The development of embryonic bone and cartilage in tissue culture

Embryonic chick long bone develops in a series of temporally controlled, cellular events and involves the integration of at least three distinctly different sets of cells: collar osteoblasts, core osteoblasts, and resorptive or osteoclastic cells. The morphology of the long bones is established by the developing cartilage rudiment or model. All of these events seem to be influenced by positional cues. The cultivation of all of these cells and their presumptive progenitor cells potentially allows a detailed analysis of their individual and collective phenotypic traits. Future studies can include how long bones form, how bone-forming and bone-resorbing cells interact, and how osteogenic cells influence each other throughout each stage of their respective developmental lineages.     

Hypermineralization of Hearing-Related Bones by a Specific Osteoblast Subtype

Auditory ossicles in the middle ear and bony labyrinth of the inner ear are highly mineralized in adult mammals. Cellular mechanisms underlying formation of dense bone during development are unknown. Here, we found that osteoblast-like cells synthesizing highly mineralized hearing-related bones produce both type I and type II collagens as the bone matrix, while conventional osteoblasts and chondrocytes primarily produce type I and type II collagens, respectively. Furthermore, these osteoblast-like cells were not labeled in a “conventional osteoblast”-specific green fluorescent protein (GFP) mouse line. Type II collagen-producing osteoblast-like cells were not chondrocytes as they express osteocalcin, localize along alizarin-labeled osteoid, and form osteocyte lacunae and canaliculi, as do conventional osteoblasts. Auditory ossicles and the bony labyrinth exhibit not only higher bone matrix mineralization but also a higher degree of apatite orientation than do long bones. Therefore, we conclude that these type II collagen-producing hypermineralizing osteoblasts (termed here auditory osteoblasts) represent a new osteoblast subtype. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

EphB4 enhances the process of endochondral ossification and inhibits remodeling during bone fracture repair

Previous reports have identified a role for the tyrosine kinase receptor EphB4 and its ligand, ephrinB2, as potential mediators of both bone formation by osteoblasts and bone resorption by osteoclasts. In the present study, we examined the role of EphB4 during bone repair after traumatic injury. We performed femoral fractures with internal fixation in transgenic mice that overexpress EphB4 under the collagen type 1 promoter (Col1‐EphB4) and investigated the bone repair process up to 12 weeks postfracture. The data indicated that Col1‐EphB4 mice exhibited stiffer and stronger bones after fracture compared with wild‐type mice. The fractured bones of Col1‐EphB4 transgenic mice displayed significantly greater tissue and bone volume 2 weeks postfracture compared with that of wild‐type mice. These findings correlated with increased chondrogenesis and mineral formation within the callus site at 2 weeks postfracture, as demonstrated by increased safranin O and von Kossa staining, respectively. Interestingly, Col1‐EphB4 mice were found to possess significantly greater numbers of clonogenic mesenchymal stromal progenitor cells (CFU‐F), with an increased capacity to form mineralized nodules in vitro under osteogenic conditions, when compared with those of the wild‐type control mice. Furthermore, Col1‐EphB4 mice had significantly lower numbers of TRAP‐positive multinucleated osteoclasts within the callus site. Taken together, these observations suggest that EphB4 promotes endochondral ossification while inhibiting osteoclast development during callus formation and may represent a novel drug target for the repair of fractured bones. © 2013 American Society for Bone and Mineral Research.     

Regulation of Sclerostin Expression in Multiple Myeloma by Dkk‐1: A Potential Therapeutic Strategy for Myeloma Bone Disease

Sclerostin is a potent inhibitor of osteoblastogenesis. Interestingly, newly diagnosed multiple myeloma (MM) patients have high levels of circulating sclerostin that correlate with disease stage and fractures. However, the source and impact of sclerostin in MM remains to be defined. Our goal was to determine the role of sclerostin in the biology of MM and its bone microenvironment as well as investigate the effect of targeting sclerostin with a neutralizing antibody (scl‐Ab) in MM bone disease. Here we confirm increased sclerostin levels in MM compared with precursor disease states like monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM. Furthermore, we found that a humanized MM xenograft mouse model bearing human MM cells (NOD‐SCID.CB17 male mice injected intravenously with 2.5 million of MM1.S‐Luc‐GFP cells) demonstrated significantly higher concentrations of mouse‐derived sclerostin, suggesting a microenvironmental source of sclerostin. Associated with the increased sclerostin levels, activated β‐catenin expression levels were lower than normal in MM mouse bone marrow. Importantly, a high‐affinity grade scl‐Ab reversed osteolytic bone disease in this animal model. Because scl‐Ab did not demonstrate significant in vitro anti‐MM activity, we combined it with the proteasome inhibitor carfilzomib. Our data demonstrated that this combination therapy significantly inhibited tumor burden and improved bone disease in our in vivo MM mouse model. In agreement with our in vivo data, sclerostin expression was noted in marrow stromal cells and osteoblasts of MM patient bone marrow samples. Moreover, MM cells stimulated sclerostin expression in immature osteoblasts while inhibiting osteoblast differentiation in vitro. This was in part regulated by Dkk‐1 secreted by MM cells and is a potential mechanism contributing to the osteoblast dysfunction noted in MM. Our data confirm the role of sclerostin as a potential therapeutic target in MM bone disease and provides the rationale for studying scl‐Ab combined with proteasome inhibitors in MM. © 2016 American Society for Bone and Mineral Research.     

A Neutralizing Antibody Targeting Oxidized Phospholipids Promotes Bone Anabolism in Chow-Fed Young Adult Mice

Oxidized phospholipids containing phosphocholine (OxPL) are pro-inflammatory lipid peroxidation products that bind to scavenger receptors (SRs), such as Scarb1, and toll-like receptors (TLRs). Excessive OxPL, as found in oxidized low-density lipoprotein (OxLDL), overwhelm these defense mechanisms and become pathogenic in atherosclerosis, nonalcoholic steatohepatitis (NASH), and osteoporosis. We previously reported that the innate IgM natural antibody E06 binds to OxPL and neutralizes their deleterious effects; expression of the single-chain (scFv) form of the antigen-binding domain of E06 (E06-scFv) as a transgene increases trabecular bone in male mice. We show herein that E06-scFv increases trabecular and cortical bone in female and male mice by increasing bone formation and decreasing osteoblast apoptosis in vivo. Homozygous E06-scFv mice have higher bone mass than hemizygous, showing a dose effect of the transgene. To investigate how OxPL restrain bone formation under physiologic conditions, we measured the levels of SRs and TLRs that bind OxPL. We found that osteoblastic cells primarily express Scarb1. Moreover, OxLDL-induced apoptosis and reduced differentiation were prevented in bone marrow–derived or calvaria-derived osteoblasts from Scarb1 knockout mice. Because Scarb1-deficient mice are reported to have high bone mass, our results suggest that E06 may promote bone anabolism in healthy young mice, at least in part, by neutralizing OxPL, which in turn promote Scarb1-mediated apoptosis of osteoblasts or osteoblast precursors. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)..     

In vivo and in vitro evidence that the high osteoblastic activity in C3H/HeJ mice compared to C57BL/6J mice is intrinsic to bone cells

Two inbred mouse strains, C3H/HeJ (C3H) and C57BL/6J (B6), displayed a profound difference in femoral peak bone density. We have previously shown that the difference could be attributed to a greater bone formation rate (BFR) that was due to a higher osteoblastic activity [measured by a mineral apposition rate (MAR)] in the C3H (high density) than B6 (low density) mice. The present study sought to determine (1) whether the BFR/MAR differences between the two mouse strains present in weight-loaded endochondral bones are also seen in less weight-loaded membranous bones and (2) whether the difference in osteoblastic activity was seen in vitro in the absence of systemic factors. To address the first objective, we performed histomorphometric measurements on the weakly loaded membranous bones (i.e., parietal bones of the calvaria) to determine if there were similar differences in MAR and BFR of membranous bones as those of highly loaded, endochondral bones. The parietal bones of adult C3H mice showed similar increases in MAR and BFR as the endochondral bones, compared to B6 mice of same age, suggesting that the differences in the MAR and BFR in the two mouse strains are probably not related to differences in mechanical strain. These findings also suggest that the gene(s) responsible for the difference in MAR between strains may not be a mechanical response gene. With respect to the second objective, we isolated osteoblasts from the parietal bones and determined their differentiation status (i.e., ALP-specific activity) and bone-forming ability (i.e., mineralized nodule formation) in vitro. Consistent with the premise that C3H osteoblasts have an intrinsic, higher differentiation status and bone-forming ability than B6 osteoblasts, osteoblasts isolated from C3H mice as compared with those from B6 mice had a significantly greater ALP-specific activity and a greater ability to form mineralized nodules in vitro in the absence of systemic factors. Because differences in ALP activity, bone-forming ability, cortical bone width, and osteoblastic activity were detected at birth, the different MAR/BFR phenotypes develop at very early life and even perhaps during embryogenesis. In conclusion, we have for the first time provided evidence that the genetic differences responsible for the observed MAR/BFR phenotype in the C3H-B6 strains are intrinsic to osteoblasts and might not depend on responses to mechanical loading and/or alterations in systemic factors.     

Characterization of Matrix-Induced Osteogenesis in Rat Calvarial Bone Defects: II. Origins of Bone-Forming Cells

Two experimental models that separated demineralized bone matrix (DBM) implants from the host bone were utilized to identify the origins of bone-forming cells in the repair of calvarial defects in rats. Rat DBM, Guanadine-HCl (Gdn-HCl) extracted insoluble residue of DBM, and Gdn-HCl extracted insoluble DBM to which the dialyzed Gdn-HCl extract was added back, were implanted in the two models which prevented cells of the adjacent host bone from participating in the repair. In addition, cells in the dura and in the subcutaneous tissue overlying the calvarial defect were locally labeled with ³H-thymidine to identify the origins of those cells that were stimulated to divide and differentiate to osteoblasts. Histological studies of the temporal events that occurred during the healing process in these defect models, combined with ³H-thymidine labeling demonstrated that the osteoblasts induced by DBM were initially derived from undifferentiated mesenchymal stem cells of the dura and later augmented by cells in the overlying connective tissue covering the defect, and not from cells in the cranial bone surrounding the circular defect. The cells of both dura and subcutaneous tissue were stimulated to proliferate and differentiate principally to osteoblasts and to a very much lesser extent to chondroblasts by DBM and by reconstituted components of DBM after Gdn-HCl extraction. Gdn-HCl-extracted insoluble DBM failed to induce bone or cartilage. These results indicate that the cytokines or other factors present in DBM are required to induce bone-forming cells derived from the dura and the overlying connective tissue for the repair of the calvarial defect. Received: 1 January 1999 / Accepted: 13 August 1999     

β‐catenin promotes bone formation and suppresses bone resorption in postnatal growing mice

Genetic studies in the mouse have demonstrated multiple roles for β‐catenin in the skeleton. In the embryo, β‐catenin is critical for the early stages of osteoblast differentiation. Postnatally, β‐catenin in mature osteoblasts and osteocytes indirectly suppresses osteoclast differentiation. However, a direct role for β‐catenin in regulating osteoblast number and/or function specifically in the postnatal life has not been demonstrated. Addressing this knowledge gap is important because low‐density lipoprotein receptor‐related protein 5 (LRP5), a coreceptor for WNT signaling proposed to function through β‐catenin, controls osteoblast number and function in postnatal mice or humans. To overcome the neonatal lethality caused by embryonic deletion of β‐catenin in early‐stage osteoblast‐lineage cells, we use the Osx‐CreER^T2 mouse strain to remove β‐catenin in Osterix (Osx)‐expressing cells by administering tamoxifen (TM) temporarily to postnatal mice. Lineage‐tracing experiments in the long bones demonstrate that Osx‐CreER^T2 targets predominantly osteoblast‐lineage cells on the bone surface, but also transient progenitors that contribute to bone marrow stromal cells and adipocytes. Deletion of β‐catenin by this strategy greatly reduces the bone formation activity of the targeted osteoblasts. However, the targeted osteoblasts rapidly turn over and are replaced by an excessive number of non‐targeted osteoblasts, causing an unexpected increase in bone formation, but an even greater increase in osteoclast number and activity produces a net effect of severe osteopenia. With time, the mutant mice also exhibit a marked increase in bone marrow adiposity. Thus, β‐catenin in postnatal Osx‐lineage cells critically regulates bone homeostasis by promoting osteoblast activity and suppressing osteoblast turnover, while restraining osteoclast and marrow fat formation. © 2013 American Society for Bone and Mineral Research.     

Conditional Deletion of Indian Hedgehog in Limb Mesenchyme Results in Complete Loss of Growth Plate Formation but Allows Mature Osteoblast Differentiation

Indian hedgehog (Ihh) is widely recognized as an essential factor for proper skeletal development. Previous in vivo studies using mutant Ihh mouse models were limited by perinatal lethality or carried out after a growth plate formed. Thus the important role of Ihh in mesenchymal cell differentiation has not been investigated. In this study, we established Prx1‐Cre;Ihh^fl/fl mice to ablate Ihh specifically in limb mesenchyme to allow us to observe the phenotype continuously from prenatal development to 3 weeks of age. Mutant mice displayed severe limb abnormalities characterized by complete lack of secondary ossification center and growth plate, indicating an essential role for Ihh in the development of these structures. Interestingly, we discovered that osteoblast differentiation and bone formation could occur in conditions of deficient Ihh. This is a novel finding that has not been observed because of the early lethality of previous Ihh mutants. Mature osteoblasts expressing osteocalcin could be detected in the center of mutant bones at postnatal day 10 (P10). Osteoclasts and blood vessel formation were also present, suggesting active bone remodeling. Histomorphometric analyses show a significant increase in osteoclast number with no major changes in bone formation rate at 3 weeks of age. Mutant long bones in the limbs were deformed, with cortices comprised of irregular woven bone. Also, there was a marked decrease in gene expression of osteoblastic and osteocytic markers. Moreover, mutant long bones displayed bone dysplasia in which we observed increased osteoclast activity and partially reduced osteoblastic and osteocytic differentiation that lead ultimately to loss of bone structures at 3 weeks of age. In summary, our data show for the first time, the presence of mature osteoblasts in long bones of the limbs despite the complete loss of growth plate formation due to Ihh deficiency. These data indicate an important function for Ihh in regulating limb mesenchymal cell differentiation. © 2015 American Society for Bone and Mineral Research.     

Ameloblastin,an Extracellular Matrix Protein,Affects Long Bone Growth and Mineralization

Matrix molecules such as the enamel‐related calcium‐binding phosphoprotein ameloblastin (AMBN) are expressed in multiple tissues, including teeth, bones, and cartilage. Here we have asked whether AMBN is of functional importance for timely long bone development and, if so, how it exerts its function related to osteogenesis. Adolescent AMBN‐deficient mice (AMBN^Δ5–6) suffered from a 33% to 38% reduction in femur length and an 8.4% shorter trunk spinal column when compared with WT controls, whereas there was no difference between adult animals. On a cellular level, AMBN truncation resulted in a shortened growth plate and a 41% to 49% reduction in the number of proliferating tibia chondrocytes and osteoblasts. Bone marrow stromal cells (BMSCs) isolated from AMBN mutant mice displayed defects in proliferation and differentiation potential as well as cytoskeleton organization. Osteogenesis‐related growth factors, such as insulin‐like growth factor 1 (IGF1) and BMP7, were also significantly (46% to 73%) reduced in AMBN‐deficient BMSCs. Addition of exogenous AMBN restored cytoskeleton structures in AMBN mutant BMSCs and resulted in a dramatic 400% to 600% increase in BMP2, BMP7, and Col1A expression. Block of RhoA diminished the effect of AMBN on osteogenic growth factor and matrix protein gene expression. Addition of exogenous BMP7 and IGF1 rescued the proliferation and differentiation potential of AMBN‐deficient BMSCs. Confirming the effects of AMBN on long bone growth, back‐crossing of mutant mice with full‐length AMBN overexpressors resulted in a complete rescue of AMBN^Δ5–6 bone defects. Together, these data indicate that AMBN affects extracellular matrix production and cell adhesion properties in the long bone growth plate, resulting in altered cytoskeletal dynamics, increased osteogenesis‐related gene expression, as well as osteoblast and chondrocyte proliferation. We propose that AMBN facilitates rapid long bone growth and an important growth spurt during the skeletogenesis of adolescent tooth‐bearing vertebrates. © 2016 American Society for Bone and Mineral Research.     

Uncovering the periosteum for skeletal regeneration: The stem cell that lies beneath

The cartilage- and bone-forming properties of the periosteum have long since been recognized. As one of the major sources of skeletal progenitor cells, the periosteum plays a crucial role not only in bone development and growth, but also during bone fracture healing. Aided by the continuous expansion of tools and techniques, we are now starting to acquire more insight into the specific role and regulation of periosteal cells. From a therapeutic point of view, the periosteum has attracted much attention as a cell source for bone tissue engineering purposes. This interest derives not only from the physiological role of the periosteum during bone repair, but is also supported by the unique properties and marked bone-forming potential of expanded periosteum-derived cells. We provide an overview of the current knowledge of periosteal cell biology, focusing on the cellular composition and molecular regulation of this remarkable tissue, as well as the application of periosteum-derived cells in regenerative medicine approaches. This article is part of a Special Issue entitled “Stem Cells and Bone”.     

Therapeutic effects of intrabone and systemic mesenchymal stem cell cytotherapy on myeloma bone disease and tumor growth

The cytotherapeutic potential of mesenchymal stem cells (MSCs) has been evaluated in various disorders including those involving inflammation, autoimmunity, bone regeneration, and cancer. Multiple myeloma (MM) is a systemic malignancy associated with induction of osteolytic lesions that often are not repaired even after prolonged remission. The aims of this study were to evaluate the effects of intrabone and systemic injections of MSCs on MM bone disease, tumor growth, and tumor regrowth in the severe combined immunodeficiency (SCID)-rab model and to shed light on the exact localization of systemically injected MSCs. Intrabone injection of MSCs, but not hematopoietic stem cells, into myelomatous bones prevented MM-induced bone disease, promoted bone formation, and inhibited MM growth. After remission was induced with melphalan treatment, intrabone-injected MSCs promoted bone formation and delayed myeloma cell regrowth in bone. Most intrabone or systemically injected MSCs were undetected 2 to 4 weeks after injection. The bone-building effects of MSCs were mediated through activation of endogenous osteoblasts and suppression of osteoclast activity. Although a single intravenous injection of MSCs had no effect on MM, sequential weekly intravenous injections of MSCs prevented MM-induced bone disease but had no effect on tumor burden. MSCs expressed high levels of anti-inflammatory (eg, HMOX1) and bone-remodeling (eg, Decorin, CYR61) mediators. In vitro, MSCs promoted osteoblast maturation and suppressed osteoclast formation, and these effects were partially prevented by blocking decorin. A subset of intravenously or intracardially injected MSCs trafficked to myelomatous bone in SCID-rab mice. Although the majority of intravenously injected MSCs were trapped in lungs, intracardially injected MSCs were mainly localized in draining mesenteric lymph nodes. This study shows that exogenous MSCs act as bystander cells to inhibit MM-induced bone disease and tumor growth and that systemically injected MSCs are attracted to bone by myeloma cells or conditions induced by MM and inhibit bone disease.     

Osteoclast‐Derived Complement Component 3a Stimulates Osteoblast Differentiation

Bone remodeling is regulated by a coupling of resorption to subsequent formation; however, the “coupling factor” and underlying mechanism are not fully understood. Here, we found that the condition medium (CM) of mature osteoclasts contains a humoral factor that stimulates the differentiation of primary osteoblasts, as determined by alkaline phosphatase (ALP) activity. We purified osteoblastogenesis‐stimulating activity from 3 L of osteoclast CM through successive ion exchange chromatographies by monitoring the ALP activity of osteoblasts and identified complement component 3 (C3). Expression of the C3 gene increased during osteoclastogenesis, and the cleavage product C3a was detected by ELISA in the CM of osteoclasts but not in that of bone marrow macrophages. The osteoblastogenesis‐stimulating activity present in osteoclast CM was inhibited by a specific antagonist of the C3a receptor (C3aR), SB290157. Conversely, the retroviral expression of C3a as well as treatment with the C3aR agonist, benzeneacetamide, stimulated osteoblast differentiation. C3 gene expression in bone was increased in the high bone turnover states of ovariectomy (OVX) or a receptor activator of NF‐κB ligand (RANKL) injection, and blocking the action of C3a with the daily administration of SB290157 resulted in the attenuation of bone formation elevated by OVX and the exacerbation of bone loss. These results suggest that osteoclast‐derived C3a functions in the relay from bone resorption to formation and may be a candidate for a coupling factor. © 2014 American Society for Bone and Mineral Research.     

Targeting of Mesenchymal Stromal Cells by Cre‐Recombinase Transgenes Commonly Used to Target Osteoblast Lineage Cells

The targeting specificity of tissue‐specific Cre‐recombinase transgenes is a key to interpreting phenotypes associated with their use. The Ocn‐Cre and Dmp1‐Cre transgenes are widely used to target osteoblasts and osteocytes, respectively. Here, we used high‐resolution microscopy of bone sections and flow cytometry to carefully define the targeting specificity of these transgenes. These transgenes were crossed with Cxcl12^gfp mice to identify Cxcl12‐abundant reticular (CAR) cells, which are a perivascular mesenchymal stromal population implicated in hematopoietic stem/progenitor cell maintenance. We show that in addition to osteoblasts, Ocn‐Cre targets a majority of CAR cells and arteriolar pericytes. Surprisingly, Dmp1‐Cre also targets a subset of CAR cells, in which expression of osteoblast‐lineage genes is enriched. Finally, we introduce a new tissue‐specific Cre‐recombinase, Tagln‐Cre, which efficiently targets osteoblasts, a majority of CAR cells, and both venous sinusoidal and arteriolar pericytes. These data show that Ocn‐Cre and Dmp1‐Cre target broader stromal cell populations than previously appreciated and may aid in the design of future studies. Moreover, these data highlight the heterogeneity of mesenchymal stromal cells in the bone marrow and provide tools to interrogate this heterogeneity. © 2016 American Society for Bone and Mineral Research.