Obesity impairs revascularization and bone healing in a mouse model of osteonecrosis.

Osteonecrosis of the femoral head (ONFH) is a devastating bone disease that is caused by a disruption of blood supply leading to necrotic cell death. Clinically, it was found that obesity has a high prevalence with ONFH. However, it remains unclear how obesity may directly affect tissue regeneration and bone healing in osteonecrosis (ON). The purpose of this study is to investigate the effects of obesity and weight loss (WL) on ON healing. In this study, we induced obesity and WL in an established surgery-induced ON mouse model via feeding a high-fat diet (HFD) and altering the diet respectively. All mice received a surgical induction of ON of distal femoral epiphysis at the age of 12 weeks. HFD was switched to normal diet (ND) after ON surgery to induce WL. Mouse body weight was recorded weekly. Mouse body composition was scanned by DEXA (Dual-energy X-ray absorptiometry) right after sacrifice at the age of 16 weeks. The distal femoral bone samples were fixed and embedded for histology such as H&E, immunohistochemistry, and TRAP staining. In this study, we found that HFD-induced obesity impaired revascularization and bone remodeling showing decreased vessel areas and reduced osteoblast and osteoclast numbers. WL could rescue obesity-induced bone healing defects. Our study is the first to test the direct effects of obesity and WL on ON bone healing. We believe our work may provide new concepts for osteonecrosis treatment in obese patients.

Local BMP2 hydrogel therapy for robust bone regeneration in a porcine model of Legg-Calvé-Perthes disease.

Legg-Calvé-Perthes disease is juvenile idiopathic osteonecrosis of the femoral head (ONFH) that has no effective clinical treatment. Previously, local injection of bone morphogenetic protein-2 (BMP2) for ONFH treatment showed a heterogeneous bone repair and a high incidence of heterotopic ossification (HO) due to the BMP2 leakage. Here, we developed a BMP2-hydrogel treatment via a transphyseal bone wash and subsequential injection of BMP2-loaded hydrogel. In vitro studies showed that a hydrogel of gelatin-heparin-tyramine retained the BMP2 for four weeks. The injection of the hydrogel can efficiently prevent leakage. With the bone wash, the injected hydrogel had a broad distribution in the head. In vivo studies on pigs revealed that the BMP2-hydrogel treatment produced a homogeneous bone regeneration without HO. It preserved the subchondral contour and restored the subchondral endochondral ossification, although it increased growth plate fusions. In summary, the study demonstrated a promising BMP2-hydrogel treatment for ONFH treatment, especially for teenagers.

Glutathione limits RUNX2 oxidation and degradation to regulate bone formation.

Reactive oxygen species (ROS) are natural products of mitochondrial oxidative metabolism and oxidative protein folding. ROS levels must be well controlled, since elevated ROS has been shown to have deleterious effects on osteoblasts. Moreover, excessive ROS is thought to underlie many of the skeletal phenotypes associated with aging and sex steroid deficiency in mice and humans. The mechanisms by which osteoblasts regulate ROS and how ROS inhibits osteoblasts are not well understood. Here, we demonstrate that de novo glutathione (GSH) biosynthesis is essential in neutralizing ROS and establish a proosteogenic reduction and oxidation reaction (REDOX) environment. Using a multifaceted approach, we demonstrate that reducing GSH biosynthesis led to acute degradation of RUNX2, impaired osteoblast differentiation, and reduced bone formation. Conversely, reducing ROS using catalase enhanced RUNX2 stability and promoted osteoblast differentiation and bone formation when GSH biosynthesis was limited. Highlighting the therapeutic implications of these findings, in utero antioxidant therapy stabilized RUNX2 and improved bone development in the Runx2+/- haplo-insufficient mouse model of human cleidocranial dysplasia. Thus, our data establish RUNX2 as a molecular sensor of the osteoblast REDOX environment and mechanistically clarify how ROS negatively impacts osteoblast differentiation and bone formation.

Aged G Protein-Coupled Receptor Kinase 3 (Grk3)-Deficient Mice Exhibit Enhanced Osteoclastogenesis and Develop Bone Lesions Analogous to Human Paget's Disease of Bone.

Paget's Disease of Bone (PDB) is a metabolic bone disease that is characterized by dysregulated osteoclast function leading to focal abnormalities of bone remodeling. It can lead to pain, fracture, and bone deformity. G protein-coupled receptor kinase 3 (GRK3) is an important negative regulator of G protein-coupled receptor (GPCR) signaling. GRK3 is known to regulate GPCR function in osteoblasts and preosteoblasts, but its regulatory function in osteoclasts is not well defined. Here, we report that Grk3 expression increases during osteoclast differentiation in both human and mouse primary cells and established cell lines. We also show that aged mice deficient in Grk3 develop bone lesions similar to those seen in human PDB and other Paget's Disease mouse models. We show that a deficiency in Grk3 expression enhances osteoclastogenesis in vitro and proliferation of hematopoietic osteoclast precursors in vivo but does not affect the osteoclast-mediated bone resorption function or cellular senescence pathway. Notably, we also observe decreased Grk3 expression in peripheral blood mononuclear cells of patients with PDB compared with age- and gender-matched healthy controls. Our data suggest that GRK3 has relevance to the regulation of osteoclast differentiation and that it may have relevance to the pathogenesis of PDB and other metabolic bone diseases associated with osteoclast activation.

Local Administration of Bone Morphogenetic Protein-2 Using a Hydrogel Carrier for Robust Bone Regeneration in a Large Animal Model of Legg-Calvé-Perthes disease.

Legg-Calvé-Perthes disease is juvenile idiopathic osteonecrosis of the femoral head (ONFH) that has no effective clinical resolutions. Previously, local injection of bone morphogenetic protein-2 (BMP2) for ONFH treatment showed a heterogeneous bone repair and a high incidence of heterotopic ossification (HO) due to the BMP2 leakage. Here, we developed a BMP2-hydrogel treatment via a transphyseal bone wash and subsequential injection of BMP2-loaded hydrogel. In vivo studies showed that a hydrogel of gelatin-heparin-tyramine retained the BMP2 for four weeks. The injection of the hydrogel can efficiently prevent leakage. With the bone wash, the injected hydrogel had a broad distribution in the head. In vivo studies on pigs revealed that the BMP2-hydrogel treatment produced a homogeneous bone regeneration without HO. It preserved the subchondral contour and restored the subchondral endochondral ossification, although it increased growth plate fusions. In summary, the study demonstrated a promising BMP2-hydrogel treatment for ONFH treatment, especially for teenagers.

Interleukin-6 signaling mediates cartilage degradation and pain in posttraumatic osteoarthritis in a sex-specific manner.

Osteoarthritis (OA) and posttraumatic OA (PTOA) are caused by an imbalance in catabolic and anabolic processes in articular cartilage and proinflammatory changes throughout the joint, leading to joint degeneration and pain. We examined whether interleukin-6 (IL-6) signaling contributed to cartilage degradation and pain in PTOA. Genetic ablation of Il6 in male mice decreased PTOA-associated cartilage catabolism, innervation of the knee joint, and nociceptive signaling without improving PTOA-associated subchondral bone sclerosis or chondrocyte apoptosis. These effects were not observed in female Il6-/- mice. Compared with wild-type mice, the activation of the IL-6 downstream mediators STAT3 and ERK was reduced in the knees and dorsal root ganglia (DRG) of male Il6-/- mice after knee injury. Janus kinases (JAKs) were critical for STAT and ERK signaling in cartilage catabolism and DRG pain signaling in tissue explants. Whereas STAT3 signaling was important for cartilage catabolism, ERK signaling mediated neurite outgrowth and the activation of nociceptive neurons. These data demonstrate that IL-6 mediates both cartilage degradation and pain associated with PTOA in a sex-specific manner and identify tissue-specific contributions of downstream effectors of IL-6 signaling, which are potential therapeutic targets for disease-modifying OA drugs.

Hypertrophic chondrocytes serve as a reservoir for marrow-associated skeletal stem and progenitor cells, osteoblasts, and adipocytes during skeletal development.

Hypertrophic chondrocytes give rise to osteoblasts during skeletal development; however, the process by which these non-mitotic cells make this transition is not well understood. Prior studies have also suggested that skeletal stem and progenitor cells (SSPCs) localize to the surrounding periosteum and serve as a major source of marrow-associated SSPCs, osteoblasts, osteocytes, and adipocytes during skeletal development. To further understand the cell transition process by which hypertrophic chondrocytes contribute to osteoblasts or other marrow associated cells, we utilized inducible and constitutive hypertrophic chondrocyte lineage tracing and reporter mouse models (Col10a1CreERT2; Rosa26fs-tdTomato and Col10a1Cre; Rosa26fs-tdTomato) in combination with a PDGFRaH2B-GFP transgenic line, single-cell RNA-sequencing, bulk RNA-sequencing, immunofluorescence staining, and cell transplantation assays. Our data demonstrate that hypertrophic chondrocytes undergo a process of dedifferentiation to generate marrow-associated SSPCs that serve as a primary source of osteoblasts during skeletal development. These hypertrophic chondrocyte-derived SSPCs commit to a CXCL12-abundant reticular (CAR) cell phenotype during skeletal development and demonstrate unique abilities to recruit vasculature and promote bone marrow establishment, while also contributing to the adipogenic lineage.

G protein-coupled receptor kinase 3 modulates mesenchymal stem cell proliferation and differentiation through sphingosine-1-phosphate receptor regulation.

BACKGROUND: The bone marrow niche supports hematopoietic cell development through intimate contact with multipotent stromal mesenchymal stem cells; however, the intracellular signaling, function, and regulation of such supportive niche cells are still being defined. Our study was designed to understand how G protein receptor kinase 3 (GRK3) affects bone marrow mesenchymal stem cell function by examining primary cells from GRK3-deficient mice, which we have previously published to have a hypercellular bone marrow and leukocytosis through negative regulation of CXCL12/CXCR4 signaling. METHODS: Murine GRK3-deficient bone marrow mesenchymal stromal cells were harvested and cultured to differentiate into three lineages (adipocyte, chondrocyte, and osteoblast) to confirm multipotency and compared to wild type cells. Immunoblotting, modified-TANGO experiments, and flow cytometry were used to further examine the effects of GRK3 deficiency on bone marrow mesenchymal stromal cell receptor signaling. Microcomputed tomography was used to determine trabecular and cortical bone composition of GRK3-deficient mice and standard ELISA to quantitate CXCL12 production from cellular cultures. RESULTS: GRK3-deficient, bone marrow-derived mesenchymal stem cells exhibit enhanced and earlier osteogenic differentiation in vitro. The addition of a sphingosine kinase inhibitor abrogated the osteogenic proliferation and differentiation, suggesting that sphingosine-1-phosphate receptor signaling was a putative G protein-coupled receptor regulated by GRK3. Immunoblotting showed prolonged ERK1/2 signaling after stimulation with sphingosine-1-phosphate in GRK3-deficient cells, and modified-TANGO assays suggested the involvement of ß-arrestin-2 in sphingosine-1-phosphate receptor internalization. CONCLUSIONS: Our work suggests that GRK3 regulates sphingosine-1-phosphate receptor signaling on bone marrow mesenchymal stem cells by recruiting ß-arrestin to the occupied GPCR to promote internalization, and lack of such regulation affects mesenchymal stem cell functionality.

Damage associated molecular patterns in necrotic femoral head inhibit osteogenesis and promote fibrogenesis of mesenchymal stem cells.

In Legg-Calvé-Perthes disease (LCPD), a loss of blood supply to the juvenile femoral head leads to extensive cell death and release of damage-associated molecular patterns (DAMPs). Over time chronic inflammatory repair process is observed with impaired bone regeneration. Increased fibrous tissue and adipose tissue are seen in the marrow space with decreased osteogenesis in a piglet model of LCPD, suggesting inhibition of osteoblastic differentiation and stimulation of fibroblastic and adipogenic differentiation of mesenchymal stem cell (MSC) during the healing process. Little is known about the DAMPs present in the necrotic femoral head and their effects on MSC differentiation. The purpose of this study was to characterize the DAMPs present in the femoral head following ischemic osteonecrosis and to determine their effects on MSC differentiation. Necrotic femoral heads were flushed with saline at 48 h, 2 weeks and 4 weeks following the induction of ischemic osteonecrosis in piglets to obtain necrotic bone fluid (NBF). Western blot analysis of the NBF revealed the presence of prototypic DAMP, high mobility group box 1 (HMGB1), and other previously described DAMPs: biglycan, 4-hydroxynonenal (4-HNE), and receptor activator of NF-κB ligand (RANKL). ELISA of the NBF revealed increasing levels of inflammatory cytokines IL1ß, IL6 and TNFα with the temporal progression of osteonecrosis. To determine the effects of NBF on MSC differentiation, we cultured primary porcine MSCs with NBF obtained by in vivo necrotic bone flushing method. NBF inhibited osteoblastic differentiation of MSCs with significantly decreased OSX expression (p = 0.008) and Von Kossa/Alizarin Red staining for mineralization. NBF also significantly increased the expression of proliferation markers Ki67 (p = 0.03) and PCNA (p < 0.0001), and fibrogenic markers Vimentin (p = 0.02) and Fibronectin (p = 0.04). Additionally, NBF treated MSC cells showed significantly elevated RANKL/OPG secretion ratio (p = 0.003) and increased expression of inflammatory cytokines IL1ß (p = 0.006) and IL6 (p < 0.0001). To specifically assess the role of DAMPs in promoting the fibrogenesis, we treated porcine fibroblasts with artificial NBF produced by bone freeze-thaw method. We found increased fibroblastic cell proliferation in an NBF dose-dependent manner. Lastly, we studied the effect of HMGB1, a prototypic DAMP, and found that HMGB1 partially contributes to MSC proliferation and fibrogenesis. In summary, our findings show that DAMPs and the inflammatory cytokines present in the necrotic femoral head inhibit osteogenesis and promote fibrogenesis of MSCs, potentially contributing to impaired bone regeneration following ischemic osteonecrosis as observed in LCPD.

Osteocytes but not osteoblasts directly build mineralized bone structures.

Bone-forming osteoblasts have been a cornerstone of bone biology for more than a century. Most research toward bone biology and bone diseases center on osteoblasts. Overlooked are the 90% of bone cells, called osteocytes. This study aims to test the hypothesis that osteocytes but not osteoblasts directly build mineralized bone structures, and that defects in osteocytes lead to the onset of hypophosphatemia rickets. The hypothesis was tested by developing and modifying multiple imaging techniques, including both in vivo and in vitro models plus two types of hypophosphatemia rickets models (Dmp1-null and Hyp, Phex mutation mice), and Dmp1-Cre induced high level of ß-catenin models. Our key findings were that osteocytes (not osteoblasts) build bone similar to the construction of a high-rise building, with a wire mesh frame (i.e., osteocyte dendrites) and cement (mineral matrices secreted from osteocytes), which is a lengthy and slow process whose mineralization direction is from the inside toward the outside. When osteoblasts fail to differentiate into osteocytes but remain highly active in Dmp-1-null or Hyp mice, aberrant and poor bone mineralization occurs, caused by a sharp increase in Wnt-ß-catenin signaling. Further, the constitutive expression of ß-catenin in osteocytes recaptures a similar osteomalacia phenotype as shown in Dmp1 null or Hyp mice. Thus, we conclude that osteocytes directly build bone, and osteoblasts with a short life span serve as a precursor to osteocytes, which challenges the existing dogma.

Minimally Invasive Necrotic Bone Washing Improves Bone Healing After Femoral Head Ischemic Osteonecrosis: An Experimental Investigation in Immature Pigs.

BACKGROUND: Ischemic osteonecrosis of the femoral head produces necrotic cell debris and inflammatory molecules in the marrow space, which elicit a chronic inflammatory repair response. The purpose of this study was to determine the effects of flushing out the necrotic cell debris and inflammatory proteins on bone repair in a piglet model of ischemic osteonecrosis. METHODS: Osteonecrosis of the femoral head of the right hindlimb was induced in 12 piglets by tying a ligature tightly around the femoral neck. One week after the surgery, 6 animals were treated with a percutaneous 3-needle bone washing procedure and non-weight-bearing (NWB) of the right hindlimb (wash group). The total saline solution wash volume was 450 mL per femoral head. Serial wash solutions were collected and analyzed. The remaining 6 animals were treated with NWB only (NWB group). At 8 weeks after the surgery, the femoral heads were assessed using radiography, micro-computed tomography (micro-CT), and histological analysis. In addition, we compared the results for these piglets with our published results for 6 piglets treated with multiple epiphyseal drilling (MED) plus NWB without bone washing (MED group). RESULTS: Necrotic cells and inflammatory proteins were present in the bone wash solution collected 1 week after ischemia induction. The protein and triglyceride concentrations decreased significantly with subsequent washing (p < 0.005). At 8 weeks after ischemia induction, the wash group had a significantly higher bone volume than the MED or NWB group (p < 0.0001). Histological bone-formation measures were also significantly increased in the wash group compared with the MED group (p = 0.002) or NWB group (p < 0.0001) while macrophage numbers were significantly decreased in the wash group. CONCLUSIONS: The percutaneous 3-needle procedure flushed out cell debris and inflammatory proteins from the necrotic femoral heads, decreased osteoclasts and macrophages, and increased bone formation following induction of ischemic osteonecrosis. CLINICAL RELEVANCE: We believe that this is the first study to investigate the concept of washing out the necrotic femoral head to improve bone healing. The minimally invasive procedure may be useful to improve the necrotic bone environment and bone repair following ischemic osteonecrosis.

Development of a murine model of ischemic osteonecrosis to study the effects of aging on bone repair.

Age at onset is one of the most important predictors of outcome following ischemic osteonecrosis (ON). Currently, there is no well-established animal model to study the effects of age on the repair process following ischemic ON. The purpose of this study was to further advance a murine model of ischemic ON using four age groups of mice to determine the effects of aging on revascularization and bone repair following ischemic ON. Ischemia was surgically induced in the distal femoral epiphysis of four age groups of skeletally immature and mature mice; juvenile (5 weeks), adolescent (12 weeks), adult (22 weeks), and middle age (52 weeks). Mice were euthanized at 2 days or 4 weeks post-ischemia surgery to evaluate the extent of ON, revascularization, and bone repair. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining showed extensive cell death in the epiphysis of all four age groups at 2 days post-ischemia surgery. At 4 weeks, the juvenile mice followed by the adolescent mice had significantly greater revascularization and repair of the necrotic marrow space, increased osteoblast and osteoclast numbers, and increased bone formation rates compared to the adult and middle-age mice. Faster revascularization and bone healing were observed in the skeletally immature mice compared to the skeletally mature mice following ischemic ON. The findings resemble the clinical observation of aging on bone repair following ischemic ON. The mouse model may serve as a useful tool to investigate the mechanisms underlying the age-related impairment of bone repair in adolescent and adult ON and to develop novel therapeutic strategies.

Anti-Interleukin-6 Therapy Decreases Hip Synovitis and Bone Resorption and Increases Bone Formation Following Ischemic Osteonecrosis of the Femoral Head.

Legg-Calvé-Perthes disease (LCPD) is a juvenile form of ischemic femoral head osteonecrosis, which produces chronic hip synovitis, permanent femoral head deformity, and premature osteoarthritis. Currently, there is no medical therapy for LCPD. Interleukin-6 (IL-6) is significantly elevated in the synovial fluid of patients with LCPD. We hypothesize that IL-6 elevation promotes chronic hip synovitis and impairs bone healing after ischemic osteonecrosis. We set out to test if anti-IL-6 therapy using tocilizumab can decrease hip synovitis and improve bone healing in the piglet model of LCPD. Fourteen piglets were surgically induced with ischemic osteonecrosis and assigned to two groups: the no treatment group (n = 7) and the tocilizumab group (15 to 20 mg/kg, biweekly intravenous injection, n = 7). All animals were euthanized 8 weeks after the induction of osteonecrosis. Hip synovium and femoral heads were assessed for hip synovitis and bone healing using histology, micro-CT, and histomorphometry. The mean hip synovitis score and the number of synovial macrophages and vessels were significantly lower in the tocilizumab group compared with the no treatment group (p < .0001, p = .01, and p < .01, respectively). Micro-CT analysis of the femoral heads showed a significantly higher bone volume in the tocilizumab group compared with the no treatment group (p = .02). The histologic assessment revealed a significantly lower number of osteoclasts per bone surface (p < .001) in the tocilizumab group compared with the no treatment group. Moreover, fluorochrome labeling showed a significantly higher percent of mineralizing bone surface (p < .01), bone formation rate per bone surface (p < .01), and mineral apposition rate (p = .04) in the tocilizumab group. Taken together, tocilizumab therapy decreased hip synovitis and osteoclastic bone resorption and increased new bone formation after ischemic osteonecrosis. This study provides preclinical evidence that tocilizumab decreases synovitis and improves bone healing in a large animal model of LCPD. © 2020 American Society for Bone and Mineral Research (ASBMR).

The CaV1.2 L-type calcium channel regulates bone homeostasis in the middle and inner ear.

Bone remodeling of the auditory ossicles and the otic capsule is highly restricted and tightly controlled by the osteoprotegerin (OPG)/receptor activator of nuclear factor kappa-Β ligand (RANKL)/receptor activator of nuclear factor kappa-Β (RANK) system. In these bony structures, a pathological decrease in OPG expression stimulates osteoclast differentiation and excessive resorption followed by accrual of sclerotic bone, ultimately resulting in the development of otosclerosis, a leading cause of deafness in adults. Understanding the signaling pathways involved in maintaining OPG expression in the ear would shed light on the pathophysiology of otosclerosis and other ear bone-related diseases. We and others previously demonstrated that Ca2+ signaling through the L-type CaV1.2 Ca2+ channel positively regulates OPG expression and secretion in long bone osteoblasts and their precursor cells in vitro and in vivo. Whether CaV1.2 regulates OPG expression in ear bones has not been investigated. We drove expression of a gain-of-function CaV1.2 mutant channel (CaV1.2TS) using Col2a1-Cre, which we found to target osteochondral/osteoblast progenitors in the auditory ossicles and the otic capsule. Col2a1-Cre;CaV1.2TS mice displayed osteopetrosis of these bones shown by µCT 3D reconstruction, histological analysis, and lack of bone sculpting, findings similar to phenotypes seen in mice with an osteoclast defect. Consistent with those observations, we found that Col2a1-Cre;CaV1.2TS mutant mice showed reduced osteoclasts in the otic capsule, upregulated mRNA expression of Opg and Opg/Rankl ratio, and increased mRNA expression of osteoblast differentiation marker genes in the otic capsule, suggesting both an anti-catabolic and anabolic effect of CaV1.2TS mutant channel contributed to the observed morphological changes of the ear bones. Further, we found that Col2a1-Cre;CaV1.2TS mice experienced hearing loss and displayed defects of body balance in behavior tests, confirming that the CaV1.2-dependent Ca2+ influx affects bone structure in the ear and consequent hearing and vestibular functions. Together, these data support our hypothesis that Ca2+ influx through CaV1.2TS promotes OPG expression from osteoblasts, thereby affecting bone modeling/remodeling in the auditory ossicles and the otic capsule. These data provide insight into potential pathological mechanisms underlying perturbed OPG expression and otosclerosis.

Interleukin-6 deletion stimulates revascularization and new bone formation following ischemic osteonecrosis in a murine model.

Legg-Calvé-Perthes disease (LCPD) is a childhood form of ischemic osteonecrosis of the femoral head which can produce a permanent femoral head deformity and early osteoarthritis. The femoral head deformity results from increased bone resorption and decreased bone formation during repair and remodeling of the necrotic femoral head. A recent study showed that a pro-inflammatory cytokine, interleukin-6 (IL-6), is significantly elevated in the synovial fluid of patients with LCPD. We hypothesized that IL-6 elevation decreases bone formation during the repair process following ischemic osteonecrosis and that IL-6 depletion will increase new bone formation. To test this hypothesis, we surgically induced ischemic osteonecrosis in the wild-type (n = 29) and IL-6 knockout (KO) mice (n = 25). The animals were assessed at 48 h, 2 weeks and 4 weeks following the induction of ischemic osteonecrosis using histologic, histomorphometric and micro-CT methods. IL-6 immunohistochemistry showed high expression of IL-6 in the osteonecrotic side of the wild-type mice at 48 h and 4 weeks following ischemic osteonecrosis, but not in the IL-6 KO mice. We also confirmed an undetectable level of IL-6 expression in the primary osteoblasts of the IL-6 KO mice compared to the readily detectable level in the wild-type mice. Furthermore, we confirmed that IL-6 deletion did not affect the extent of bone necrosis in the IL-6 KO mice compared to the wild-type mice by performing histologic and terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) assessments at 2 weeks following the induction of ischemia. Both groups had the same extent of ischemic osteonecrosis and absence of repair at 2 weeks. At 4 weeks, the necrotic epiphyses showed a significant increase in the extent of revascularization in the IL-6 KO mice compared to the wild-type mice (p = 0.001). In addition, a significantly greater recovery of the hematopoietic bone marrow was observed in the osteonecrotic side of the IL-6 KO mice compared to the wild-type mice (p < 0.01). Vascular endothelial growth factor (VEGF) immunohistochemistry showed regionally increased staining in the areas of repair in the osteonecrosis side of IL-6 KO mice compared to the wild-type mice at 4 weeks following ischemic osteonecrosis. Micro-CT assessment of the wild-type mice at 4 weeks showed a significant decrease in the percent bone volume (p < 0.01) in the osteonecrotic side compared to the control side. In contrast, IL-6 KO mice showed significantly increased bone volume in the osteonecrotic side compared to the osteonecrotic side of WT mice (p < 0.001). No significant difference in the bone volume percentage was found between the control side of the wild-type and the IL-6 KO mice. Histomorphometric analysis at 4 weeks revealed increased osteoblast number/bone surface (p < 0.001), bone formation rate (BFR) (p = 0.0001), and mineral apposition rate (MAR) (p < 0.0001) in the osteonecrotic side of the IL-6 KO mice compared to the wild-type mice. The number of osteoclast/bone surface was also increased in the IL-6 KO mice compared to the wild-type mice (p < 0.0001). No significant difference was observed between the control side of the wild-type and IL-6 KO mice with regards to the number of osteoblast or osteoclast/bone surface, BFR, and MAR. We next obtained primary osteoblasts from IL-6 KO mice and showed they expressed a significantly higher level of RANKL/OPG than wild-type mice (p = 0.001) in hypoxia culture condition. Taken together, the findings indicate that IL-6 deletion stimulates revascularization and new bone formation following ischemic osteonecrosis. This study provides new evidence that therapeutic strategies to block IL-6 may be beneficial for bone healing following ischemic osteonecrosis.

Increased Ca2+ signaling through CaV1.2 promotes bone formation and prevents estrogen deficiency-induced bone loss.

While the prevalence of osteoporosis is growing rapidly with population aging, therapeutic options remain limited. Here, we identify potentially novel roles for CaV1.2 L-type voltage-gated Ca2+ channels in osteogenesis and exploit a transgenic gain-of-function mutant CaV1.2 to stem bone loss in ovariectomized female mice. We show that endogenous CaV1.2 is expressed in developing bone within proliferating chondrocytes and osteoblasts. Using primary BM stromal cell (BMSC) cultures, we found that Ca2+ influx through CaV1.2 activates osteogenic transcriptional programs and promotes mineralization. We used Prx1-, Col2a1-, or Col1a1-Cre drivers to express an inactivation-deficient CaV1.2 mutant in chondrogenic and/or osteogenic precursors in vivo and found that the resulting increased Ca2+ influx markedly thickened bone not only by promoting osteogenesis, but also by inhibiting osteoclast activity through increased osteoprotegerin secretion from osteoblasts. Activating the CaV1.2 mutant in osteoblasts at the time of ovariectomy stemmed bone loss. Together, these data highlight roles for CaV1.2 in bone and demonstrate the potential dual anabolic and anticatabolic therapeutic actions of tissue-specific CaV1.2 activation in osteoblasts.

Essential Roles of Bone Morphogenetic Protein-1 and Mammalian Tolloid-like 1 in Postnatal Root Dentin Formation.

INTRODUCTION: Mutations in the proteinase bone morphogenetic protein-1 (BMP1) were recently identified in patients with osteogenesis imperfecta, which can be associated with type 1 dentinogenesis imperfecta. BMP1 is co-expressed in various tissues and has overlapping activities with the closely related proteinase mammalian tolloid-like 1 (TLL1). In this study we investigated whether removing the overlapping activities of BMP1 and TLL1 affects the mineralization of tooth root dentin. METHODS: Floxed alleles of the BMP1 and TLL1 genes were excised via ubiquitously expressed Cre induced by tamoxifen treatment beginning at 3 days of age (harvested at 3 weeks of age) or beginning at 4 weeks of age (harvested at 8 weeks of age). Multiple techniques, including x-ray analysis, double-labeling with calcein and alizarin red stains for measurement of dentin formation rate, and histologic and immunostaining assays, were used to analyze the dentin phenotype. RESULTS: BMP1/TLL1 double knockout mice displayed short and thin root dentin, defects in dentin mineralization, and delayed tooth eruption. Molecular mechanism studies revealed accumulation of collagens in dentin and a sharp reduction in non-collagenous proteins such as dentin matrix protein 1 and dentin sialophosphoprotein. Furthermore, we found a strong reduction in tartrate-resistant acid phosphatase, which is likely caused by defects in bone cells. CONCLUSIONS: BMP1/TLL1 appear to play crucial roles in maintaining extracellular matrix homeostasis essential to root formation and dentin mineralization.

HES factors regulate specific aspects of chondrogenesis and chondrocyte hypertrophy during cartilage development.

RBPjκ-dependent Notch signaling regulates multiple processes during cartilage development, including chondrogenesis, chondrocyte hypertrophy and cartilage matrix catabolism. Select members of the HES- and HEY-families of transcription factors are recognized Notch signaling targets that mediate specific aspects of Notch function during development. However, whether particular HES and HEY factors play any role(s) in the processes during cartilage development is unknown. Here, for the first time, we have developed unique in vivo genetic models and in vitro approaches demonstrating that the RBPjκ-dependent Notch targets HES1 and HES5 suppress chondrogenesis and promote the onset of chondrocyte hypertrophy. HES1 and HES5 might have some overlapping function in these processes, although only HES5 directly regulates Sox9 transcription to coordinate cartilage development. HEY1 and HEYL play no discernable role in regulating chondrogenesis or chondrocyte hypertrophy, whereas none of the HES or HEY factors appear to mediate Notch regulation of cartilage matrix catabolism. This work identifies important candidates that might function as downstream mediators of Notch signaling both during normal skeletal development and in Notch-related skeletal disorders.

NOTCH signaling in skeletal progenitors is critical for fracture repair.

Fracture nonunions develop in 10%-20% of patients with fractures, resulting in prolonged disability. Current data suggest that bone union during fracture repair is achieved via proliferation and differentiation of skeletal progenitors within periosteal and soft tissues surrounding bone, while bone marrow stromal/stem cells (BMSCs) and other skeletal progenitors may also contribute. The NOTCH signaling pathway is a critical maintenance factor for BMSCs during skeletal development, although the precise role for NOTCH and the requisite nature of BMSCs following fracture is unknown. Here, we evaluated whether NOTCH and/or BMSCs are required for fracture repair by performing nonstabilized and stabilized fractures on NOTCH-deficient mice with targeted deletion of RBPjk in skeletal progenitors, maturing osteoblasts, and committed chondrocytes. We determined that removal of NOTCH signaling in BMSCs and subsequent depletion of this population result in fracture nonunion, as the fracture repair process was normal in animals harboring either osteoblast- or chondrocyte-specific deletion of RBPjk. Together, this work provides a genetic model of a fracture nonunion and demonstrates the requirement for NOTCH and BMSCs in fracture repair, irrespective of fracture stability and vascularity.