How faithfully does intramembranous bone regeneration recapitulate embryonic skeletal development?

Postnatal intramembranous bone regeneration plays an important role during a wide variety of musculoskeletal regeneration processes such as fracture healing, joint replacement and dental implant surgery, distraction osteogenesis, stress fracture healing, and repair of skeletal defects caused by trauma or resection of tumors. The molecular basis of intramembranous bone regeneration has been interrogated using rodent models of most of these conditions. These studies reveal that signaling pathways such as Wnt, TGFß/BMP, FGF, VEGF, and Notch are invoked, reminiscent of embryonic development of membranous bone. Discoveries of several skeletal stem cell/progenitor populations using mouse genetic models also reveal the potential sources of postnatal intramembranous bone regeneration. The purpose of this review is to compare the underlying molecular signals and progenitor cells that characterize embryonic development of membranous bone and postnatal intramembranous bone regeneration.

CHIP regulates skeletal development and postnatal bone growth.

C terminus of Hsc70-interacting protein (CHIP) is a chaperone-dependent and U-box containing E3 ubiquitin ligase. In previous studies, we found that CHIP regulates the stability of multiple tumor necrosis factor receptor-associated factor proteins in bone cells. In Chip global knockout (KO) mice, nuclear factor-κB signaling is activated, osteoclast formation is increased, osteoblast differentiation is inhibited, and bone mass is decreased in postnatal Chip KO mice. To determine the role of Chip in different cell types at different developmental stages, we created Chipflox/flox mice. We then generated Chip conditional KO mice ChipCMV and ChipOsxER and demonstrated defects in skeletal development and postnatal bone growth in Chip conditional KO mice. Our findings indicate that Chip conditional KO mice could serve as a critical reagent for further investigations of functions of CHIP in bone cells and in other cell types.

Biomechanics of Implant Fixation in Osteoporotic Bone.

PURPOSE OF REVIEW: The purpose of this review is to critically evaluate the current literature regarding implant fixation in osteoporotic bone. RECENT FINDINGS: Clinical studies have not only demonstrated the growing prevalence of osteoporosis in patients undergoing total joint replacement (TJR) but may also indicate a significant gap in screening and treatment of this comorbidity. Osteoporosis negatively impacts bone in multiple ways beyond the mere loss of bone mass, including compromising skeletal regenerative capacity, architectural deterioration, and bone matrix quality, all of which could diminish implant fixation. Recent findings both in preclinical animal models and in clinical studies indicate encouraging results for the use of osteoporosis drugs to promote implant fixation. Implant fixation in osteoporotic bone presents an increasing clinical challenge that may be benefitted by increased screening and usage of osteoporosis drugs.

Loss of Intestinal Alkaline Phosphatase Leads to Distinct Chronic Changes in Bone Phenotype.

BACKGROUND: The gut is becoming increasingly recognized as the source of various systemic diseases, and recently, it has been linked to bone metabolism via the so-called gut-bone axis. The microbiome and gut-derived mediators are thought to impact upon bone metabolism, and administration of probiotics has been shown to have beneficial effects in bone. The gut brush border enzyme intestinal alkaline phosphatase (IAP) plays an important role in controlling calcium absorption, inhibiting lipopolysaccharides, and other inflammatory mediators responsible for endotoxemia and appears to preserve the normal gut microbiota. Interestingly, IAP-deficient mice (AKP3-/-) also display a significant decrease in fecal Lactobacillus, the genus shown to be beneficial to bone. MATERIALS AND METHODS: IAP mRNA levels in mouse bone were measured using quantitative real-time polymerase chain reaction. Femurs of IAP-knockout (KO) and wild-type (WT) mice were analyzed by microcomputed tomography and histopathology. Serum levels of alkaline phosphatase, calcium, and phosphorus were measured. Target cell response upon exposure to serum from IAP-KO and WT mice was quantified using primary bone marrow macrophages. RESULTS: IAP was not significantly expressed in bones of WT or KO animals. IAP (alkaline phosphatase 3) expression in bone was vanishingly low compared to the duodenum (bone versus duodenum, 56.9 ± 17.7 versus 25,430.3 ± 10,884.5 relative expression, P = 0.01). Bone histology of younger IAP-KO and WT animals was indistinguishable, whereas older IAP-deficient mice showed a distinctly altered phenotype on histology and computed tomography scan. Younger KO mice did not display any abnormal levels in blood chemistry. Older IAP-KO animals showed an isolated increase in serum alkaline phosphatase levels reflecting an environment of active bone formation (IAP-WT versus IAP-KO, 80 ± 27.4 U/I versus 453 ± 107.5 U/I, P = 0.004). There was no significant difference in serum calcium or phosphorus levels between KO and WT mice. Serum from IAP-KO mice induced a significantly higher inflammatory target cell response. CONCLUSIONS: Through its multiple functions, IAP seems to play a crucial role in connecting the gut to the bone. IAP deficiency leads to chronic changes in bone formation, most likely through dysbiosis and systemic dissemination of proinflammatory mediators.

In vivo cyclic compression causes cartilage degeneration and subchondral bone changes in mouse tibiae.

OBJECTIVE: Alterations in the mechanical loading environment in joints may have both beneficial and detrimental effects on articular cartilage and subchondral bone, and may subsequently influence the development of osteoarthritis (OA). Using an in vivo tibial loading model, the aim of this study was to investigate the adaptive responses of cartilage and bone to mechanical loading and to assess the influence of load level and duration. METHODS: Cyclic compression at peak loads of 4.5N and 9.0N was applied to the left tibial knee joint of adult (26-week-old) C57BL/6 male mice for 1, 2, and 6 weeks. Only 9.0N loading was utilized in young (10-week-old) mice. Changes in articular cartilage and subchondral bone were analyzed by histology and micro-computed tomography. RESULTS: Mechanical loading promoted cartilage damage in both age groups of mice, and the severity of joint damage increased with longer duration of loading. Metaphyseal bone mass increased with loading in young mice, but not in adult mice, whereas epiphyseal cancellous bone mass decreased with loading in both young and adult mice. In both age groups, articular cartilage thickness decreased, and subchondral cortical bone thickness increased in the posterior tibial plateau. Mice in both age groups developed periarticular osteophytes at the tibial plateau in response to the 9.0N load, but no osteophyte formation occurred in adult mice subjected to 4.5N peak loading. CONCLUSION: This noninvasive loading model permits dissection of temporal and topographic changes in cartilage and bone and will enable investigation of the efficacy of treatment interventions targeting joint biomechanics or biologic events that promote OA onset and progression.

Skeletal consequences of preterm birth in pigs as a model for preterm infants.

Preterm birth affects about 10% of all live births with many resultant health challenges, including metabolic bone disease of prematurity (MBDP), which is characterized by elevated alkaline phosphatase, suppressed phosphate, and deficient skeletal development. Because of the lack of an animal model, very little is known about bone structure, strength, and quality after preterm birth. This study investigated the utility of a pig model to replicate clinical features of preterm birth, including MBDP, and sought to determine if early postnatal administration of IGF-1 was an effective treatment. Preterm pigs, born by caesarean section at 90% gestation, were reared in intensive care facilities (respiratory, thermoregulatory, and nutritional support) and compared with sow-reared term pigs born vaginally. Preterm pigs were systemically treated with vehicle or IGF-1 (recombinant human IGF-1/BP-3, 2.25 mg/kg/d). Tissues were collected at postnatal days 1, 5, and 19 (the normal weaning period in pigs). Most bone-related outcomes were affected by preterm birth throughout the study period, whereas IGF-1 supplementation had almost no effect. By day 19, alkaline phosphatase was elevated, phosphate and calcium were reduced, and the bone resorption marker C-terminal crosslinks of type I collagen was elevated in preterm pigs compared to term pigs. Preterm pigs also had decrements in femoral cortical cross-sectional properties, consistent with reduced whole-bone strength. Thus, the preterm pig model replicates many features of preterm bone development in infants, including features of MBDP, and allows for direct interrogation of skeletal tissues, enhancing the field's ability to examine underlying mechanisms.

Premature birth interrupts a critical period of skeletal development as the majority of fetal bone mineral accumulation occurs during the last gestational trimester, leaving preterm infants at increased risk for low bone mineral density and fractures. Although there are some data on growth in bone mass in preterm infants, very little is known about bone structural properties, quality, and strength during development after preterm birth. In this study, we sought to evaluate the pig as a model for postnatal skeletal development after premature birth. Preterm pigs born after approximately 90% of the full gestation period were compared to full-term control pigs through day 19 of life. Levels of 2 blood markers used to diagnose osteoporosis of prematurity were replicated in the pig model. Bone properties related to strength were reduced even when accounting for their smaller body size, possibly suggesting elevated fracture risk in preterm infants. Based on the similarities between the preterm pig model and preterm human infants, the pig model may prove to be useful to study factors and interventions affecting postnatal bone development after preterm birth.

Cells transiently expressing periostin are required for intramedullary intramembranous bone regeneration.

Intramembranous bone regeneration plays an important role in fixation of intramedullary implants used in joint replacement and dental implants used in tooth replacement. Despite widespread recognition of the importance of intramembranous bone regeneration in these clinical procedures, the underlying mechanisms have not been well explored. A previous study that examined transcriptomic profiles of regenerating bone from the marrow space showed that increased periostin gene expression preceded increases in several osteogenic genes. We therefore sought to determine the role of cells transiently expressing periostin in intramedullary intramembranous bone regeneration. We used a genetic mouse model that allows tamoxifen-inducible fluorescent labeling of periostin expressing cells. These mice underwent ablation of the bone marrow cavity through surgical disruption, a well-established intramembranous bone regeneration model. We found that in intact bones, fluorescently labeled cells were largely restricted to the periosteal surface of cortical bone and were absent in bone marrow. However, following surgical disruption of the bone marrow cavity, cells transiently expressing periostin were found within the regenerating tissue of the bone marrow compartment even though the cortical bone remained intact. The source of these cells is likely heterogenous, including cells occupying the periosteal surface as well as pericytes and endothelial cells within the marrow cavity. We also found that diphtheria toxin-mediated depletion of cells transiently expressing periostin at the time of surgery impaired intramembranous bone regeneration in mice. These data suggest a critical role of periostin expressing cells in intramedullary intramembranous bone regeneration and may lead to novel therapeutic interventions to accelerate or enhance implant fixation.

Acute systemic macrophage depletion in osteoarthritic mice alleviates pain-related behaviors and does not affect joint damage.

Background: Osteoarthritis (OA) is a painful degenerative joint disease and a leading source of years lived with disability globally due to inadequate treatment options. Neuroimmune interactions reportedly contribute to OA pain pathogenesis. Notably, in rodents, macrophages in the DRG are associated with onset of persistent OA pain. Our objective was to determine the effects of acute systemic macrophage depletion on pain-related behaviors and joint damage using surgical mouse models in both sexes. Methods: We depleted CSF1R+ macrophages by treating male macrophage Fas-induced apoptosis (MaFIA) transgenic mice 8- or 16-weeks post destabilization of the medial meniscus (DMM) with AP20187 or vehicle control (10 mg/kg i.p., 1x/day for 5 days), or treating female MaFIA mice 12 weeks post partial meniscectomy (PMX) with AP20187 or vehicle control. We measured pain-related behaviors 1-3 days before and after depletion, and, 3-4 days after the last injection we examined joint histopathology and performed flow cytometry of the dorsal root ganglia (DRGs). In a separate cohort of male 8-week DMM mice or age-matched naïve vehicle controls, we conducted DRG bulk RNA-sequencing analyses after the 5-day vehicle or AP20187 treatment. Results: Eight- and 16-weeks post DMM in male mice, AP20187-induced macrophage depletion resulted in attenuated mechanical allodynia and knee hyperalgesia. Female mice showed alleviation of mechanical allodynia, knee hyperalgesia, and weight bearing deficits after macrophage depletion at 12 weeks post PMX. Macrophage depletion did not affect the degree of cartilage degeneration, osteophyte width, or synovitis in either sex. Flow cytometry of the DRG revealed that macrophages and neutrophils were reduced after AP20187 treatment. In addition, in the DRG, only MHCII+ M1-like macrophages were significantly decreased, while CD163+MHCII- M2-like macrophages were not affected in both sexes. DRG bulk RNA-seq revealed that Cxcl10 and Il1b were upregulated with DMM surgery compared to naïve mice, and downregulated in DMM after acute macrophage depletion. Conclusions: Acute systemic macrophage depletion reduced the levels of pro-inflammatory macrophages in the DRG and alleviated pain-related behaviors in established surgically induced OA in mice of both sexes, without affecting joint damage. Overall, these studies provide insight into immune cell regulation in the DRG during OA.

Clearing-enabled light sheet microscopy as a novel method for three-dimensional mapping of the sensory innervation of the mouse knee.

A major barrier that hampers our understanding of the precise anatomic distribution of pain sensing nerves in and around the joint is the limited view obtained from traditional two dimensional (D) histological approaches. Therefore, our objective was to develop a workflow that allows examination of the innervation of the intact mouse knee joint in 3D by employing clearing-enabled light sheet microscopy. We first surveyed existing clearing protocols (SUMIC, PEGASOS, and DISCO) to determine their ability to clear the whole mouse knee joint, and discovered that a DISCO protocol provided the most optimal transparency for light sheet microscopy imaging. We then modified the DISCO protocol to enhance binding and penetration of antibodies used for labeling nerves. Using the pan-neuronal PGP9.5 antibody, our protocol allowed 3D visualization of innervation in and around the mouse knee joint. We then implemented the workflow in mice intra-articularly injected with nerve growth factor (NGF) to determine whether changes in the nerve density can be observed. Both 3D and 2D analytical approaches of the light sheet microscopy images demonstrated quantifiable changes in midjoint nerve density following 4 weeks of NGF injection in the medial but not in the lateral joint compartment. We provide, for the first time, a comprehensive workflow that allows detailed and quantifiable examination of mouse knee joint innervation in 3D.

Zucker Diabetic-Sprague Dawley Rats Have Impaired Peri-Implant Bone Formation, Matrix Composition, and Implant Fixation Strength.

An increasing number of patients with type 2 diabetes (T2DM) will require total joint replacement (TJR) in the next decade. T2DM patients are at increased risk for TJR failure, but the mechanisms are not well understood. The current study used the Zucker Diabetic-Sprague Dawley (ZDSD) rat model of T2DM with Sprague Dawley (SPD) controls to investigate the effects of intramedullary implant placement on osseointegration, peri-implant bone structure and matrix composition, and fixation strength at 2 and 10 weeks post-implant placement. Postoperative inflammation was assessed with circulating MCP-1 and IL-10 2 days post-implant placement. In addition to comparing the two groups, stepwise linear regression modeling was performed to determine the relative contribution of glucose, cytokines, bone formation, bone structure, and bone matrix composition on osseointegration and implant fixation strength. ZDSD rats had decreased peri-implant bone formation and reduced trabecular bone volume per total volume compared with SPD controls. The osseointegrated bone matrix of ZDSD rats had decreased mineral-to-matrix and increased crystallinity compared with SPD controls. Osseointegrated bone volume per total volume was not different between the groups, whereas implant fixation was significantly decreased in ZDSD at 2 weeks but not at 10 weeks. A combination of trabecular mineral apposition rate and postoperative MCP-1 levels explained 55.6% of the variance in osseointegration, whereas cortical thickness, osseointegration mineral apposition rate, and matrix compositional parameters explained 69.2% of the variance in implant fixation strength. The results support the growing recognition that both peri-implant structure and matrix composition affect implant fixation and suggest that postoperative inflammation may contribute to poor outcomes after TJR surgeries in T2DM patients. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Colon epithelial cell-specific Bmal1 deletion impairs bone formation in mice.

The circadian clock system regulates multiple metabolic processes, including bone metabolism. Previous studies have demonstrated that both central and peripheral circadian signaling regulate skeletal growth and homeostasis in mice. Disruption in central circadian rhythms has been associated with a decline in bone mineral density in humans and the global and osteoblast-specific disruption of clock genes in bone tissue leads to lower bone mass in mice. Gut physiology is highly sensitive to circadian disruption. Since the gut is also known to affect bone remodeling, we sought to test the hypothesis that circadian signaling disruption in colon epithelial cells affects bone. We therefore assessed structural, functional, and cellular properties of bone in 8 week old Ts4-Cre and Ts4-Cre;Bmal1fl/fl (cBmalKO) mice, where the clock gene Bmal1 is deleted in colon epithelial cells. Axial and appendicular trabecular bone volume was significantly lower in cBmalKO compared to Ts4-Cre 8-week old mice in a sex-dependent fashion, with male but not female mice showing the phenotype. Similarly, the whole bone mechanical properties were deteriorated in cBmalKO male mice. The tissue level mechanisms involved suppressed bone formation with normal resorption, as evidenced by serum markers and dynamic histomorphometry. Our studies demonstrate that colon epithelial cell-specific deletion of Bmal1 leads to failure to acquire trabecular and cortical bone in male mice.

Activation of canonical Wnt signaling accelerates intramembranous bone regeneration in male mice.

Canonical Wnt signaling plays an important role in skeletal development, homeostasis, and both endochondral and intramembranous repair. While studies have demonstrated that the inhibition of Wnt signaling impairs intramembranous bone regeneration, how its activation affects intramembranous bone regeneration has been underexplored. Therefore, we sought to determine the effects of activation of canonical Wnt signaling on intramembranous bone regeneration by using the well-established marrow ablation model. We hypothesized that mice with a mutation in the Wnt ligand coreceptor gene Lrp5 would have accelerated intramembranous bone regeneration. Male and female wild-type and Lrp5-mutant mice underwent unilateral femoral bone marrow ablation surgery in the right femur at 4 weeks of age. Both the left intact and right operated femurs were assessed at Days 3, 5, 7, 10, and 14. The intact femur of Lrp5 mutant mice of both sexes had higher bone mass than wild-type littermates, although to a greater degree in males than females. Overall, the regenerated bone volume in Lrp5 mutant male mice was 1.8-fold higher than that of littermate controls, whereas no changes were observed between female Lrp5 mutant and littermate control mice. In addition, the rate of intramembranous bone regeneration (from Day 3 to Day 7) was higher in Lrp5 mutant male mice compared to their same-sex littermate controls with no difference in the females. Thus, activation of canonical Wnt signaling increases bone mass in intact bones of both sexes, but accelerates intramembranous bone regeneration following an injury challenge only in male mice.

Sclerostin antibody improves phosphate metabolism hormones, bone formation rates, and bone mass in adult Hyp mice.

X-linked hypophosphatemia (XLH) is caused by a loss-of-function mutation in the phosphate regulating gene with homology to endopeptidase located on the X chromosome (PHEX). Loss of functional PHEX results in elevated fibroblast growth factor 23 (FGF23), impaired phosphate reabsorption, and inhibited skeletal mineralization. Sclerostin, a protein produced primarily by osteocytes, suppresses bone formation by antagonizing canonical Wnt-signaling and is reported to be elevated in XLH patients. Our previous study reported that a monoclonal antibody to sclerostin (Scl-Ab) decreases FGF23 and increases phosphate and bone mass in growing Hyp mice (XLH murine model). In the current study, we investigated the efficacy of Scl-Ab in treating XLH pathophysiology in adult Hyp mice that are past the period of rapid skeletal growth (12 and 20-weeks old). We hypothesized that Scl-Ab would not only increase bone formation, bone strength and bone mass, but would also normalize phosphate regulating hormones, FGF23, parathyroid hormone (PTH), and vitamin 1,25(OH)2D. Scl-Ab treatment increased cortical area, trabecular bone volume fraction, trabecular bone formation rate, and the bending moment in both sexes of both age groups. Scl-Ab treatment suppressed circulating levels of intact FGF23 and c-term FGF23 in treated male and female wild-type and Hyp mice of both age groups and improved both vitamin 1,25(OH)2D and PTH. Scl-Ab treated Hyp mice also showed evidence of increased renal expression of the sodium-phosphate co-transporter, NPT2a, specifically in the female Hyp mice. Our study suggests that Scl-Ab treatment can improve several skeletal and metabolic pathologies associated with XLH, further establishes the role of sclerostin in the regulation of FGF23 and provides evidence that Scl-Ab can improve phosphate regulation by targeting the bone-renal axis.

The relative contribution of bone microarchitecture and matrix composition to implant fixation strength in rats.

Bone microarchitectural parameters significantly contribute to implant fixation strength but the role of bone matrix composition is not well understood. To determine the relative contribution of microarchitecture and bone matrix composition to implant fixation strength, we placed titanium implants in 12-week-old intact Sprague-Dawley rats, ovariectomized-Sprague-Dawley rats, and Zucker diabetic fatty rats. We assessed bone microarchitecture by microcomputed tomography, bone matrix composition by Raman spectroscopy, and implant fixation strength at 2, 6, and 10 weeks postimplantation. A stepwise linear regression model accounted for 83.3% of the variance in implant fixation strength with osteointegration volume/total volume (50.4%), peri-implant trabecular bone volume fraction (14.2%), cortical thickness (9.3%), peri-implant trabecular crystallinity (6.7%), and cortical area (2.8%) as the independent variables. Group comparisons indicated that osseointegration volume/total volume was significantly reduced in the ovariectomy group at Week 2 (~28%) and Week 10 (~21%) as well as in the diabetic group at Week 10 (~34%) as compared with the age matched Sprague-Dawley group. The crystallinity of the trabecular bone was significantly elevated in the ovariectomy group at Week 2 (~4%) but decreased in the diabetic group at Week 10 (~3%) with respect to the Sprague-Dawley group. Our study is the first to show that bone microarchitecture explains most of the variance in implant fixation strength, but that matrix composition is also a contributing factor. Therefore, treatment strategies aimed at improving bone-implant contact and peri-implant bone volume without compromising matrix quality should be prioritized.

Intramembranous bone regeneration in diversity outbred mice is heritable.

There are over one million cases of failed bone repair in the U.S. annually, resulting in substantial patient morbidity and societal costs. Multiple candidate genes affecting bone traits such as bone mineral density have been identified in human subjects and animal models using genome-wide association studies (GWAS). This approach for understanding the genetic factors affecting bone repair is impractical in human subjects but could be performed in a model organism if there is sufficient variability and heritability in the bone regeneration response. Diversity Outbred (DO) mice, which have significant genetic diversity and have been used to examine multiple intact bone traits, would be an excellent possibility. Thus, we sought to evaluate the phenotypic distribution of bone regeneration, sex effects and heritability of intramembranous bone regeneration on day 7 following femoral marrow ablation in 47 12-week old DO mice (23 males, 24 females). Compared to a previous study using 4 inbred mouse strains, we found similar levels of variability in the amount of regenerated bone (coefficient of variation of 86 % v. 88 %) with approximately the same degree of heritability (0.42 v. 0.49). There was a trend toward more bone regeneration in males than females. The amount of regenerated bone was either weakly or not correlated with bone mass at intact sites, suggesting that the genetic factors responsible for bone regeneration and intact bone phenotypes are at least partially independent. In conclusion, we demonstrate that DO mice exhibit variation and heritability of intramembranous bone regeneration that will be suitable for future GWAS.

Alcohol and Circadian Disruption Minimally Impact Bone Properties in Two Cohorts of Male Mice While Between-Cohort Differences Predominate: Association With Season of Birth?

Many lifestyle factors affect bone. Sleep deprivation increases risk for fractures and alcohol consumption can lead to alterations in the skeleton. How combined exposure to these two risk factors affects bone is unclear. Thus, we sought to determine the effects of circadian rhythm disruption and chronic alcohol intake on bone structure and mechanical properties in mice. A total of 120 male C57BL/6J mice were used in two cohorts of 60 mice each because of limited availability of light-tight housing cabinets. One cohort was born in winter and the other in summer. Mice were randomly assigned to circadian disruption (weekly shifting of the light/dark cycle) and control (no shifting) groups beginning at 8 to 12 weeks of age for 12 weeks at which time mice were administered an alcohol-containing or control diet for an additional 10 weeks. Bone structure and mechanical properties of the femur were assessed by micro-computed tomography and three-point bending, respectively. The initial data analysis revealed a likely cohort effect. Thus, we used a three-way analysis of variance to assess the effects of circadian rhythm disruption, alcohol intake, and cohort. Circadian rhythm disruption alone had minimal effects on bone structure and mechanical properties. Alcohol intake reduced body mass and had minimal effects on cortical bone regardless of circadian disruption. Alcohol intake resulted in higher trabecular bone volume, but these beneficial effects were blunted when circadian rhythm was disrupted. Cohort significantly affected body size, many cortical bone structure outcomes, some trabecular bone structure outcomes, and tissue-level material properties. Thus, cohort had the predominant effect on bone structure and mechanical properties in this study, with chronic alcohol intake and environmental circadian disruption having less consistent effects. The data indicate that season of birth may affect skeletal phenotypes and that studies requiring multiple cohorts should determine if a cohort effect exists. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

RNAseq and RNA molecular barcoding reveal differential gene expression in cortical bone following hindlimb unloading in female mice.

Disuse-induced bone loss is seen following spinal cord injury, prolonged bed rest, and exposure to microgravity. We performed whole transcriptomic profiling of cortical bone using RNA sequencing (RNAseq) and RNA molecular barcoding (NanoString) on a hindlimb unloading (HLU) mouse model to identify genes whose mRNA transcript abundances change in response to disuse. Eleven-week old female C57BL/6 mice were exposed to ambulatory loading or HLU for 7 days (n = 8/group). Total RNA from marrow-flushed femoral cortical bone was analyzed on HiSeq and NanoString platforms. The expression of several previously reported genes associated with Wnt signaling and metabolism was altered by HLU. Furthermore, the increased abundance of transcripts, such as Pfkfb3 and Mss51, after HLU imply these genes also have roles in the cortical bone's response to altered mechanical loading. Our study demonstrates that an unbiased approach to assess the whole transcriptomic profile of cortical bone can reveal previously unidentified mechanosensitive genes and may eventually lead to novel targets to prevent disuse-induced osteoporosis.

Phosphate restriction impairs mTORC1 signaling leading to increased bone marrow adipose tissue and decreased bone in growing mice.

Bone marrow stromal cells (BMSCs) are multipotent cells that differentiate into cells of the osteogenic and adipogenic lineage. A striking inverse relationship between bone marrow adipose tissue (BMAT) and bone volume is seen in several conditions, suggesting that differentiation of BMSCs into bone marrow adipocytes diverts cells from the osteogenic lineage, thereby compromising the structural and mechanical properties of bone. Phosphate restriction of growing mice acutely decreases bone formation, blocks osteoblast differentiation and increases BMAT. Studies performed to evaluate the cellular and molecular basis for the effects of acute phosphate restriction demonstrate that it acutely increases 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and inhibits mammalian target of rapamycin complex 1 (mTORC1) signaling in osteoblasts. This is accompanied by decreased expression of Wnt10b in BMSCs. Phosphate restriction also promotes expression of the key adipogenic transcription factors, peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT-enhancer binding protein α (CEBPα), in CXCL12 abundant reticular (CAR) cells, which represent undifferentiated BMSCs and are the main source of BMAT and osteoblasts in the adult murine skeleton. Consistent with this, lineage tracing studies reveal that the BMAT observed in phosphate-restricted mice is of CAR cell origin. To determine whether circumventing the decrease in mTORC1 signaling in maturing osteoblasts attenuates the osteoblast and BMAT phenotype, phosphate-restricted mice with OSX-CreERT2 -mediated haploinsufficiency of the mTORC1 inhibitor, TSC2, were generated. TSC2 haploinsufficiency in preosteoblasts/osteoblasts normalized bone volume and osteoblast number in phosphate-restricted mice and attenuated the increase in BMAT observed. Thus, acute phosphate restriction leads to decreased bone and increases BMAT by impairing mTORC1 signaling in osterix-expressing cells. © 2021 American Society for Bone and Mineral Research (ASBMR).

Implant surface alters compartmental-specific contributions to fixation strength in rats.

Mechanical fixation of the implant to host bone is an important contributor to orthopedic implant survivorship. The relative importance of bone-implant contact, trabecular bone architecture, and cortical bone geometry to implant fixation strength has never been directly tested, especially in the settings of differential implant surface properties. Thus, using a rat model where titanium rods were placed into the intramedullary canal of the distal femur, we determined the relative contribution of bone-implant contact and peri-implant bone architecture to the fixation strength in implants with different surface roughness: highly polished and smooth (as-received) and dual acid-etched (DAE) implants. Using a training set that maximized variance in implant fixation strength, we initially examined correlation between implant fixation strength and outcome parameters from microcomputed tomography and found that osseointegration volume per total volume (OV/TV), trabecular bone volume per total volume (BV/TV), and cortical thickness (Ct.Th) were the single best compartment-specific predictors of fixation strength. We defined separate regression models to predict implant fixation strength for as-received and DAE implants. When the training set models were applied to independent validation sets, we found strong correlations between predicted and experimentally measured implant fixation strength, with r2 = .843 in as received and r2 = .825 in DAE implants. Interestingly, for as-received implants, OV/TV explained more of the total variance in implant fixation strength than the other variables, whereas in DAE implants, Ct.Th had the most explanatory power, suggesting that surface topography of implants affects which bone compartment is most important in providing implant fixation strength.

Dose-dependent skeletal deficits due to varied reductions in mechanical loading in rats.

Reduced skeletal loading leads to marked bone loss. Animal models of hindlimb suspension are widely used to assess alterations in skeleton during the course of complete unloading. More recently, the effects of partial unloading on the musculoskeletal system have been interrogated in mice and rats, revealing dose-dependent effects of partial weight bearing (PWB) on the skeleton and skeletal muscle. Here, we extended these studies to determine the structural and functional skeletal alterations in 14-week-old male Wister rats exposed to 20%, 40%, 70%, or 100% of body weight for 1, 2, or 4 weeks (n = 11-12/group). Using in vivo pQCT, we found that trabecular bone density at the proximal tibia declined in proportion to the degree of unloading and continued progressively with time, without evidence of a plateau by 4 weeks. Ex vivo measurements of trabecular microarchitecture in the distal femur by microcomputed tomography revealed deficits in bone volume fraction, 2 and 4 weeks after unloading. Histologic analyses of trabecular bone in the distal femur revealed the decreased osteoblast number and mineralizing surface in unloaded rats. Three-point bending of the femoral diaphysis indicated modest or no reductions in femoral stiffness and estimated modulus due to PWB. Our results suggest that this rat model of PWB leads to trabecular bone deterioration that is progressive and generally proportional to the degree of PWB, with minimal effects on cortical bone.