The role of morphogens in endochondral ossification

Summary The formation of bone occurs normally by one of two developmental processes: intramembranous or endochondral ossification. Endochondral ossification occurs in the morphogenesis of the limb buds and growth plates, and in the regeneration of bone following injury (fracture callus). Two classes of diffusible morphogen-like molecules (MLMs) involved in limb development are the bone morphogenetic proteins (BMPs) and retinoic acid (RA). These MLMs are associated, respectively, with the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA) of the primitive limb bud. They function as potent regulators of pattern formation and are involved in tissue proliferation and differentiation. The presence of endochondral ossification in fracture callus suggests a role for MLMs in that process as well. To date, virtually nothing is known about the role of morphogens in the regeneration of bone (fracture healing). In this article, we review the current knowledge of MLMs in bone formation and propose a theory on their role in fracture healing. We hypothesize that MLMs involved in fracture healing may also express spatial and temporal information. A more complete understanding of the role of morphogens in both limb development and fracture healing is of major importance to practicing orthopedists and their patients.     

F‐spondin regulates chondrocyte terminal differentiation and endochondral bone formation

This study examines the role of F‐spondin, an extracellular matrix protein of osteoarthritic cartilage, during chondrocyte maturation in embryonic growth plate cartilage. In chick tibia, F‐spondin expression localized to the hypertrophic and calcified zones of the growth plate. Functional studies using tibial organ cultures indicated that F‐spondin inhibited (～35%, p = 0.02), and antibodies to F‐spondin increased (～30%, p < 0.1) longitudinal limb growth relative to untreated controls. In cell cultures, induction of chondrocyte maturation, by retinoic acid (RA) or transforming growth factor (TGF)‐β treatment led to a significant upregulation of F‐spondin (p < 0.05). F‐spondin transfection increased mineral deposition, alkaline phosphatase (AP) and matrix metalloproteinase (MMP)‐13 mRNA levels (p < 0.05), and AP activity following RA stimulation, compared to mock transfected controls. Using AP as a differentiation marker we then investigated the mechanism of F‐spondin promaturation effects. Blocking endogenous F‐spondin via its thrombospondin (TSR) domain inhibited RA induced AP activity 40% compared to controls (p < 0.05). The stimulatory effect of F‐spondin on AP expression was also inhibited following depletion of TGF‐β from culture supernatants. Our findings indicate that F‐spondin is expressed in embryonic cartilage, where it has the capacity to enhance chondrocyte terminal differentiation and mineralization via interactions in its TSR domain and TGF‐β dependent pathways. Published by Wiley Periodicals, Inc. J Orthop Res 28:1323–1329, 2010     

The critical role of the epidermal growth factor receptor in endochondral ossification

Loss of epidermal growth factor receptor (EGFR) activity in mice alters growth plate development, impairs endochondral ossification, and retards growth. However, the detailed mechanism by which EGFR regulates endochondral bone formation is unknown. Here, we show that administration of an EGFR-specific small-molecule inhibitor, gefitinib, into 1-month-old rats for 7 days produced profound defects in long bone growth plate cartilage characterized by epiphyseal growth plate thickening and massive accumulation of hypertrophic chondrocytes. Immunostaining demonstrated that growth plate chondrocytes express EGFR, but endothelial cells and osteoclasts show little to no expression. Gefitinib did not alter chondrocyte proliferation or differentiation and vascular invasion into the hypertrophic cartilage. However, osteoclast recruitment and differentiation at the chondro-osseous junction were attenuated owing to decreased RANKL expression in the growth plate. Moreover, gefitinib treatment inhibited the expression of matrix metalloproteinases (MMP-9, -13, and -14), increased the amount of collagen fibrils, and decreased degraded extracellular matrix products in the growth plate. In vitro, the EGFR ligand transforming growth factor α (TGF-α) strongly stimulated RANKL and MMPs expression and suppressed osteoprotegerin (OPG) expression in primary chondrocytes. In addition, a mouse model of cartilage-specific EGFR inactivation exhibited a similar phenotype of hypertrophic cartilage enlargement. Together our data demonstrate that EGFR signaling supports osteoclastogenesis at the chondro-osseous junction and promotes chondrogenic expression of MMPs in the growth plate. Therefore, we conclude that EGFR signaling plays an essential role in the remodeling of growth plate cartilage extracellular matrix into bone during endochondral ossification.     

Wnt-mediated reciprocal regulation between cartilage and bone development during endochondral ossification

The role of Wnt signaling is extensively studied in skeletal development and postnatal bone remodeling, mostly based on the genetic approaches of β-catenin manipulation. However, given their independent function, a requirement for β-catenin is not the same as that for Wnt. Here, we investigated the effect of Wnt proteins in both tissues through generating cartilage- or bone-specific Wls null mice, respectively. Depletion of Wls by Col2-Cre, which would block Wnt secretion in the chondrocytes and perichondrium, delayed chondrocyte hypertrophy in the growth plate and impaired perichondrial osteogenesis. Loss of Wls in chondrocytes also disturbed the proliferating chondrocyte morphology and division orientation, which was similar to the defect observed in Wnt5a null mice. On the other hand, inactivation of Wls in osteoblasts by Col1-Cre resulted in a shorter hypertrophic zone and an increase of TRAP positive cell number in the chondro-osseous junction of growth plate, coupled with a decrease in bone mass. Taken together, our studies reveal that Wnt proteins not only modulate differentiation and cellular communication within populations of chondrocytes, but also mediate the cross regulation between the chondrocytes and osteoblasts in growth plate.     

A role for the BMP antagonist chordin in endochondral ossification.

Bone morphogenetic proteins (BMPs) are ubiquitous regulators of cellular growth and differentiation. A variety of processes modulate BMP activity, including negative regulation by several distinct binding proteins. One such BMP antagonist chordin has a role in axis determination and neural induction in the early embryo. In this study, a role for chordin during endochondral ossification has been investigated. During limb development, Chordin expression was detected only at the distal ends of the skeletal elements. In cultured embryonic sternal chondrocytes, Chordin expression was related inversely to the stages of maturation. Further, treating cultured chondrocytes with chordin interfered with maturation induced by treatment with BMP-2. These results suggest that chordin may negatively regulate chondrocyte maturation and limb growth in vivo. To address this hypothesis, chordin protein was expressed ectopically in Hamburger-Hamilton (HH) stage 25-27 embryonic chick limbs. The phenotypic changes and alteration of gene expression in treated limbs revealed that overexpression of chordin protein delayed chondrocyte maturation in developing skeletal elements. In summary, these findings strongly support a role for chordin as a negative regulator of endochondral ossification.     

Changes in lipids during matrix: induced endochondral bone formation

The changes in lipids occurring during the process of endochondral ossification have been characterized by studying the discrete phases of matrix-induced endochondral bone formation in the rat. Calcium-acidic phospholipid-phosphate complexes were shown to increase in concentration during cartilage calcification (day 9) and to peak in content during early bone formation (days 11-13), the times during which the rate of mineral deposition, as indicated by the change in ash weight was greatest. These data support the hypothesis that the calcium-acidic phospholipid-phosphate complexes play a role in the in vivo initiation of hydroxyapatite deposition. The overall lipid composition of the induced matrix newly formed cartilage (days 7-9) was comparable to that of normal cartilage, with the phospholipid composition matching that of chondrocyte plasma membranes. Times of vascular invasion and formation of marrow cavities were marked by elevated total lipid and triglyceride contents.     

Changes in lipids during matrix: Induced endochondral bone formation

Summary The changes in lipids occurring during the process of endochondral ossification have been characterized by studying the discrete phases of matrix-induced endochondral bone formation in the rat. Calcium-acidic phospholipid-phosphate complexes were shown to increase in concentration during cartilage calcification (day 9) and to peak in content during early bone formation (day 11–13), the times during which the rate of mineral deposition, as indicated by the change in ash weight was greatest. These data support the hypothesis that the calcium-acidic phospholipid-phosphate complexes play a role in thein vivo initiation of hydroxyapatite deposition. The overall lipid composition of the induced matrix newly formed cartilage (days 7–9) was comparable to that of normal cartilage, with the phospholipid composition matching that of chondrocyte plasma membranes. Times of vascular invasion and formation of marrow cavities were marked by elevated total lipid and triglyceride contents.     

The role of three genetically distinct collagen types in endochondral ossification and calcification of cartilage.

The role of three genetically distinct collagen types in the formation of endochondral bone and in calcification and resorption of cartilage has been assessed. Using antibodies specific to types I, II and III collagen we have demonstrated in the embryonic chick tibia that endochondral bone formation began with deposition of type III collagen in lacunae of hypertropic chondrocytes by invading bone-marrow-derived cells. This was followed by the deposition of type I collagen, which is the collagenous constituent of endochondral osteoid. At later stages of development endochondral osteoid was found in the epiphysial growth plate in apparently intact lacunae of hypertrophic chondrocytes; this indicated that the latter might contribute to the synthesis of osteoid type I collagen. Immuno-histological staining for collagen types, and von Kossa staining for calcium phosphate on parallel sections, demonstrated that type I and type II collagen matrices were substrates for calcification. Endochondral bone (with type I collagen) was found on scaffolding of both uncalcified and calcified cartilage (with type II collagen), indicating that calcification of endochondral osteoid and of the underlying cartilage occurred independentyl. Spicules of endochondral cancellous bone of a four-week-old chick contained a core of calcified type II collagen.     

The glycosylation profile of osteoadherin alters during endochondral bone formation

Endochondral bone formation involves the dynamic interplay between the cells and their extracellular environment to facilitate the deposition of a calcified matrix. Numerous molecules are involved within this process, including collagens and non-collagenous proteins, and their post-translational modifications have been shown to effect their biomolecular interactions. Osteoadherin (OSAD), a keratin sulfate (KS)-substituted small leucine-rich proteoglycan has been isolated from mineralized tissues and is considered to be a mineralized tissue-specific protein. However, to date, information is limited concerning the dynamic expression and role of this proteoglycan during bone formation and the biomineralization process. The current study aimed to examine the dynamic expression of this protein throughout mouse metatarsal long bone development, from the cartilage anlagen (E15) to the fully formed bone (Adult). Using quantitative gene expression analysis we observed that OSAD was produced with the onset of mineralization and the formation of the ossification center. This finding was reflected in the localization studies, using both light and electron microscopy, and showed that initial OSAD localization was restricted to the endosteal surfaces of the diaphysis and forming metaphysis. Furthermore, we analyzed protein extracts, both mineral and non-mineral associated fractions, and showed that OSAD was substituted with varying patterns of glycosylation during bone development. Sequential enzymatic digestions of the non-mineral bound protein extracts demonstrated that OSAD lacked any KS chains throughout all development stages. Whereas, in the mineral bound fractions, with long bone maturation the substitution with KS became more apparent with development. Therefore, it can be concluded that different pools of OSAD are produced during endochondral bone formation and these may have specific roles in directing the mineralization process.     

Microarray analysis of gene expression during the inflammation and endochondral bone formation stages of rat femur fracture repair

Microarray analysis of gene expression was performed in the healing femur fractures of 13-week-old male rats during the inflammatory stage of repair, at 3 days post-fracture, and the endochondral bone formation stage of repair, at 11 days post-fracture. Multiple replicate pairs of fracture tissues paired with unfractured tissues, and unfractured control bones that had the stabilizing K-wire were introduced. This approach normalized the marrow contributions to the RNA repertoire. We identified 6555 genes with significant changes in expression in fracture tissues at 3 days and 11 days healing. The repertoire of growth factor genes expressed was also surprisingly restricted at both post-fracture intervals. The large number of Expressed Sequence Tags (ESTs) expressed at both post-fracture times indicates that several molecular pathways yet to be identified regulate fracture repair. The number of genes expressed during immune responses and inflammatory processes was restricted with higher expression largely during the early post-fracture analysis. Several of the genes identified in this study have been associated with regulation of cell and extracellular matrix interactions during scarless healing of fetal skin wounds. These observations suggest that these genes might also regulate the scarless healing characteristic of bone regeneration by similar mechanisms.     

Role of proteoglycans in endochondral ossification: Inhibition of calcification

Summary Proteoglycans from bovine nasal septa and from the Swarm rat chondrosarcoma were isolated as aggregates (PGC) and as monomers (PGS). Portions of the PGC preparations were degraded with cathepsin D or chondroitinase AC. Chondroitin sulfates were isolated by differential precipitation from alkaline digests of the PGS from bovine nasal septa. The effects of these preparations at concentrations up to 2 mg/ml on the precipitation of tricalcium phosphatein vitro at pH 7.8 in 16 hours at 25°C were ascertained. To this end, the amounts of calcium and phosphate in the precipitates and in the supernates were determined. The PGC preparations were found to be very effective inhibitors; in the presence of 2 mg/ml, precipitate did not form. The PGS preparations were less effective than the PGC preparations; in the presence of 2 mg/ml, about 20% as much calcium phosphate precipitated as in their absence. The chondroitinase AC-degraded preparations at concentrations equivalent to 2 mg/ml of the PGC preparations were approximately as effective as the PGS preparations. On the other hand, the cathepsin D-degraded PGC preparations and the chondroitin sulfate chains were relatively poor inhibitors. Although the viscosity of the solutions may have influenced the rate at which the precipitates settled to the bottom of the bubes, the amounts of the tricalcium phosphate formed were related to the composition and concentration of the proteoglycan preparations.     

Loss of ephrinB1 in osteogenic progenitor cells impedes endochondral ossification and compromises bone strength integrity during skeletal development

The EphB receptor tyrosine kinase family and their ephrinB ligands have been implicated as mediators of skeletal development and bone homeostasis in humans, where mutations in ephrinB1 contribute to frontonasal dysplasia and coronal craniosynostosis. In mouse models, ephrinB1 has been shown to be a critical factor mediating osteoblast function. The present study examined the functional importance of ephrinB1 during endochondral ossification using the Cre recombination system with targeted deletion of ephrinB1 (EfnB1^fl/fl) in osteogenic progenitor cells, under the control of the osterix (Osx:Cre) promoter. The Osx:EfnB1^−/− mice displayed aberrant bone growth during embryonic and postnatal skeletal development up to 4 weeks of age, when compared to the Osx:Cre controls. Furthermore, compared to the Osx:Cre control mice, the Osx:EfnB1^−/− mice exhibited significantly weaker and less rigid bones, with a reduction in trabecular/ cortical bone formation, reduced trabecular architecture and a reduction in the size of the growth plates at the distal end of the femora from newborn through to 4 weeks of age. The aberrant bone formation correlated with increased numbers of tartrate resistant acid phosphatase positive osteoclasts and decreased numbers of bone lining osteoblasts in 4 week old Osx:EfnB1^−/− mice, compared to Osx:Cre control mice. Taken together, these observations demonstrate the importance of ephrinB1 signalling between cells of the skeleton required for endochondral ossification.     

Mechanical stresses and endochondral ossification in the chondroepiphysis

In 1911, Gebhardt used a photoelastic model to relate mechanical stresses to the ossification pattern of the chondroepiphysis. Pauwels later conducted a photoelastic study using the same model geometry to develop a theory that the secondary ossific nucleus originates at a position of high-magnitude hydrostatic pressure where the shear stresses are zero. We conducted two-dimensional finite element analyses of the model used by Gebhardt and Pauwels. We demonstrate that Pauwels's photoelastic results are correct but are based on the imposition of incorrect boundary conditions. When more realistic boundary conditions were used, the finite element results changed dramatically. These results suggest that (a) the ossific nucleus appears in an area of high shear (deviatoric) stresses; (b) the edge of the advancing ossification front (zone of Ranvier or ossification grove) also experiences high shear stresses; and (c) the joint surface, where articular cartilage forms, is exposed to high-magnitude hydrostatic compression. These findings support the theory proposed by Carter and associates that intermittently applied shear stresses (or strain energy) promote endochondral ossification and that intermittently applied hydrostatic compression inhibits or prevents cartilage degeneration and ossification.     

Unique roles of phosphorus in endochondral bone formation and osteocyte maturation

The mechanisms by which inorganic phosphate (P_i) homeostasis controls bone biology are poorly understood. Here we used Dmp1 null mice, a hypophosphatemic rickets/osteomalacia model, combined with a metatarsal organ culture and an application of neutralizing fibroblast growth factor 23 (FGF‐23) antibodies to gain insight into the roles of P_i in bone biology. We showed (1) that abnormal bone remodeling in Dmp1 null mice is due to reduced osteoclast number, which is secondary to a reduced ratio of RANKL/OPG expressed by osteoclast supporting cells and (2) that osteoblast extracellular matrix mineralization, growth plate maturation, secondary ossification center formation, and osteoblast differentiation are phosphate‐dependent. Finally, a working hypothesis is proposed to explain how phosphate and DMP1 control osteocyte maturation. © 2011 American Society for Bone and Mineral Research.     

Pinealectomy in a broiler chicken model impairs endochondral ossification and induces rapid cancellous bone loss

Background contextAdolescent idiopathic scoliosis (AIS) in humans is a lateral curvature of the spine often associated with osteopenia. It has recently been accepted that the development of AIS is closely associated with spinal overgrowth. Pinealectomy (PNX) in a chicken model consistently induces scoliosis with anatomic features similar to human AIS; however, the mechanism of PNX inducing scoliosis is poorly understood.PurposeThis experimental study attempts to improve the understanding of the mechanisms underlying the onset of scoliosis in a PNX broiler chicken model.MethodsA histomorphometric study was performed to analyze longitudinal bone growth and cancellous bone remodeling before the development of scoliosis. Static and dynamic parameters in cancellous bone and chondro-osseous junction of the 7th thoracic vertebral body at 9 days after hatching were compared between PNX chickens (n=9) and control chickens with no surgery (n=5).ResultsPNX resulted in a rapid and marked loss of cancellous bone volume (7.9±0.9% vs. 14.2±1.8%, mean±SD, p<.0005) and profoundly disrupted trabecular structure with increases in dynamic formative parameters, such as mineralizing surface, mineralization apposition rate, and adjusted appositional rate. In the chondro-osseous junction, activated osteoclasts phagocytized degenerating chondrocytes, leaving a minimal amount of cartilage matrix and activated osteoblasts, losing their scaffolding for bone formation, and directly covering the hypertrophic zone cells. The osteoid surface and thickness in the chondro-osseous junction were significantly increased in PNX chickens (43.1±14.2% vs. 11.6±5.7% and 4.1±0.2μm vs. 2.9±0.4 μm). In the subjacent cartilage regions being protected from further resorption, abundant labeled cartilage remained with higher cellularity.ConclusionsIt is known that fast-growing birds have a unique paradigm of rapid bone elongation with minimal metaphyseal bone production. A bone-forming surface exists at the front of cartilage ossification in the growth plate; therefore, papillae of hypertrophic chondrocytes become included between the trabeculae of metaphyseal bone and the overall thickness of the growth plate increases considerably in addition to distal expansion. Our results indicate that the unique mechanism for rapid bone elongation in chicken is more pronounced after PNX. PNX also induces high turnover osteoporosis, which may also contribute to the development of scoliosis in the chicken.     

Influence of estrogen and progesterone on matrix-induced endochondral bone formation

Summary The influence of estradiol and progesterone, alone or in combination, on the discrete phases of matrix-induced endochondral bone formation was investigated. Administration of estradiol and progesterone in combination increased mesenchymal cell proliferation, as indicated by [³H] thymidine incorporaton into acid precipitable material. However, ornithine decarboxylase (ODC) activity was significantly suppressed by the combination of estradiol and progesterone. Also, this treatment did not influence the³⁵SO₄ incorporation into proteoglycans on day 7. Mineralization of newly induced bone was quantitated by alkaline phosphatase,⁴⁵Ca incorporation into bone mineral and calcium content, and was found to be significantly increased by progesterone alone and in combination with estradiol in both matrix-induced plaques and tibial metaphysis. These results demonstrated the stimulatory role of progesterone in combination with estradiol in bone formation and mineralization.     

Influence of estrogen and progesterone on matrix-induced endochondral bone formation

The influence of estradiol and progesterone, alone or in combination, on the discrete phases of matrix-induced endochondral bone formation was investigated. Administration of estradiol and progesterone in combination increased mesenchymal cell proliferation, as indicated by [3H] thymidine incorporation into acid precipitable material. However, ornithine decarboxylase (ODC) activity was significantly suppressed by the combination of estradiol and progesterone. Also, this treatment did not influence the 35SO4 incorporation into proteoglycans on day 7. Mineralization of newly induced bone was quantitated by alkaline phosphatase, 45Ca incorporation into bone mineral and calcium content, and was found to be significantly increased by progesterone alone and in combination with estradiol in both matrix-induced plaques and tibial metaphysis. These results demonstrated the stimulatory role of progesterone in combination with estradiol in bone formation and mineralization.