Recombinant human bone morphogenetic protein-2 enhances expression of interleukin-6 and transforming growth factor-β1 genes in normal human osteoblast-like cells

The process of recombinant human bone morphogenetic protein-2 (rhBMP-2)-induced endochondral ossification involves (1) the proliferation and differentiation of mesenchymal cells into chondroblasts and osteoblasts; (2) the production and maturation of cartilage and bone matrix; and (3) the differentiation of circulating osteoclast precursor cells into osteoclasts. Currently the molecular mechanisms of these complex sequential events are unknown. It seemed reasonable to us to assume that communication between cells through soluble mediators during bone induction by rhBMP-2 may play an important role in the sequential differentiation of chondroblasts, osteoblasts, and osteoclasts. We have therefore used a human osteoblast-like initial transfectant cell line (HOBIT) to study the effect of rhBMP-2 on gene expression of interleukin-6 (IL-6) and transforming growth factor-β1 (TGF-β1), both of which affect osteogenesis and ostoeclastogenesis. Our results have demonstrated that rhBMP-2 acts on HOBIT cells to stimulate expression of IL-6 and TGF-β1 genes and the production of IL-6. Enhancement of gene expression of IL-6 and TGF-β1 by rhBMP-2 was both sensitive (half maximal effect at approximately 10 ng/ml) and potent (maximum induction was approximately four and threefold greater than controls, respectively). Time course studies showed that the induction of TGF-β1 and IL-6 mRNA occurs within short periods—4 and 8 hours after exposure to rhBMP-2, respectively. Interestingly, these effects, however, were not accompanied by the mitogenic action of rhBMP-2. It suggests that rhBMP-2 enhances IL-6 and TGF-β1 production during osteogenesis and at least in part mediates the complex sequential differentiation of chondroblasts, osteoblasts, and osteoclasts during rhBMP-2-induced endochondral ossification. © 1994 wiley-Liss, Inc.     

Heparanase mRNA expression during fracture repair in mice

Bone fracture healing takes place through endochondral ossification where cartilaginous callus is replaced by bony callus. Vascular endothelial growth factor (VEGF) is a requisite for endochondral ossification, where blood vessel invasion of cartilaginous callus is crucial. Heparanase is an endoglucuronidase that degrades heparan sulfate proteoglycans (HSPG) and releases heparin-binding growth factors including VEGF as an active form. To investigate the role of heparanase in VEGF recruitment during fracture healing, the expression of heparanase mRNA and VEGF, and vessel formation were examined in mouse fractured bone. On days 5 and 7 after the fracture, when mesenchymal cells proliferated and differentiated into chondrocytes, heparanase mRNA was detected in osteo(chondro)clasts and their precursors, but not in the inflammatory phase (day 3). On day 10, both VEGF and HSPG were produced by hypertrophic chondrocytes of the cartilaginous callus and by osteoblasts of the bony callus; numerous osteo(chondro)clasts resorbing the cartilage expressed strong heparanase signals. Adjacent to the cartilage resorption sites, angiogenesis with CD31-positive endothelial cells and osteogenesis with osteonectin-positive osteoblasts were observed. On days 14 and 21, osteoclasts in the woven bone tissue expressed heparanase mRNA. These data suggest that by producing heparanase osteo(chondro)clasts contribute to the recruitment of the active form of VEGF. Thus osteo(chondro)clasts may promote local angiogenesis as well as callus resorption in endochondral ossification during fracture healing.     

Chondrocytes Transdifferentiate into Osteoblasts in Endochondral Bone during Development,Postnatal Growth and Fracture Healing in Mice

One of the crucial steps in endochondral bone formation is the replacement of a cartilage matrix produced by chondrocytes with bone trabeculae made by osteoblasts. However, the precise sources of osteoblasts responsible for trabecular bone formation have not been fully defined. To investigate whether cells derived from hypertrophic chondrocytes contribute to the osteoblast pool in trabecular bones, we genetically labeled either hypertrophic chondrocytes by Col10a1-Cre or chondrocytes by tamoxifen-induced Agc1-CreERT2 using EGFP, LacZ or Tomato expression. Both Cre drivers were specifically active in chondrocytic cells and not in perichondrium, in periosteum or in any of the osteoblast lineage cells. These in vivo experiments allowed us to follow the fate of cells labeled in Col10a1-Cre or Agc1-CreERT2 -expressing chondrocytes. After the labeling of chondrocytes, both during prenatal development and after birth, abundant labeled non-chondrocytic cells were present in the primary spongiosa. These cells were distributed throughout trabeculae surfaces and later were present in the endosteum, and embedded within the bone matrix. Co-expression studies using osteoblast markers indicated that a proportion of the non-chondrocytic cells derived from chondrocytes labeled by Col10a1-Cre or by Agc1-CreERT2 were functional osteoblasts. Hence, our results show that both chondrocytes prior to initial ossification and growth plate chondrocytes before or after birth have the capacity to undergo transdifferentiation to become osteoblasts. The osteoblasts derived from Col10a1-expressing hypertrophic chondrocytes represent about sixty percent of all mature osteoblasts in endochondral bones of one month old mice. A similar process of chondrocyte to osteoblast transdifferentiation was involved during bone fracture healing in adult mice. Thus, in addition to cells in the periosteum chondrocytes represent a major source of osteoblasts contributing to endochondral bone formation in vivo.     

Wnt/beta-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis

Chondrocytes and osteoblasts are two primary cell types in the skeletal system that are differentiated from common mesenchymal progenitors. It is believed that osteoblast differentiation is controlled by distinct mechanisms in intramembranous and endochondral ossification. We have found that ectopic canonical Wnt signaling leads to enhanced ossification and suppression of chondrocyte formation. Conversely, genetic inactivation of beta-catenin, an essential component transducing the canonical Wnt signaling, causes ectopic formation of chondrocytes at the expense of osteoblast differentiation during both intramembranous and endochondral ossification. Moreover, inactivation of beta-catenin in mesenchymal progenitor cells in vitro causes chondrocyte differentiation under conditions allowing only osteoblasts to form. Our results demonstrate that beta-catenin is essential in determining whether mesenchymal progenitors will become osteoblasts or chondrocytes regardless of regional locations or ossification mechanisms. Controlling Wnt/beta-catenin signaling is a common molecular mechanism underlying chondrocyte and osteoblast differentiation and specification of intramembranous and endochondral ossification.     

Growth cartilage calcification and formation of bone trabeculae are late and dissociated events in the endochondral ossification of <Emphasis Type="Italic">Rana catesbeiana</Emphasis>

Endochondral ossification in the growth cartilage of long bones from the bullfrog Rana catesbeiana was examined. In stage-46 tadpoles and 1-year-old animals, the hypertrophic cartilage had a smooth contact with the bone marrow and the matrix showed no calcification or endochondral bone formation. In spite of showing no aspects of calcification, the chondrocytes exhibited alkaline phosphatase activity and some of them died by apoptosis. However, matrix calcification and endochondral ossification were observed in 2-year-old bullfrogs. Calcium deposits appeared as isolated or coalesced spherical structures in the extracellular matrix of hypertrophic cartilage. Bone trabeculae were restricted to the central area at the sites where the hypertrophic cartilage surface was exposed to the bone marrow. Cartilage matrix calcification and the formation of bone trabeculae were not dependent on each other. Osteoclasts were involved in calcified matrix resorption. These results demonstrate that the calcification of hypertrophic cartilage and the deposition of bone trabeculae are late events in R. catesbeiana and do not contribute to the development and growth of long bones in adults. These processes may play a role in reinforcing bony structures as the bullfrog gains weight in adulthood. In addition, the deposition of bone trabeculae is not dependent on cartilage matrix calcification.     

Leukaemia inhibitory factor (lif) is expressed in hypertrophic chondrocytes and vascular sprouts during osteogenesis

Avascular cartilage is replaced by highly vascularized bone tissue during endochondral ossification, a process involving capillary invasion of calcified hypertrophic cartilage in association with apoptosis of hypertrophic chondrocytes, degradation of cartilage matrix and deposition of bone matrix. All of these events are closely controlled, especially by cytokines and growth factors. Leukaemia inhibitory factor (LIF), a member of the gp130 cytokine family, is involved in osteoarticular tissue metabolism and might participate in osteogenesis. Immunohistochemical staining showed that LIF is expressed in hypertrophic chondrocytes and vascular sprouts of cartilage and bone during rat and human osteogenesis. LIF is also present in osteoblasts but not in osteoclasts. Observations in a rat endochondral ossification model were confirmed by studies of human cartilage biopsies from foetuses with osteogenesis imperfecta. LIF was never detected in adult articular chondrocytes and bone-marrow mesenchymal cells. These results and other data in the literature suggest that LIF is involved in the delicate balance between the rate of formation of calcified cartilage and its vascularization for bone development.     

Fibromodulin is expressed by both chondrocytes and osteoblasts during fetal bone development

Fibromodulin, a keratan-sulfate proteoglycan, was first isolated in articular cartilage and tendons. We have identified fibromodulin as a gene regulated during BMP-2-induced differentiation of a mouse prechondroblastic cell line. Because expression of fibromodulin during endochondral bone formation has not been studied, we examined whether selected cells of the chondrocytic and osteoblastic lineage expressed fibromodulin. Fibromodulin mRNA was detected in conditionally immortalized murine bone marrow stromal cells, osteoblasts, and growth plate chondrocytes, as well as in primary murine calvarial osteoblasts. We, therefore, investigated the temporo-spatial expression of fibromodulin in vivo during endochondral bone formation by in situ hybridization. Fibromodulin was first detected at 15.5 days post coitus (dpc) in the perichondrium and proliferating chondrocytes. Fibromodulin mRNA was also detected at 15.5 dpc in the bone collar and periosteum. At later time points fibromodulin was expressed in the primary spongiosa and the endosteum. To determine whether fibromodulin was expressed during intramembranous bone formation as well, in situ hybridization was performed on calvariae. Fibromodulin mRNA was present in calvarial osteoblasts from 15.5 dpc. These results demonstrate that fibromodulin is developmentally expressed in cartilage and bone cells during endochondral and intramembranous ossification. These findings suggest that this extracellular matrix protein plays a role in both endochondral and intramembranous bone formation.     

Role of CTGF/HCS24/ecogenin in skeletal growth control

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) is a multifunctional growth factor for chondrocytes, osteoblasts, and vascular endothelial cells. CTGF/Hcs24 promotes the proliferation and maturation of growth cartilage cells and articular cartilage cells in culture and hypertrophy of growth cartilage cells in culture. The factor also stimulates the proliferation and differentiation of cultured osteoblastic cells. Moreover, CTGF/Hcs24 promotes the adhesion, proliferation, and migration of vascular endothelial cells, as well as induces tube formation by the cells and strong angiogenesis in vivo. Because angiogenesis is critical for the replacement of cartilage with bone at the final stage of endochondral ossification and because gene expression of CTGF/Hcs24 predominates in hypertrophic chondrocytes in the physiological state, a major physiological role for this factor should be the promotion of the entire process of endochondral ossification, with the factor acting on the above three types of cells as a paracrine factor. Thus, CTGF/Hcs24 should be called "ecogenin: endochondral ossification genetic factor." In addition to hypertrophic chondrocytes, osteoblasts activated by various stimuli including wounding also express a significantly high level of CTGF/Hcs24. These findings in conjunction with in vitro findings about osteoblasts mentioned above suggest the involvement of CTGF/Hcs24 in intramembranous ossification and bone modeling/remodeling. Because angiogenesis is also critical for intramembranous ossification and bone remodeling, CTGF/Hcs24 expressed in endothelial cells activated by various stimuli including wounding may also play important roles in direct bone formation. In conclusion, although the most important physiological role of CTGF/Hcs24 is ecogenin action, the factors also play important roles in skeletal growth and modeling/remodeling via its direct action on osteoblasts under both physiological and pathological conditions.     

The expression of the fibrillar collagen genes during fracture healing: heterogeneity of the matrices and differentiation of the osteoprogenitor cells

The cells that express the genes for the fibrillar collagens, types I, II, III and V, during callus development in rabbit tibial fractures healing under stable and unstable mechanical conditions were localized. The fibroblast-like cells in the initial fibrous matrix express types I, III and V collagen mRNAs. Osteoblasts, and osteocytes in the newly formed membranous bone under the periosteum, express the mRNAs for types I, III and V collagens, but osteocytes in the mature trabeculae express none of these mRNAs. Cartilage formation starts at 7 days in calluses forming under unstable mechanical conditions. The differentiating chondrocytes express both types I and II collagen mRNAs, but later they cease expression of type I collagen mRNA. Both types I and II collagens were located in the cartilaginous areas. The hypertrophic chondrocytes express neither type I, nor type II, collagen mRNA. Osteocalcin protein was located in the bone and in some cartilaginous regions. At 21 days, irrespective of the mechanical conditions, the callus consists of a layer of bone; only a few osteoblasts lining the cavities now express type I collagen mRNA.We suggest that osteoprogenitor cells in the periosteal tissue can differentiate into either osteoblasts or chondrocytes and that some cells may exhibit an intermediate phenotype between osteoblasts and chondrocytes for a short period. The finding that hypertrophic chondrocytes do not express type I collagen mRNA suggests that they do not transdifferentiate into osteoblasts during endochondral ossification in fracture callus.     

BMP-2, BMP-4, and PDGF-bb stimulate chemotactic migration of primary human mesenchymal progenitor cells

For bone development, remodeling, and repair; the recruitment of mesenchymal progenitor cells (MPC) and their differentiation to osteoblasts is mandatory. The process of migration is believed to be regulated in part by growth factors stored within the bone matrix and released by bone resorption. In this study, primary human MPCs and to osteoblasts differentiated progenitor cells were examined for chemotaxis in response to human basic fibroblast growth factor (rhbFGF), human transforming growth factor beta 1 (rhTGF-beta1), human platelet derived growth factor bb (rhPDGF-bb), human bone morphogenetic protein-2 (rhBMP-2), and recombinant bone morphogenetic protein-4 of Xenopus laevis (rxBMP-4) from 0.001 to 1.0 ng/ml each. The results of migration were expressed as a chemotactic index (CI). Migration of primary human progenitor cells was stimulated by rhBMP-2, rxBMP-4, and rhPDGF-bb in a dose-dependent manner. The increase of CI was up to 3.5-fold for rhBMP-2, 3.6-fold for rxBMP-4, and up to 22-fold for rhPDGF-bb, whereas rhTGF-beta1 and rhbFGF did not stimulate cell migration in the concentration range tested. In contrast differentiated progenitor cells behave similar to primary human osteoblasts. RhBMP-2, rhPDGF-bb, and rhTGF-beta1 stimulated the migration from 2.2 to 2.4-fold each, while rxBMP-4 and rhbFGF reached only a CI of 1.7-1.6. The effect of rhBMP-2, rxBMP-4, and rhPDGF-bb as chemoattractive proteins for primary human MPC, including the change in response to growth factors after differentiation suggests a functional role for recruitment of MPCs during bone development and remodeling, as well as fracture healing.     

Ihh signaling is directly required for the osteoblast lineage in the endochondral skeleton

Indian hedgehog (Ihh) is indispensable for development of the osteoblast lineage in the endochondral skeleton. In order to determine whether Ihh is directly required for osteoblast differentiation, we have genetically manipulated smoothened (Smo), which encodes a transmembrane protein that is essential for transducing all Hedgehog (Hh) signals. Removal of Smo from perichondrial cells by the Cre-LoxP approach prevents formation of a normal bone collar and also abolishes development of the primary spongiosa. Analysis of chimeric embryos composed of wild-type and Smo(n/n) cells indicates that Smo(n/n) cells fail to contribute to osteoblasts in either the bone collar or the primary spongiosa but generate ectopic chondrocytes. In order to assess whether Ihh is sufficient to induce bone formation in vivo, we have analyzed the bone collar in the long bones of embryos in which Ihh was artificially expressed in all chondrocytes by the UAS-GAL4 bigenic system. Although ectopic Ihh does not induce overt ossification along the entire cartilage anlage, it promotes progression of the bone collar toward the epiphysis, suggesting a synergistic effect between ectopic Ihh and endogenous factors such as the bone morphogenetic proteins (BMPs). In keeping with this model, Hh signaling is further found to be required in BMP-induced osteogenesis in cultures of a limb-bud cell line. Taken together, these results demonstrate that Ihh signaling is directly required for the osteoblast lineage in the developing long bones and that Ihh functions in conjunction with other factors such as BMPs to induce osteoblast differentiation. We suggest that Ihh acts in vivo on a potential progenitor cell to promote osteoblast and prevent chondrocyte differentiation.     

L-Maf,a downstream target of Pax6, is essential for chick lens development

Vascular endothelial growth factor (VEGF)-mediated angiogenesis is an important part of bone formation. To clarify the role of VEGF isoforms in endochondral bone formation, we examined long bone development in mice expressing exclusively the VEGF120 isoform (VEGF120/120 mice). Neonatal VEGF120/120 long bones showed a completely disturbed vascular pattern, concomitant with a 35% decrease in trabecular bone volume, reduced bone growth and a 34% enlargement of the hypertrophic chondrocyte zone of the growth plate. Surprisingly, embryonic hindlimbs at a stage preceding capillary invasion exhibited a delay in bone collar formation and hypertrophic cartilage calcification. Expression levels of marker genes of osteoblast and hypertrophic chondrocyte differentiation were significantly decreased in VEGF120/120 bones. Furthermore, inhibition of all VEGF isoforms in cultures of embryonic cartilaginous metatarsals, through the administration of a soluble receptor chimeric protein (mFlt-1/Fc), retarded the onset and progression of ossification, suggesting that osteoblast and/or hypertrophic chondrocyte development were impaired. The initial invasion by osteoclasts and endothelial cells into VEGF120/120 bones was retarded, associated with decreased expression of matrix metalloproteinase-9. Our findings indicate that expression of VEGF164 and/or VEGF188 is important for normal endochondral bone development, not only to mediate bone vascularization but also to allow normal differentiation of hypertrophic chondrocytes, osteoblasts, endothelial cells and osteoclasts.     

Signaling Cascades Governing Cdc42-Mediated Chondrogenic Differentiation and Mensenchymal Condensation

Endochondral ossification consists of successive steps of chondrocyte differentiation, including mesenchymal condensation, differentiation of chondrocytes, and hypertrophy followed by mineralization and ossification. Loss-of-function studies have revealed that abnormal growth plate cartilage of the Cdc42 mutant contributes to the defects in endochondral bone formation. Here, we have investigated the roles of Cdc42 in osteogenesis and signaling cascades governing Cdc42-mediated chondrogenic differentiation. Though deletion of Cdc42 in limb mesenchymal progenitors led to severe defects in endochondral ossification, either ablation of Cdc42 in limb preosteoblasts or knockdown of Cdc42 in vitro had no obvious effects on bone formation and osteoblast differentiation. However, in Cdc42 mutant limb buds, loss of Cdc42 in mesenchymal progenitors led to marked inactivation of p38 and Smad1/5, and in micromass cultures, Cdc42 lay on the upstream of p38 to activate Smad1/5 in bone morphogenetic protein-2-induced mesenchymal condensation. Finally, Cdc42 also lay on the upstream of protein kinase B to transactivate Sox9 and subsequently induced the expression of chondrocyte differential marker in transforming growth factor-β1-induced chondrogenesis. Taken together, by using biochemical and genetic approaches, we have demonstrated that Cdc42 is involved not in osteogenesis but in chondrogenesis in which the BMP2/Cdc42/Pak/p38/Smad signaling module promotes mesenchymal condensation and the TGF-β/Cdc42/Pak/Akt/Sox9 signaling module facilitates chondrogenic differentiation.     

Distinguishing the contributions of the perichondrium, cartilage, and vascular endothelium to skeletal development

During the initiation of endochondral ossification three events occur that are inextricably linked in time and space: chondrocytes undergo terminal differentiation and cell death, the skeletal vascular endothelium invades the hypertrophic cartilage matrix, and osteoblasts differentiate and begin to deposit a bony matrix. These developmental programs implicate three tissues, the cartilage, the perichondrium, and the vascular endothelium. Due to their intimate associations, the interactions among these three tissues are exceedingly difficult to distinguish and elucidate. We developed an ex vivo system to unlink the processes initiating endochondral ossification and establish more precisely the cellular and molecular contributions of the three tissues involved. In this ex vivo system, the renal capsule of adult mice was used as a host environment to grow skeletal elements. We first used a genetic strategy to follow the fate of cells derived from the perichondrium and from the vasculature. We found that the perichondrium, but not the host vasculature, is the source of both trabecular and cortical osteoblasts. Endothelial cells residing within the perichondrium are the first cells to participate in the invasion of the hypertrophic cartilage matrix, followed by endothelial cells derived from the host environment. We then combined these lineage analyses with a series of tissue manipulations to address how the absence of the perichondrium or the vascular endothelium affected skeletal development. We show that although the perichondrium influences the rate of chondrocytes maturation and hypertrophy, it is not essential for chondrocytes to undergo late hypertrophy. The perichondrium is crucial for the proper invasion of blood vessels into the hypertrophic cartilage and both the perichondrium and the vasculature are essential for endochondral ossification. Collectively, these studies clarify further the contributions of the cartilage, perichondrium, and vascular endothelium to long bone development.     

Conversion of vascular endothelial cells into multipotent stem-like cells

Mesenchymal stem cells can give rise to several cell types, but varying results depending on isolation methods and tissue source have led to controversies about their usefulness in clinical medicine. Here we show that vascular endothelial cells can transform into multipotent stem-like cells by an activin-like kinase-2 (ALK2) receptor-dependent mechanism. In lesions from individuals with fibrodysplasia ossificans progressiva (FOP), a disease in which heterotopic ossification occurs as a result of activating ALK2 mutations, or from transgenic mice expressing constitutively active ALK2, chondrocytes and osteoblasts expressed endothelial markers. Lineage tracing of heterotopic ossification in mice using a Tie2-Cre construct also suggested an endothelial origin of these cell types. Expression of constitutively active ALK2 in endothelial cells caused endothelial-to-mesenchymal transition and acquisition of a stem cell-like phenotype. Similar results were obtained by treatment of untransfected endothelial cells with the ligands transforming growth factor-β2 (TGF-β2) or bone morphogenetic protein-4 (BMP4) in an ALK2-dependent manner. These stem-like cells could be triggered to differentiate into osteoblasts, chondrocytes or adipocytes. We suggest that conversion of endothelial cells to stem-like cells may provide a new approach to tissue engineering.     

Perlecan modulates VEGF signaling and is essential for vascularization in endochondral bone formation

Perlecan (Hspg2) is a heparan sulfate proteoglycan expressed in basement membranes and cartilage. Perlecan deficiency (Hspg2(-/-)) in mice and humans causes lethal chondrodysplasia, which indicates that perlecan is essential for cartilage development. However, the function of perlecan in endochondral ossification is not clear. Here, we report the critical role of perlecan in VEGF signaling and angiogenesis in growth plate formation. The Hspg2(-/-) growth plate was significantly wider but shorter due to severely impaired endochondral bone formation. Hypertrophic chondrocytes were differentiated in Hspg2(-/-) growth plates; however, removal of the hypertrophic matrix and calcified cartilage was inhibited. Although the expression of MMP-13, CTGF, and VEGFA was significantly upregulated in Hspg2(-/-) growth plates, vascular invasion into the hypertrophic zone was impaired, which resulted in an almost complete lack of bone marrow and trabecular bone. We demonstrated that cartilage perlecan promoted activation of VEGF/VEGFR by binding to the VEGFR of endothelial cells. Expression of the perlecan transgene specific to the cartilage of Hspg2(-/-) mice rescued their perinatal lethality and growth plate abnormalities, and vascularization into the growth plate was restored, indicating that perlecan in the growth plate, not in endothelial cells, is critical in this process. These results suggest that perlecan in cartilage is required for activating VEGFR signaling of endothelial cells for vascular invasion and for osteoblast migration into the growth plate. Thus, perlecan in cartilage plays a critical role in endochondral bone formation by promoting angiogenesis essential for cartilage matrix remodeling and subsequent endochondral bone formation.     

A biochemical strategy for simulation of endochondral and intramembranous ossification

Following the assumption that parathyroid hormone related protein and Indian hedgehog form a biochemical regulatory loop for the endochondral process and bone morphogenetic protein 2 and Noggin in the intramembranous process, this paper implements these regulatory mechanisms. For this purpose, we use a set of reaction–diffusion equations that are widely used in morphogenesis, in which biochemical factors are assumed to be secreted by precursor cells, mesenchymal cells and chondrocytes, in endochondral and intramembranous ossification, respectively. The solution leads to the so-called Turing patterns, which represent these processes of ossification in a very approximate way.