Craniosynostosis has been associated with fibroblast growth factors (FGFs) and their receptors. The purpose of this study was to quantitatively determine the effect of FGF2 on rat calvarial osteoblasts and a rat cranial suture formation model.
Design:
Fetal rat calvarial osteoblasts were cultured with and without FGF2. Cell attachment and proliferation was determined by alamarBlue dye assay and cell morphology by toluidine-blue staining. In rat calvarial organ culture, postnatal day 15 rat calvariae with dura mater were placed in serum-free media with and without FGF2. A unique quantitative analysis of suture fusion was developed by obtaining measurements of suture bridging in histological serial sections at progressive stages of fusion.
Results:
Attachment for cells treated with FGF2 was similar to control. In contrast, proliferation was higher for cells treated with FGF2 while maintaining an osteoblastic morphology. After 5 days in organ culture, FGF2-treated posterior frontal sutures showed a dramatic increase in fusion, compared with untreated controls. This increased fusion was maintained throughout days 7 and 10 in culture. Also, fusion was enhanced on the dural side of the suture, as is normally observed in vivo, and the normal tissue architecture was maintained.
Conclusions:
These results indicate that FGF2 can promote rat osteoblast attachment and normal cell morphology as well as induce cell proliferation. In calvarial organ culture, FGF2 treatment produced an enhanced suture fusion. These results provide further support for a critical role for FGF2 in cranial suture development. These studies also present a new quantitative approach to evaluating the effect of suture-perturbing growth factors on cranial suture fusion.
AhmedS.A., GogalRMJr., WalshJ.E.. A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assay.J Immunol Methods.1994; 170: 211–224.
BairdA., WalickeP.A.. Fibroblast growth factors.Br Med Bull.1989; 45: 438–452.
4.
BellowsC.G., AubinJ.E., HeerscheJ.N., AntoszM.E.. Mineralized bone nodules formed in vitro from enzymatically released rat calvaria cell populations.Calcif Tissue Int.1986; 38: 143–154.
CanalisE., CentrellaM., McCarthyT.. Effects of basic fibroblast growth factor on bone formation in vitro.J Clin Invest.1988; 81: 1572–1577.
8.
CoffinJ.D., FlorkiewiczR.Z., NeumannJ., Mert-HopkinsT., DornG.W., LightfootP., GermanR., HowlesP.N., KierA., O'TooleB.A.. Abnormal bone growth and selective translational regulation in basic fibroblast growth factor (FGF-2) transgenic mice.Mol Biol Cell.1995; 6: 1861–1873.
9.
DebiaisF., HottM., GrauletA.M., MarieP.J.. The effects of fibroblast growth factor-2 on human neonatal calvaria osteoblastic cells are differentiation stage specific.J Bone Miner Res.1998; 13: 645–654.
FragaleA., TartagliaM., BernardiniS., Di StasiA., Di RoccoC., VelardiF., TetiA., BettagliaP., MigliaccioS.. Decreased proliferation and altered differentiation in osteoblasts from genetically and clinically distinct craniosynostotic disorders.Am J Pathol.1999; 154: 1465–1477.
12.
GlobusR.K., PlouetJ., GospodarowiczD.. Cultured bovine bone cells synthesize basic fibroblast growth factor and store it in their extracellular matrix.Endocrinology.1989; 124: 1539–1547.
13.
GreenwaldJ.A., MehraraB.J., SpectorJ.A., WarrenS., CriseraF., FagenholzP., BouletreauP., LongakerM.. Regional differentiation of cranial suture-associated dura mater in vivo and in vitro: implications for suture fusion and patency.J Bone Miner Res.2000; 15: 2413–2430.
14.
GreenwaldJ.A., MehraraB.J., SpectorJ.A., WarrenS., FagenholzP., SmithL., BouletreauP., CriseraF., UenoH., LongakerM.. In vivo modulation of FGF biological activity alters cranial suture fate.Am J Pathol.2001; 158: 441–452.
15.
GronowiczG.A., DeromeM.E.. Synthetic peptide containing Arg-Gly-Asp inhibits bone formation and resorption in a mineralizing organ culture system of fetal rat parietal bones.J Bone Miner Res.1994; 9: 193–201.
16.
HurleyM.M., AbreuC., GronowiczG., KawaguchiH., LorenzoJ.. Expression and regulation of basic fibroblast growth factor mRNA levels in mouse osteoblastic MC3T3-E1 cells.J Biol Chem.1994; 269: 9392–9396.
17.
IsekiS., WilkieA.O., HeathJ.K., IshimaruT., EtoK., Morriss-KayG.M.. Fgfr2 and osteopontin domains in the developing skull vault are mutually exclusive and can be altered by locally applied FGF2.Development.1997; 124: 3375–3384.
18.
IsekiS., WilkieA.O., Morriss-KayG.M.. Fgfr1 and Fgfr2 have distinct differentiation- and proliferation-related roles in the developing mouse skull vault.Development.1999; 126: 5611–5620.
19.
JohnsonD.R., HauserI.A., VollR.E., EmmrichF.. Arterial and venular endothelial cell costimulation of cytokine secretion by human T cell clones.J Leukoc Biol.1998; 63: 612–619.
20.
KatohR., UristM.R.. Surface adhesion and attachment factors in bone morphogenetic protein-induced chondrogenesis in vitro.Clin Orthop.1993: 295–304.
21.
KimH.J., RiceD.P., KettunenP.J., ThesleffI.. FGF-, BMP- and Shh-mediated signalling pathways in the regulation of cranial suture morphogenesis and calvarial bone development.Development.1998; 125: 1241–1251.
22.
LajeunieE., CatalaM., RenierD.. Craniosynostosis: from a clinical description to an understanding of bone formation of the skull.Childs Nerv Syst.1999; 15: 676–680.
LomriA., LemonnierJ., HottM., de ParsevalN., LajeunieE., MunnichA., RenierD., MarieP.. Increased calvaria cell differentiation and bone matrix formation induced by fibroblast growth factor receptor 2 mutations in Apert syndrome.J Clin Invest.1998; 101: 1310–1317.
25.
MansukhaniA., BellostaP., SahniM., BasilicoC.. Signaling by fibroblast growth factors (FGF) and fibroblast growth factor receptor 2 (FGFR2)-activating mutations blocks mineralization and induces apoptosis in osteoblasts.J Cell Biol.2000; 149: 1297–1308.
26.
MehraraB.J., GreenwaldJ., ChinG.S., DudziakM., SagriogluJ., SteinbrechD., SaadehP., GittesG., LongakerM.. Regional differentiation of rat cranial suture-derived dural cells is dependent on association with fusing and patent cranial sutures.Plast Reconstr Surg.1999; 104: 1003–1013.
27.
MehraraB.J., MackoolR.J., McCarthyJ.G., GittesG.K., LongakerM.T.. Immunolocalization of basic fibroblast growth factor and fibroblast growth factor receptor-1 and receptor-2 in rat cranial sutures.Plast Reconstr Surg.1998; 102: 1805–1817; discussion 1818–1820.
28.
MonteroA., OkadaY., TomitaM., ItoM., TsurukamiH., NakamuraT., DoetschmanT., CoffinJ., HurleyM.. Disruption of the fibroblast growth factor-2 gene results in decreased bone mass and bone formation.J Clin Invest.2000; 105: 1085–1093.
29.
MossM.L.. Growth of the calvaria in the rat: the determination of osseous morphology.Am J Anat.1962; 94: 333.
30.
MostD., LevineJ.P., ChangJ., SungJ., McCarthyJ., SchendelS., LongakerM.. Studies in cranial suture biology: up-regulation of transforming growth factor-beta1 and basic fibroblast growth factor mRNA correlates with posterior frontal cranial suture fusion in the rat.Plast Reconstr Surg.1998; 101: 1431–1440.
OppermanL.A., AdabK., GakungaP.T.. Transforming growth factor-beta 2 and TGF-beta 3 regulate fetal rat cranial suture morphogenesis by regulating rates of cell proliferation and apoptosis.Dev Dyn.2000; 219: 237–247.
35.
OppermanL.A., NolenA.A., OgleR.C.. TGF-beta 1, TGF-beta 2, and TGF-beta 3 exhibit distinct patterns of expression during cranial suture formation and obliteration in vivo and in vitro [see comments].J Bone Miner Res.1997; 12: 301–310.
36.
OppermanL.A., PassarelliR.W., MorganE.P., ReintjesM., OgleR.C.. Cranial sutures require tissue interactions with dura mater to resist osseous obliteration in vitro.J Bone Miner Res.1995; 10: 1978–1987.
37.
OppermanL.A., PersingJ.A., SheenR., OgleR.C.. In the absence of periosteum, transplanted fetal and neonatal rat coronal sutures resist osseous obliteration.J Craniofac Surg.1994; 5: 327–332.
38.
OppermanL.A., SweeneyT.M., RedmonJ., PersingJ.A., OgleR.C.. Tissue interactions with underlying dura mater inhibit osseous obliteration of developing cranial sutures.Dev Dyn.1993; 198: 312–322.
39.
RiceD.P., KimH.J., ThesleffI.. Apoptosis in murine calvarial bone and suture development.Eur J Oral Sci.1999; 107: 265–275.
40.
RothD.A., LongakerM.T., McCarthyJ.G., RosenD., McMullenH., LevineJ., SungJ., GoldL.. Studies in cranial suture biology: part I. Increased immunoreactivity for TGF-beta isoforms (beta 1, beta 2, and beta 3) during rat cranial suture fusion.J Bone Miner Res.1997; 12: 311–321.
41.
ScuttA., BertramP.. Basic fibroblast growth factor in the presence of dexamethasone stimulates colony formation, expansion, and osteoblastic differentiation by rat bone marrow stromal cells.Calcif Tissue Int.1999; 64: 69–77.
42.
ThompsonD., JonesB., HaywardR., HarknessW.. Assessment and treatment of craniosynostosis.Br J Hosp Med.1994; 52: 17–24.
43.
WilkieA.O.. Craniosynostosis: genes and mechanisms.Hum Mol Genet.1997; 6: 1647–1656.
44.
XuJ., LiuZ., OrnitzD.M.. Temporal and spatial gradients of Fgf8 and Fgf17 regulate proliferation and differentiation of midline cerebellar structures.Development.2000; 127: 1833–1843.
