Osteocytes and Bone Diseases

For many centuries, the osteoblast is considered to be responsible for bone formation. It is also believed that an imbalance of osteoblasts (weak) and osteoclasts (strong) is the main cause for bone diseases such as osteomalacia and osteoporosis and periodontal diseases, globally the most prevalent dental disease. Most studies are aimed at these two surface cells, although neither of them penetrates into the deep bone matrix. Osteocytes, terminally differentiated osteoblasts, are buried deep in the bone and account for up to 95% of all bone cells. Due to the high mineral density around osteocytes, most people consider them “quiescent” cells. Only recent research revealed more profound and important roles of osteocytes, such as mechano-sensors. In this study, we developed the innovative “FITC-Imaris technique”, which combines FITC (Fluorescein isothiocyanate), confocal microscopy and Imaris software. With this method, we could visualize the 3-D morphology of embedded osteocytesand statistically quantitate the osteocyte structure cell surface total cell volume, and dendrite numbers. We examined Dmp1 (dentin matrix protein 1) mutant mice, an established osteomalacia animal model, and showed both significant morphological and statistical differences in the osteocyte structure between the Dmp1-null mice and their age-matched control littermates, suggesting a high correlation between osteocytes and osteomalacia. Then, we studied periostin knockout mice, a periodontal disease mouse model, and found that osteocyte’ morphological and pathological changes are closely linked to alveolar bone loss. Monoclonal anti-SOST antibody) restores not only bone loss, but also osteocyte morphology, suggesting osteocytes may be responsible for bone loss in periostin mutant mice. Lastly, we examined OVX rats as an osteoporosis model and showed that Ocys failed to maintain their shape, dendrite number and size in response to estrogen deficiency. Abnormalities in blood vessel morphology and bone matrices also developed, resulting in osteoporotic changes in both compact and trabecular bone. Similarly, administering SOST antibody normalized osteocyte morphology and recovered bone loss from osteoporosis. Altogether, we demonstrated that Ocy maturation was directly linked to a slow mineralization process. Minerals were constantly “pumped” via Ocy-dendrites to the surrounding matrix and to the bone surface. These study results expanded our understanding of how osteocytes regulate bone development and mineralization. These findings have clinical relevance, as SOST antibody improved bone phenotypes in all osteomalacia, periodontal disease and osteoporosis animal models, holding great potential for treating human bone disease

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

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

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

Osteocytes and Bone Diseases

For many centuries, the osteoblast is considered to be responsible for bone formation. It is also believed that an imbalance of osteoblasts (weak) and osteoclasts (strong) is the main cause for bone diseases such as osteomalacia and osteoporosis and periodontal diseases, globally the most prevalent dental disease. Most studies are aimed at these two surface cells, although neither of them penetrates into the deep bone matrix. Osteocytes, terminally differentiated osteoblasts, are buried deep in the bone and account for up to 95% of all bone cells. Due to the high mineral density around osteocytes, most people consider them “quiescent” cells. Only recent research revealed more profound and important roles of osteocytes, such as mechano-sensors. In this study, we developed the innovative “FITC-Imaris technique”, which combines FITC (Fluorescein isothiocyanate), confocal microscopy and Imaris software. With this method, we could visualize the 3-D morphology of embedded osteocytesand statistically quantitate the osteocyte structure cell surface total cell volume, and dendrite numbers. We examined Dmp1 (dentin matrix protein 1) mutant mice, an established osteomalacia animal model, and showed both significant morphological and statistical differences in the osteocyte structure between the Dmp1-null mice and their age-matched control littermates, suggesting a high correlation between osteocytes and osteomalacia. Then, we studied periostin knockout mice, a periodontal disease mouse model, and found that osteocyte’ morphological and pathological changes are closely linked to alveolar bone loss. Monoclonal anti-SOST antibody) restores not only bone loss, but also osteocyte morphology, suggesting osteocytes may be responsible for bone loss in periostin mutant mice. Lastly, we examined OVX rats as an osteoporosis model and showed that Ocys failed to maintain their shape, dendrite number and size in response to estrogen deficiency. Abnormalities in blood vessel morphology and bone matrices also developed, resulting in osteoporotic changes in both compact and trabecular bone. Similarly, administering SOST antibody normalized osteocyte morphology and recovered bone loss from osteoporosis. Altogether, we demonstrated that Ocy maturation was directly linked to a slow mineralization process. Minerals were constantly “pumped” via Ocy-dendrites to the surrounding matrix and to the bone surface. These study results expanded our understanding of how osteocytes regulate bone development and mineralization. These findings have clinical relevance, as SOST antibody improved bone phenotypes in all osteomalacia, periodontal disease and osteoporosis animal models, holding great potential for treating human bone disease

The Structure Features and Improving Effects of Polysaccharide from Astragalus membranaceus on Antibiotic-Associated Diarrhea

Astragalus membranaceus (Astragalus) is often used as a medical and food resource in China. The present study was designed to investigate the features and effects of polysaccharide from Astragalus membranaceus (WAP) on rats with antibiotic-associated diarrhea (AAD). WAP was mainly composed of glucose, galactose, arabinose and glacturonic acid, with glucan, arabinogalactan and RG-I regions, and it showed loosely irregular sheet conformation. WAP decreased the inflammatory cell infiltration of colon in AAD rats, increased propionate and butyrate production, improved metabolic levels, adjusted the diversity and composition of gut microbiota, increased the relative abundance of Pseudomonas, and decreased the relative abundance of Allobaculum and Coprococcus. In conclusion, WAP contained different types of polysaccharide regions and sheet three-dimensional conformation, while it ameliorated AAD by recovering the colon structure, adjusting the gut microbiota, and improving the SCFAs levels. The results can provide some data basis for natural products to alleviate the side effects related to antibiotics

The Protective Effects of Ginseng Polysaccharides and Their Effective Subfraction against Dextran Sodium Sulfate-Induced Colitis

Polysaccharides from Panax ginseng are natural carbohydrates with multiple activities. However, little was known about its functions on colitis. In this study, we aim to investigate the protective effects of ginseng polysaccharides and its effective subfraction on dextran sodium sulfate (DSS)-induced colitis. Water soluble ginseng polysaccharides (WGP) were obtained from dry ginseng root, then purified to neutral fraction (WGPN) and acidic fraction (WGPA) by ion exchange chromatography. An animal model was constructed with male Wistar rats, which were treated with a normal diet (con group), DSS (DSS group), WGP (WGP group), WGPN (WGPN group), and WGPA (WGPA group), respectively. Both WGP and WGPA alleviated the colitis symptoms and colon structure changes of colitis rats. They decreased the disease activity index (DAI) scores and improved colon health; reduced colon damage and recovered the intestinal barrier via regulating the tight-junction-related proteins (ZO-1 and Occludin); downregulated inflammatory cytokines (IL-1β, IL-2, IL-6, and IL-17) and inhibited the TLR4/MyD88/NF-κB-signaling pathway in the colon; regulated the diversity and composition of gut microbiota, especially the relative abundance of Ruminococcus; enhanced the production of SCFAs. In conclusion, WGP exerted a protective effect against colitis with its acidic fraction (WGPA) as an effective fraction. The results support the utilization and investigation of ginseng polysaccharides as a potential intervention strategy for the prevention of colitis

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

Abstract 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