in G610c/+. As they age, Brtl/+ mice show severe bone resorption in both the maxilla and mandible (P < .05). By contrast, G610c/+ mice experience mandibular resorption consistently across all ages, but maxillary resorption is only evident at 6 mo (P < .05). Western blot shows high osteoclastic activities in the Brtl/+ maxilla. Both models exhibit delayed pre-functional eruptions of the third molars (P < .05), which are similar to those observed in some bisphosphonate-treated OI subjects. Our study shows that the Brtl/+ and G610c/+ mice display clear features found in type IV OI patients; both show age-related changes in the craniofacial growth phenotype. Therefore, understanding the craniofacial features of these models and how they age will allow us to select the most accurate mouse model, mouse age, and bone structure for the specific craniofacial bone treatment of differing OI groups.

Parathyroid hormone and trabectedin have differing effects on macrophages and stress fracture repair.

Stress fractures occur as a result of repeated mechanical stress on bone and are commonly found in the load-bearing lower extremities. Macrophages are key players in the immune system and play an important role in bone remodeling and fracture healing. However, the role of macrophages in stress fractures has not been adequately addressed. We hypothesize that macrophage infiltration into a stress fracture callus site promotes bone healing. To test this, a unilateral stress fracture induction model was employed in which the murine ulna of four-month-old, C57BL/6 J male mice was repeatedly loaded with a pre-determined force until the bone was displaced a distance below the threshold for complete fracture. Mice were treated daily with parathyroid hormone (PTH, 50 µg/kg/day) starting two days before injury and continued until 24 h before euthanasia either four or six days after injury, or treated with trabectedin (0.15 mg/kg) on the day of stress fracture and euthanized three or seven days after injury. These treatments were used due to their established effects on macrophages. While macrophages have been implicated in the anabolic effects of PTH, trabectedin, an FDA approved chemotherapeutic, compromises macrophage function and reduces bone mass. At three- and four-days post injury, callus macrophage numbers were analyzed histologically. There was a significant increase in macrophages with PTH treatment compared to vehicle in the callus site. By one week of healing, treatments differentially affected the bony callus as analyzed by microcomputed tomography. PTH enhanced callus bone volume. Conversely, callus bone volume was decreased with trabectedin treatment. Interestingly, concurrent treatment with PTH and trabectedin rescued the reduction observed in the callus with trabectedin treatment alone. This study reports on the key involvement of macrophages during stress fracture healing. Given these observed outcomes on macrophage physiology and bone healing, these findings may be important for patients actively receiving either of these FDA-approved therapeutics.

Sclerostin antibody enhances implant osseointegration in bone with Col1a1 mutation.

We evaluated the potential of sclerostin antibody (SclAb) therapy to enhance osseointegration of dental and orthopaedic implants in a mouse model (Brtl/+) mimicking moderate to severe Osteogenesis Imperfecta (OI). To address the challenges in achieving stable implant integration in compromised bone conditions, our aim was to determine the effectiveness of sclerostin antibody (SclAb) at improving bone-to-implant contact and implant fixation strength. Utilizing a combination of micro-computed tomography, mechanical push-in testing, immunohistochemistry, and Western blot analysis, we observed that SclAb treatment significantly enhances bone volume fraction (BV/TV) and bone-implant contact (BIC) in Brtl/+ mice, suggesting a normalization of bone structure toward WT levels. Despite variations in implant survival rates between the maxilla and tibia, SclAb treatment consistently improved implant stability and resistance to mechanical forces, highlighting its potential to overcome the inherent challenges of OI in dental and orthopaedic implant integration. These results suggest that SclAb could be a valuable therapeutic approach for enhancing implant success in compromised bone conditions.

The effects of zoledronic acid in the bone and vasculature support of hematopoietic stem cell niches.

Hematopoietic stem cells (HSC) are maintained in a tightly regulated bone microenvironment constituted by a rich milieu of cells. Bone cells such as osteoblasts are associated with niche maintenance as regulators of the endosteal microenvironment. Bone remodeling also plays a role in HSC mobilization although it is poorly defined. The effects of zoledronic acid (ZA), a potent bisphosphonate that inhibits bone resorption, were investigated on bone marrow cell populations focusing on HSCs, and the endosteal and vascular niches in bone. ZA treatment significantly increased bone volume and HSCs in both young and adult mice (4 week and 4 month old, respectively). ZA increased vessel numbers with no overall change in vascular volume in bones of young and had no effect on vasculature in adult mice. Since both young and adult mice had increased HSCs and bone mass with differing vasculature responses, this suggests that ZA indirectly supports HSCs via the osteoblastic niche and not the vascular niche. Additionally, gene expression in Lin- cells demonstrated increased expression of self-renewal-related genes Bmi1 and Ink4a suggesting a role of ZA in the modulation of cell commitment and differentiation toward a long-term self-renewing cell. Genes that support the osteoblastic niche, BMP2 and BMP6 were also augmented in ZA treated mice. In conclusion, ZA-induced HSC expansion occurs independent of the vascular niche via indirect modulation of the osteoblastic niche.

Bone dysplasia in Hutchinson-Gilford progeria syndrome is associated with dysregulated differentiation and function of bone cell populations.

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder affecting tissues of mesenchymal origin. Most individuals with HGPS harbor a de novo c.1824C > T (p.G608G) mutation in the gene encoding lamin A (LMNA), which activates a cryptic splice donor site resulting in production of the toxic "progerin" protein. Clinical manifestations include growth deficiency, lipodystrophy, sclerotic dermis, cardiovascular defects, and bone dysplasia. Here we utilized the LmnaG609G knock-in (KI) mouse model of HGPS to further define mechanisms of bone loss associated with normal and premature aging disorders. Newborn skeletal staining of KI mice revealed altered rib cage shape and spinal curvature, and delayed calvarial mineralization with increased craniofacial and mandibular cartilage content. MicroCT analysis and mechanical testing of adult femurs indicated increased fragility associated with reduced bone mass, recapitulating the progressive bone deterioration that occurs in HGPS patients. We investigated mechanisms of bone loss in KI mice at the cellular level in bone cell populations. Formation of wild-type and KI osteoclasts from marrow-derived precursors was inhibited by KI osteoblast-conditioned media in vitro, suggesting a secreted factor(s) responsible for decreased osteoclasts on KI trabecular surfaces in vivo. Cultured KI osteoblasts exhibited abnormal differentiation characterized by reduced deposition and mineralization of extracellular matrix with increased lipid accumulation compared to wild-type, providing a mechanism for altered bone formation. Furthermore, quantitative analyses of KI transcripts confirmed upregulation of adipogenic genes both in vitro and in vivo. Thus, osteoblast phenotypic plasticity, inflammation and altered cellular cross-talk contribute to abnormal bone formation in HGPS mice.