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.

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.

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.

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).

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.

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.

Acute Phosphate Restriction Impairs Bone Formation and Increases Marrow Adipose Tissue in Growing Mice.

Phosphate plays a critical role in chondrocyte maturation and skeletal mineralization. Studies examining the consequences of dietary phosphate restriction in growing mice demonstrated not only the development of rickets, but also a dramatic decrease in bone accompanied by increased marrow adipose tissue (MAT). Thus studies were undertaken to determine the effects of dietary phosphate restriction on bone formation and bone marrow stromal cell (BMSC) differentiation. Acute phosphate restriction of 28-day-old mice profoundly inhibited bone formation within 48 hours. It also resulted in increased mRNA expression of the early osteolineage markers Sox9 and Runt-related transcription factor 2 (Runx2), accompanied by decreased expression of the late osteolineage markers Osterix and Osteocalcin in BMSCs and osteoblasts, suggesting that phosphate restriction arrests osteoblast differentiation between Runx2 and Osterix. Increased expression of PPARγ and CEBPα, key regulators of adipogenic differentiation, was observed within 1 week of dietary phosphate restriction and was followed by a 13-fold increase in MAT at 3 weeks of phosphate restriction. In vitro phosphate restriction did not alter BMSC osteogenic or adipogenic colony formation, implicating aberrant paracrine or endocrine signaling in the in vivo phenotype. Because BMP signaling regulates the transition between Runx2 and Osterix, this pathway was interrogated. A dramatic decrease in pSmad1/5/9 immunoreactivity was observed in the osteoblasts of phosphate-restricted mice on day 31 (d31) and d35. This was accompanied by attenuated expression of the BMP target genes Id1, KLF10, and Foxc2, the latter of which promotes osteogenic and angiogenic differentiation while impairing adipogenesis. A decrease in expression of the Notch target gene Hey1, a BMP-regulated gene that governs angiogenesis, was also observed in phosphate-restricted mice, in association with decreased metaphyseal marrow vasculature. Whereas circulating phosphate levels are known to control growth plate maturation and skeletal mineralization, these studies reveal novel consequences of phosphate restriction in the regulation of bone formation and osteoblast differentiation. © 2016 American Society for Bone and Mineral Research.

The effects of PTH, loading and surgical insult on cancellous bone at the bone-implant interface in the rabbit.

Enhancing the quantity and quality of cancellous bone with anabolic pharmacologic agents may lead to more successful outcomes of non-cemented joint replacements. Using a novel rabbit model of cancellous bone loading, we examined two specific questions regarding bone formation at the bone-implant interface: (1) does the administration of intermittent PTH, a potent anabolic agent, and mechanical loading individually and combined enhance the peri-implant cancellous bone volume fraction; and, (2) does surgical trauma enhance the anabolic effect of PTH on peri-implant bone volume fraction. In this model, PTH enhanced peri-implant bone volume fraction by 30% in loaded bone, while mechanical loading alone increased bone volume fraction modestly (+10%). Combined mechanical loading and PTH treatment had no synergistic effect on any cancellous parameters. However, a strong combined effect was found in bone volume fraction with combined surgery and PTH treatment (+34%) compared to intact control limbs. Adaptive changes in the cancellous bone tissue included increased ultimate stress and enhanced remodeling activity. The number of proliferative osteoblasts increased as did their expression of pro-collagen 1 and PTH receptor 1, and the number of TRAP positive osteoclasts also increased. In summary, both loading and intermittent PTH treatment enhanced peri-implant bone volume, and surgery and PTH treatment had a strong combined effect. This finding is of clinical importance since enhancing early osseointegration in the post-surgical period has numerous potential benefits.