Neuropeptide Y is expressed by osteocytes and can inhibit osteoblastic activity

Osteocytes are the most abundant osteoblast lineage cells within the bone matrix. They respond to mechanical stimulation and can participate in the release of regulatory proteins that can modulate the activity of other bone cells. We hypothesize that neuropeptide Y (NPY), a neurotransmitter with regulatory functions in bone formation, is produced by osteocytes and can affect osteoblast activity. To study the expression of NPY by the osteoblast lineage cells, we utilized transgenic mouse models in which we can identify and isolate populations of osteoblasts and osteocytes. The Col2.3GFP transgene is active in osteoblasts and osteocytes, while the DMP1 promoter drives green fluorescent protein (GFP) expression in osteocytes. Real‐time PCR analysis of RNA from the isolated populations of cells derived from neonatal calvaria showed higher NPY mRNA in the preosteocytes/osteocytes fraction compared to osteoblasts. NPY immunostaining confirmed the strong expression of NPY in osteocytes (DMP1GFP⁺), and lower levels in osteoblasts. In addition, the presence of NPY receptor Y1 mRNA was detected in cavaria and long bone, as well as in primary calvarial osteoblast cultures, whereas Y2 mRNA was restricted to the brain. Furthermore, NPY expression was reduced by 30–40% in primary calvarial cultures when subjected to fluid shear stress. In addition, treatment of mouse calvarial osteoblasts with exogenous NPY showed a reduction in the levels of intracellular cAMP and markers of osteoblast differentiation (osteocalcin, BSP, and DMP1). These results highlight the potential regulation of osteoblast lineage differentiation by local NPY signaling. J. Cell. Biochem. 108: 621–630, 2009. © 2009 Wiley‐Liss, Inc.     

Matrix metalloproteinases (MMPs) safeguard osteoblasts from apoptosis during transdifferentiation into osteocytes: MT1-MMP maintains osteocyte viability

Osteoblasts undergo apoptosis or differentiate into either osteocytes or bone-lining cells after termination of bone matrix synthesis. In this study, we investigated the role of matrix metalloproteinases (MMPs) in differentiation of osteoblasts, bone formation, transdifferentiation into osteocytes, and osteocyte apoptosis. This was accomplished by using calvarial sections from the MT1-MMP-deficient mouse and by culture of the mouse osteoblast cell line MC3T3-E1 and primary mouse calvarial osteoblasts. We found that a synthetic matrix metalloprotease inhibitor, GM6001, strongly inhibited bone formation in vitro of both primary osteoblasts and MC3T3 cells by approximately 75%. To further investigate at which level of osteoblast differentiation MMP inhibition was attenuating osteoblast function, we found that neither preosteoblast nor mature osteoblast activity was affected. In contrast, cell survival of osteoblasts forced to transdifferentiate into osteocytes in 3D type I collagen gels were inhibited by more than 50% when exposed to 10 microM GM6001 and to Tissue Inhibitor of Metalloproteinase-2 (TIMP-2), a natural MT1-MMP inhibitor. This shows the importance of MMPs in safeguarding osteoblasts from apoptosis when transdifferentiating into osteocytes. By examination of osteoblasts and osteocytes embedded in calvarial bone in the MT1-MMP deficient mice, we found that MT1-MMP deficient mice had 10-fold higher levels of apoptotic osteocytes than wild-type controls. We have previously shown that MT1-MMP activates latent Transforming Growth Factorbeta (TGF-beta). These findings strongly suggest that MT1-MMP-activated TGF-beta maintains osteoblast survival during transdifferentiation into osteocytes, and maintains mature osteocyte viability. Thus, the interrelationship of MMPs and TGF-beta may play an important role in bone formation and maintenance.     

Osteocytes subjected to pulsating fluid flow regulate osteoblast proliferation and differentiation

Osteocytes are thought to orchestrate bone remodeling, but it is unclear exactly how osteocytes influence neighboring bone cells. Here, we tested whether osteocytes, osteoblasts, and periosteal fibroblasts subjected to pulsating fluid flow (PFF) produce soluble factors that modulate the proliferation and differentiation of cultured osteoblasts and periosteal fibroblasts. We found that osteocyte PFF conditioned medium (CM) inhibited bone cell proliferation, and osteocytes produced the strongest inhibition of proliferation compared to osteoblasts and periosteal fibroblasts. The nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) attenuated the inhibitory effects of osteocyte PFF CM, suggesting that a change in NO release is at least partially responsible for the inhibitory effects of osteocyte PFF CM. Furthermore, osteocyte PFF CM stimulated osteoblast differentiation measured as increased alkaline phosphatase activity, and l-NAME decreased the stimulatory effects of osteocyte PFF CM on osteoblast differentiation. We conclude that osteocytes subjected to PFF inhibit proliferation but stimulate differentiation of osteoblasts in vitro via soluble factors and that the release of these soluble factors was at least partially dependent on the activation of a NO pathway in osteocytes in response to PFF. Thus, the osteocyte appears to be more responsive to PFF than the osteoblast or periosteal fibroblast with respect to the production of soluble signaling molecules affecting osteoblast proliferation and differentiation.     

Regulation of human cranial osteoblast phenotype by FGF-2, FGFR-2 and BMP-2 signaling

The formation of cranial bone requires the differentiation of osteoblasts from undifferentiated mesenchymal cells. The balance between osteoblast recruitment, proliferation, differentiation and apoptosis in sutures between cranial bones is essential for calvarial bone formation. The mechanisms that control human osteoblasts during normal calvarial bone formation and premature suture ossification (craniosynostosis) begin to be understood. Our studies of the human calvaria osteoblast phenotype and calvarial bone formation showed that premature fusion of the sutures in non-syndromic and syndromic (Apert syndrome) craniosynostoses results from precocious osteoblast differentiation. We showed that Fibroblast Growth Factor-2 (FGF-2), FGF receptor-2 (FGFR-2) and Bone Morphogenetic Protein-2 (BMP-2), three essential factors involved in skeletal development, regulate the proliferation, differentiation and apoptosis in human calvaria osteoblasts. Mechanisms that induce the differentiated osteoblast phenotype have also been identified in human calvaria osteoblasts. We demonstrated the implication of molecules (N-cadherin, Il-1) and signaling pathways (src, PKC) by which these local factors modulate human calvaria osteoblast differentiation and apoptosis. The identification of these essential signaling molecules provides new insights into the pathways controlling the differentiated osteoblast phenotype, and leads to a more comprehensive view in the mechanisms that control normal and premature cranial ossification in humans.     

Cell-to-cell interactions in bone

Bone development (modeling) occurs by migration, aggregation, and condensation of immature osteo/chondroprogenitor cells to form the cartilaginous anlage. This process requires precisely controlled cell-cell interactions. Likewise, bone remodeling in the adult skeleton is a dynamic process that requires coordinated cellular activities among osteoblasts, osteocytes, and osteoclasts to maintain bone homeostasis. The cooperative nature of both bone modeling and remodeling requires tightly regulated mechanisms of intercellular recognition and communication that permit the cells to sort and migrate, synchronize activity, equalize hormonal responses, and diffuse locally generated signals. Osteoblasts and osteocytes achieve these interactions through cadherin-based adherens junctions as well as by formation of communicating junctions, gap junctions. This review examines the current knowledge of how direct cell-to-cell interactions modulate osteoblast function.     

Matrix metalloproteinase-dependent activation of latent transforming growth factor-beta controls the conversion of osteoblasts into osteocytes by blocking osteoblast apoptosis

Upon termination of bone matrix synthesis, osteoblasts either undergo apoptosis or differentiate into osteocytes or bone lining cells. In this study, we investigated the role of matrix metalloproteinases (MMPs) and growth factors in the differentiation of osteoblasts into osteocytes and in osteoblast apoptosis. The mouse osteoblast cell line MC3T3-E1 and primary mouse calvarial osteoblasts were either grown on two-dimensional (2-D) collagen-coated surfaces, where they morphologically resemble flattened, cuboidal bone lining cells, or embedded in three-dimensional (3-D) collagen gels, where they resemble dendritic osteocytes constituting a network of cells. When MC3T3-E1 osteoblasts were grown in a 3-D matrix in the presence of an MMP inhibitor (GM6001), the cell number was dose-dependently reduced by approximately 50%, whereas no effect was observed on a 2-D substratum. In contrast, the murine mature osteocyte cell line, MLO-Y4, was unaffected by GM6001 under all culture conditions. According to TUNEL assay, the osteoblast apoptosis was increased 2.5-fold by 10 microm GM6001. To investigate the mechanism by which MMPs mediate the survival of osteoblasts, we examined the effect of GM6001 on MC3T3-E1 osteoblasts in the presence of extracellular matrix components and growth factors, including tenascin, fibronectin, laminin, collagenase-cleaved collagen, gelatin, parathyroid hormone, basic fibroblast growth factor, vascular epidermal growth factor, insulin-like growth factor, interleukin-1, and latent and active transforming growth factor-beta (TGF-beta). Only active TGF-beta, but not latent TGF-beta or other agents tested, restored cell number and apoptosis to control levels. Furthermore, we found that the membrane type MMP, MT1-MMP, which is produced by osteoblasts, could activate latent TGF-beta and that antibodies neutralizing endogenous TGF-beta led to a similar decrease in cell number as GM6001. Whereas inhibitors of other protease families did not induce osteoblast apoptosis, an inhibitor of the p44/42 mitogen-activated protein kinase showed the same but non-synergetic effect as GM6001. These findings suggest that MMP-activated TGF-beta maintains osteoblast survival during trans-differentiation into osteocytes by a p44/42-dependent pathway.     

Pannexin 3 functions as an ER Ca(2+) channel, hemichannel, and gap junction to promote osteoblast differentiation

The pannexin proteins represent a new gap junction family. However, the cellular functions of pannexins remain largely unknown. Here, we demonstrate that pannexin 3 (Panx3) promotes differentiation of osteoblasts and ex vivo growth of metatarsals. Panx3 expression was induced during osteogenic differentiation of C2C12 cells and primary calvarial cells, and suppression of this endogenous expression inhibited differentiation. Panx3 functioned as a unique Ca(2+) channel in the endoplasmic reticulum (ER), which was activated by purinergic receptor/phosphoinositide 3-kinase (PI3K)/Akt signaling, followed by activation of calmodulin signaling for differentiation. Panx3 also formed hemichannels that allowed release of ATP into the extracellular space and activation of purinergic receptors with the subsequent activation of PI3K-Akt signaling. Panx3 also formed gap junctions and propagated Ca(2+) waves between cells. Blocking the Panx3 Ca(2+) channel and gap junction activities inhibited osteoblast differentiation. Thus, Panx3 appears to be a new regulator that promotes osteoblast differentiation by functioning as an ER Ca(2+) channel and a hemichannel, and by forming gap junctions.     

Osteoblasts stimulated with pulsed electromagnetic fields increase HUVEC proliferation via a VEGF‐A independent mechanism

The clinically beneficial effect of low frequency pulsed electromagnetic fields (ELF‐PEMF) on bone healing has been described, but the exact mechanism of action remains unclear. A recent study suggests that there is a direct autocrine mitogenic effect of ELF‐PEMF on angiogenesis. The hypothesis of this study is that ELF‐PEMF also has an indirect effect on angiogenesis by manipulation of vascular endothelial growth factor (VEGF)‐A‐based paracrine intercellular communication with neighboring osteoblasts. Conditioned media experiments measured fetal rat calvarial cell (FRC) and human umbilical vein endothelial cell (HUVEC) proliferation using tritiated thymidine uptake. We demonstrate that ELF‐PEMF (15 Hz, 1.8 mT, for 8 h) has an indirect effect on the proliferation rate of both endothelial cells and osteoblasts in vitro by altering paracrine mediators. Conditioned media from osteoblast cells stimulated with ELF‐PEMF increased endothelial proliferation 54‐fold, whereas media from endothelial cells stimulated with ELF‐PEMF did not affect osteoblast proliferation. We examined the role of the pro‐angiogenic mediator VEGF‐A in the mitogenic effect of ELF‐PEMF‐stimulated osteoblast media on endothelial cells. The production of VEGF‐A by FRC as measured by ELISA was not changed by exposure to PEMF, and blocking experiments demonstrated that the ELF‐PEMF‐induced osteoblast‐derived endothelial mitogen observed in these studies was not VEGF‐A, but some other soluble angiogenic mediator. Bioelectromagnetics 30:189–197, 2009. © 2008 Wiley‐Liss, Inc.     

Identification of kaempferol‐regulated proteins in rat calvarial osteoblasts during mineralization by proteomics

Kaempferol, a flavonoid, promotes osteoblast mineralization in vitro and bone formation in vivo; however, its mechanism of action is yet unknown. We adopted proteomic approach to identify the differential effect of kaempferol on rat primary calvarial osteoblasts during mineralization. The primary rat calvarial osteoblasts were treated with kaempferol (5.0 μM) for 9 days under mineralizing condition that resulted in significant increase in alkaline phosphatase activity and mineralization of the cells. Further, 2‐D analysis of the kaempferol‐treated osteoblast lysates revealed 18 differentially expressed proteins (nine upregulated and nine downregulated) on the basis of >/<2.0‐fold as cut‐off (p<0.01) that were then identified by MALDI‐TOF MS. These included cytoskeletal proteins, intracellular signaling protein, chaperone, extracellular matrix protein, and proteins involved in glycolysis and cell–matrix interactions. Proteomics data were confirmed by Western blotting and quantitative real‐time PCR by randomly selecting two upregulated and two downregulated proteins. Western blot analysis confirmed upregulation of HSP‐70 and cytokeratin‐14 levels, and downregulation of aldose reductase and caldesmon expression. We further demonstrated that kaempferol treatment inhibits aldose reductase activity in osteoblasts indicating an altered cellular metabolism by decelerating polyol pathway that was associated with the kaempferol‐induced osteoblast mineralization. In conclusion, this is a first comprehensive study on the differential regulation of proteins by kaempferol in primary osteoblast, which would further help to elucidate the role of the identified proteins in the process of osteoblast mineralization.     

Human skeletal growth factor stimulates collagen synthesis and inhibits proliferation in a clonal osteoblast cell line (MC3T3-E1)

Human skeletal growth factor (hSGF), an 11-kD polypeptide purified from human bone, has been proposed to be a local regulator of bone formation. To investigate the underlying cellular mechanisms in an in vitro model system, we examined the effects of hSGF on proliferation and collagen synthesis in cells of the clonal osteoblast cell line MC3T3-E1. This line was isolated from newborn mouse calvarial cells and retains many characteristics of mature osteoblasts (Sudo, H., et al., (1984) J. Cell Biol. 96:191). A 14-hr treatment with hSGF increased noncollagenous protein synthesis to 215% of unstimulated controls and increased collagen synthesis to 630% of controls as determined by [3H]proline incorporation and high-pressure liquid chromatographic separation of [3H]proline and [3H]hydroxyproline in acid hydrolysates of trichloroacetic acid-insoluble protein. HSGF did not increase cell number over a 48-hr period and caused a reversible inhibition of DNA synthesis. Half-maximal hSGF concentration for stimulation of [3H]proline incorporation and inhibition of [3H]thymidine incorporation was 100 ng/ml. HSGF also inhibited DNA synthesis in cells stimulated by serum. In contrast, hSGF stimulated both collagen synthesis and DNA synthesis in primary cultures of chick embryo bone cells, which may be developmentally less mature than MC3T3-E1 cells. The results suggest that hSGF directly stimulated mature osteoblast matrix synthetic activity and that hSGF has differential effects on proliferation of osteoblast progenitor cells and mature osteoblasts.     

Mechanically stimulated osteocytes regulate osteoblastic activity via gap junctions

The strong correlation between a bone's architectural properties and the mechanical forces that it experiences has long been attributed to the existence of a cell that not only detects mechanical load but also structurally adapts the bone matrix to counter it. One of the most likely cellular candidates for such a "mechanostat" is the osteocyte, which resides within the mineralized bone matrix and is perfectly situated to detect mechanically induced signals. However, as osteocytes can neither form nor resorb bone, it has been hypothesized that they orchestrate mechanically induced bone remodeling by coordinating the actions of cells residing on the bone surface, such as osteoblasts. To investigate this hypothesis, we developed a novel osteocyte-osteoblast coculture model that mimics in vivo systems by permitting us to expose osteocytes to physiological levels of fluid shear while shielding osteoblasts from it. Our results show that osteocytes exposed to a fluid shear rate of 4.4 dyn/cm² rapidly increase the alkaline phosphatase activity of the shielded osteoblasts and that osteocytic-osteoblastic physical contact is a prerequisite. Furthermore, both functional gap junctional intercellular communication and the mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2 signaling pathway are essential components in the osteoblastic response to osteocyte communicated mechanical signals. By utilizing other nonosteocytic coculture models, we also show that the ability to mediate osteoblastic alkaline phosphatase levels in response to the application of fluid shear is a phenomena unique to osteocytes and is not reproduced by other mesenchymal cell types. osteocyte; osteoblast; fluid-flow; coculture; mechanical stimulation; gap junction; intercellular communication     

Impaired Bone Homeostasis in Amyotrophic Lateral Sclerosis Mice with Muscle Atrophy

There is an intimate relationship between muscle and bone throughout life. However, how alterations in muscle functions in disease impact bone homeostasis is poorly understood. Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease characterized by progressive muscle atrophy. In this study we analyzed the effects of ALS on bone using the well established G93A transgenic mouse model, which harbors an ALS-causing mutation in the gene encoding superoxide dismutase 1. We found that 4-month-old G93A mice with severe muscle atrophy had dramatically reduced trabecular and cortical bone mass compared with their sex-matched wild type (WT) control littermates. Mechanically, we found that multiple osteoblast properties, such as the formation of osteoprogenitors, activation of Akt and Erk1/2 pathways, and osteoblast differentiation capacity, were severely impaired in primary cultures and bones from G93A relative to WT mice; this could contribute to reduced bone formation in the mutant mice. Conversely, osteoclast formation and bone resorption were strikingly enhanced in primary bone marrow cultures and bones of G93A mice compared with WT mice. Furthermore, sclerostin and RANKL expression in osteocytes embedded in the bone matrix were greatly up-regulated, and β-catenin was down-regulated in osteoblasts from G93A mice when compared with those of WT mice. Interestingly, calvarial bone that does not load and long bones from 2-month-old G93A mice without muscle atrophy displayed no detectable changes in parameters for osteoblast and osteoclast functions. Thus, for the first time to our knowledge, we have demonstrated that ALS causes abnormal bone remodeling and defined the underlying molecular and cellular mechanisms.     

Effects of insulin and insulin‐like growth factor 1 on osteoblast proliferation and differentiation: differential signalling via Akt and ERK

Insulin and insulin‐like growth factor 1 (IGF‐1) are evolutionarily conserved hormonal signalling molecules, which influence a wide array of physiological functions including metabolism, growth and development. Using genetic mouse studies, both insulin and IGF‐1 have been shown to be anabolic agents in osteoblasts and bone development primarily through the activation of Akt and ERK signalling pathways. In this study, we examined the temporal signalling actions of insulin and IGF‐1 on primary calvarial osteoblast growth and differentiation. First, we observed that the IGF‐1 receptor expression decreases whereas insulin receptor expression increases during osteoblast differentiation. Subsequently, we show that although both insulin and IGF‐1 promote osteoblast differentiation and mineralization in vitro, IGF‐1, but not insulin, can induce osteoblast proliferation. The IGF‐1‐induced osteoblast proliferation was mediated via both MAPK and Akt pathways because the IGF‐1‐mediated cell proliferation was blocked by U0126, an MEK/MAPK inhibitor, or LY294002, a PI3‐kinase inhibitor. Osteocalcin, an osteoblast‐specific protein whose expression corresponds with osteoblast differentiation, was increased in a dose‐ and time‐dependent manner after insulin treatment, whereas it was decreased with IGF‐1 treatment. Moreover, insulin treatment dramatically induced osteocalcin promoter activity, whereas IGF‐1 treatment significantly inhibited it, indicating direct effect of insulin on osteocalcin synthesis. Copyright © 2012 John Wiley & Sons, Ltd.     

Ectopic Msx2 overexpression inhibits and Msx2 antisense stimulates calvarial osteoblast differentiation.

Msx2 is believed to play a role in regulating bone development, particularly in sutures of cranial bone. In this study we investigated the effects of retroviral-mediated overexpression of Msx2 mRNA, in both sense and antisense orientations, on primary cultured chick calvarial osteoblasts. Unregulated overexpression of sense mRNA produced high levels of Msx2 protein throughout the culture period, preventing the expected fall as the cells differentiate. The continued high expression of Msx2 prevented osteoblastic differentiation and mineralization of the extracellular matrix. In contrast, expression of antisense Msx2 RNA decreased proliferation and accelerated differentiation. In other studies, we showed that the Msx2 promoter was widely expressed during the proliferative phase of mouse calvarial osteoblast cultures but was preferentially downregulated in osteoblastic nodules. These results support a model in which Msx2 prevents differentiation and stimulates proliferation of cells at the extreme ends of the osteogenic fronts of the calvariae, facilitating expansion of the skull and closure of the suture.     

Gap junctional regulation of signal transduction in bone cells

The role of gap junctions, particularly that of connexin43 (Cx43), has become an area of increasing interest in bone physiology. An abundance of studies have shown that Cx43 influences the function of osteoblasts and osteocytes, which ultimately impacts bone mass acquisition and skeletal homeostasis. However, the molecular details underlying how Cx43 regulates bone are only coming into focus and have proven to be more complex than originally thought. In this review, we focus on the diverse molecular mechanisms by which Cx43 gap junctions and hemichannels regulate cell signaling pathways, gene expression, mechanotransduction and cell survival in bone cells. This review will highlight key signaling factors that have been identified as downstream effectors of Cx43 and the impact of these pathways on distinct osteoblast and osteocyte functions.     

Mechanotransduction activates α₅β₁ integrin and PI3K/Akt signaling pathways in mandibular osteoblasts

It is unclear how bone cells at different sites detect mechanical loading and how site-specific mechanotransduction affects bone homeostasis. To differentiate the anabolic mechanical responses of mandibular cells from those of calvarial and long bone cells, we isolated osteoblasts from C57B6J mouse bones, cultured them for 1week, and subjected them to therapeutic low intensity pulsed ultrasound (LIPUS). While the expression of the marker proteins of osteoblasts and osteocytes such as alkaline phosphatase and FGF23, as well as Wnt1 and β-catenin, was equally upregulated, the expression of mandibular osteoblast messages related to bone remodeling and apoptosis differed from that of messages of other osteoblasts, in that the messages encoding the pro-remodeling protein RANKL and the anti-apoptotic protein Bcl-2 were markedly upregulated from the very low baseline levels. Blockage of the PI3K and α(5)β(1) integrin pathways showed that the mandibular osteoblast required mechanotransduction downstream of α(5)β(1) integrin to upregulate expression of the proteins β-catenin, p-Akt, Bcl-2, and RANKL. Mandibular osteoblasts thus must be mechanically loaded to preserve their capability to promote remodeling and to insure osteoblast survival, both of which maintain intact mandibular bone tissue. In contrast, calvarial Bcl-2 is fully expressed, together with ILK and phosphorylated mTOR, in the absence of LIPUS. The antibody blocking α(5)β(1) integrin suppressed both the baseline expression of all calvarial proteins examined and the LIPUS-induced expression of all mandibular proteins examined. These findings indicate that the cellular environment, in addition to the tridermic origin, determines site-specific bone homeostasis through the remodeling and survival of osteoblastic cells. Differentiated cells of the osteoblastic lineage at different sites transmit signals through transmembrane integrins such as α(5)β(1) integrin in mandibular osteoblasts, whose signaling may play a major role in controlling bone homeostasis.     

Age-related changes in the biomolecular mechanisms of calvarial osteoblast biology affect fibroblast growth factor-2 signaling and osteogenesis

The ability of immature animals to orchestrate successful calvarial ossification has been well described. This capacity is markedly attenuated in mature animals and humans greater than 2 years of age. Few studies have investigated biological differences between juvenile and adult osteoblasts that mediate successful osteogenesis. To identify possible mechanisms for this clinical observation, we investigated cellular and molecular differences between primary osteoblasts derived from juvenile (2-day-old) and adult (60-day-old) rat calvaria. Data demonstrated that juvenile osteoblasts contain a subpopulation of less differentiated cells as observed by spindle-like morphology and decreased osteocalcin production. Juvenile, compared with adult, osteoblasts showed increased proliferation and adhesion. Furthermore, following rhFGF-2 stimulation juvenile osteoblasts increased expression of collagen I alpha 1 (5-fold), osteopontin (13-fold), and osteocalcin (16-fold), compared with relatively unchanged adult osteoblasts. Additionally, juvenile osteoblasts organized and produced more matrix proteins and formed 41-fold more bone nodules. Alternatively, adult osteoblasts produced more FGF-2 and preferentially translated the high molecular weight (22 kDa) form. Although adult osteoblasts transcribed more FGF-R1 and juvenile osteoblasts transcribed more FGF-R2 at baseline levels, juvenile osteoblasts translated more FGF-R1 and -R2 and showed increased phosphorylation. Collectively, these findings begin to explain why juvenile, but not adult, osteoblasts successfully heal calvarial defects.