Search

Background: Bone transport osteogenesis (BTO), distraction of a free portion of bone across a defect, offers an autologous solution to large cranial defects that may allow treatment without permanent hardware implantation. This study establishes a sheep model to evaluate the feasibility and distraction kinetics of BTO., Methods: Subtotal cranial defects (3.5 × 3.5 cm) were created in 10 young adult sheep and a transport segment (3.5 × 2 cm) traversed the defect at varying distraction rates (0, 0.5, 1.0, and 1.5 mm/day) using semi-buried cranial distractors. After a 6-week consolidation period, sheep were euthanized and the resultant bone was analyzed by CT, histology, and mechanical testing., Results: Gross examination, histology, and 3D CT revealed that control animals had fibrous nonunion whereas distraction animals had ossified defects with fibrous nonunion at the distal docking site. There was one premature consolidation in the 0.5 mm/day group. The volume of bony regenerate in the 0.5, 1.0, and 1.5 mm/day distraction rate groups was statistically indistinct (P = 0.16). The mean flexural moduli (MPa) of non-decalcified samples from the control cranium, transport segment, and bone regenerate were found to be 4.50 ± 4.9, 6.17 ± 2.1, and 4.14 ± 4.8, respectively (P = 0.24)., Conclusions: This experiment provides proof of concept for BTO for large calvarial defects in a sheep model. Distraction at a rate of 0.5 mm per day may place individuals at higher risk for premature consolidation, but distraction rates did not have significant effects on regenerate quantity or quality. Future work will include the use of curvilinear distraction devices for 3-dimensional contour.

Background: Commercially available recombinant human bone morphogenetic protein 2 (rhBMP2) has demonstrated efficacy in bone regeneration, but not without significant side effects. The authors used rhBMP2 encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres placed in a rabbit cranial defect model to test whether low-dose, sustained delivery can effectively induce bone regeneration., Methods: The rhBMP2 was encapsulated in 15% PLGA using a double-emulsion, solvent extraction/evaporation technique, and its release kinetics and bioactivity were tested. Two critical-size defects (10 mm) were created in the calvaria of New Zealand white rabbits (5 to 7 months of age, male and female) and filled with a collagen scaffold containing either (1) no implant, (2) collagen scaffold only, (3) PLGA-rhBMP2 (0.1 μg per implant), or (4) free rhBMP2 (0.1 μg per implant). After 6 weeks, the rabbits were killed and defects were analyzed by micro-computed tomography, histology, and finite element analysis., Results: The rhBMP2 delivered by means of bioactive PLGA microspheres resulted in higher volumes and surface area coverage of new bone than an equal dose of free rhBMP2 by micro-computed tomography (p=0.025 and p=0.025). Finite element analysis indicated that the mechanical competence using the regional elastic modulus did not differ with rhBMP2 exposure (p=0.70). PLGA-rhBMP2 did not demonstrate heterotopic ossification, craniosynostosis, or seroma formation., Conclusions: Sustained delivery by means of PLGA microspheres can significantly reduce the rhBMP2 dose required for de novo bone formation. Optimization of the delivery system may be a key to reducing the risk for recently reported rhBMP2-related adverse effects.

Background: Infant dura mater plays a critical role in calvarial development. This investigation examines the expression of nitric oxide synthase isoforms in the craniofacial skeleton and the influence of nitric oxide signaling on the growth and differentiation of fetal dural and calvarial bone cells., Methods: Sections of fetal and adult calvaria were evaluated for endothelial and inducible nitric oxide synthase expression by immunohistochemistry. Primary fetal (E18) murine dural cell and calvarial osteoblast cultures were treated with 1 microM or 10 microM DETA-NONOate, a nitric oxide donor compound, or 1 mM N-monomethyl-l-arginine (l-NMMA), a nitric oxide synthase inhibitor. Controls were left untreated. Cell proliferation was measured at 48 hours, and mRNA transcripts for Runx2, alkaline phosphatase, and osteopontin were measured by reverse transcription and quantitative real-time polymerase chain reaction at 2 to 18 days. Experiments were performed in triplicate., Results: Fetal, but not adult, dural cells express endothelial nitric oxide synthase. DETA-NONOate stimulated osteoblast mitogenesis by 16 percent (p < 0.05) but did not affect proliferation of dural cells. l-NMMA inhibited proliferation of dural cells and calvarial osteoblasts by 35 percent (p < 0.01) and 17 percent (p = 0.05), respectively. Exogenous nitric oxide increased dural cell transcription of Runx2, alkaline phosphatase (p = 0.03), and osteopontin (p = 0.09) and calvarial osteoblast transcription of Runx2 (p = 0.02) and osteopontin (p < 0.01). Fetal calvarial osteoblasts and dural cells treated with l-NMMA demonstrated reduced transcription of Runx2 and alkaline phosphatase (p < 0.05)., Conclusions: Fetal dural cells and calvarial osteoblasts express endothelial nitric oxide synthase. Nitric oxide enhances proliferation and differentiation of fetal dural cells and calvarial osteoblasts. These results suggest that endothelial nitric oxide synthase-derived nitric oxide may play an important role in development of the fetal craniofacial skeleton.

Background: The higher prevalence of metopic and sagittal suture synostosis in male infants suggests a role for androgens in early craniofacial development. These experiments characterize the influence of androgen stimulation on growth and differentiation of fetal dural and calvarial bone cells and on cranial suture fusion., Methods: Primary murine fetal (E18) dural cells and calvarial osteoblasts were isolated and cultured. Cells were treated for 48 hours with 5alpha-dihydrotestosterone (0 to 1000 nM). Cell proliferation was examined by nonradioactive proliferation assay; mRNA expression of alkaline phosphatase, transforming growth factor (TGF)-beta1, and the bone matrix proteins osteopontin, osteocalcin, and type 1 collagen was determined by reverse-transcriptase polymerase chain reaction. In separate experiments, intact fetal calvariae were grown in tissue culture with 10 nM 5alpha-dihydrotestosterone for 7 and 14 days and then examined histologically., Results: Androgen stimulation at 5 nM increased proliferation of fetal dural cells by 46.0 percent and of fetal calvarial osteoblasts by 20.5 percent. Dural expression of osteopontin, osteocalcin, and type 1 collagen was enhanced by 5alpha-dihydrotestosterone, as was that of TGF-beta1 and alkaline phosphatase. Androgen stimulation increased calvarial osteoblast expression of alkaline phosphatase and TGF-beta1 but induced little change in expression of osteocalcin, osteopontin, and type 1 collagen. In tissue culture, 5alpha-dihydrotestosterone stimulated osteoid formation and fusion of sagittal sutures., Conclusions: Androgen stimulation of dural cells and osteoblasts isolated from fetal calvaria promotes cell proliferation and osteoblastic differentiation and can induce cranial suture fusion. These results suggest that sex steroid hormone signaling may stimulate sutural osteogenesis by means of osteodifferentiation of dural cells, thus explaining the male prevalence of nonsyndromic craniosynostosis.

Background: Proteins within the transforming growth factor (TGF)-beta family play a central role in both normal and pathologic calvarial morphogenesis. Previous work has suggested differential functions of the TGF-beta isoforms in these processes. Little is known, however, about effects of TGF-betas on the underlying dura. Furthermore, studies on the effects of TGF-beta isoforms on osteoblasts have been conflicting. The purpose of this study was to determine the effect of TGF-beta isoforms, specifically TGF-beta1 and TGF-beta3, on fetal calvarial osteoblast and dural cell differentiation, proliferation, and apoptosis., Methods: Primary cultures of fetal calvarial osteoblasts and dural cells were established from embryonic day-18 CD-1 mice. Cells were treated for 48 hours with TGF-beta1 or TGF-beta3. Northern blot analysis, cell counts, and apoptosis assays were performed., Results: In dural cells, TGF-beta1 stimulated the expression of early osteodifferentiation genes and resulted in a slight decrease in cell number and no effect on apoptosis. Similar results were observed in osteoblasts. TGF-beta3 had little or no effect on the genes studied in both cell types but resulted in increased apoptosis and concomitant decreases in cell number in both cell types., Conclusions: This study demonstrates that dural cells respond to TGF-beta and that this response is isoform-specific. TGF-beta1 stimulates osteodifferentiation of previously uncommitted cells in the dura. It also stimulates early events in bone matrix deposition and has little effect on late markers of bone differentiation in osteoblasts and dural cells. Both isoforms result in decreases in cell number. TGF-beta3 results in greater decreases in cell number and isoform-specific stimulation of apoptosis in both dural cells and calvarial osteoblasts.

Calvarial bone is formed by the intramembranous bone-forming process, which involves many signaling molecules. The constitutive activation of the fibroblast growth factor (FGF) signaling pathway accelerates osteoblast differentiation and results in premature cranial suture closure. Bone morphogenetic protein (BMP) signaling pathways, which involve the downstream transcription factors Dlx5 and Msx2, are also involved in the bone-forming processes. However, the relationships between these two main signaling cascades are still unclear. We found that FGF2 treatment of developing bone fronts stimulated Bmp2 gene expression but that BMP2 treatment could not induce Fgf2 expression. Moreover, the disruption of the Runx2 gene completely eliminated the expression of Bmp2 and its downstream genes Dlx5 and Msx2 in the developing primordium of bone, while the expression of Fgf2 was maintained. In addition, cultured Runx2-/- cells expressed very low baseline levels of Bmp2 that were up-regulated by transfection with a Runx2-expressing plasmid. These levels in turn were markedly elevated by FGF2 treatment. FGF2 treatment also strongly enhanced the Bmp2 expression in MC3T3-E1 cells, whose endogenous Runx2 gene is intact and which express Bmp2 at low baseline levels as well. These results indicate that Runx2 is an important mediator of the expression of Bmp2 in response to FGF stimulation in cranial bone development., (Copyright 2005 Wiley-Liss, Inc.)

Systemically administered fibroblast growth factors (FGFs) show anabolic effects on bone formation in animals, whereas in vitro cell culture studies have demonstrated that FGFs block mineralized bone nodule formation. These apparently contradictory outcomes indicate that the nature of FGF action is complex and that the biological effect of FGFs may depend on the differentiation stage of osteoblasts, interaction with other cytokines, or the length and mode of exposure to factors. Thus, we have utilized primary calvarial bone cell populations at different maturation phases to determine their responses to 2, FGF-9, and BMP-2, the factors expressed in bone. FGF-2 and FGF-9 stimulated proliferation of the cell populations consisting of more mature osteoblasts, but not those with undifferentiated precursor cells. Continuous treatment with FGF-2/-9 inhibited expression of several osteoblast marker genes and mineralization. However, brief pretreatment with FGF-2/-9 or sequential treatment with FGF-2/-9 followed by BMP-2 led to marked stimulation of mineralization, suggesting that FGFs enhance the intrinsic osteogenic potential. Furthermore, FGF-2 and FGF-9 increased expression of other osteogenic factors BMP-2 and TGFbeta-1. Meanwhile, blocking endogenous FGF signaling, using a virally transduced dominant-negative FGF receptor (FgfR), resulted in drastically reduced expression of the BMP-2 gene, demonstrating for the first time that endogenous FGF/FgfR signaling is a positive upstream regulator of the BMP-2 gene in calvarial osteoblasts. In contrast, expression of a BMP antagonist noggin was inhibited by FGF-2 and FGF-9. Thus, collective data from this study suggest that FGF/FgfR signaling enhances the intrinsic osteogenic potential by selectively expanding committed osteogenic cell populations as well as inversely regulating BMP-2 and noggin gene expression.

Growth and development is the net result of environmental modulation of genetic inheritance. Mesenchymal cells differentiate into chondrogenic, osteogenic, and fibrogenic cells: the first 2 are chiefly responsible for endochondral ossification, and the last 2 for sutural growth. Cells are influenced by genes and environmental cues to migrate, proliferate, differentiate, and synthesize extracellular matrix in specific directions and magnitudes, ultimately resulting in macroscopic shapes such as the nose and the chin. Mechanical forces, the most studied environmental cues, readily modulate bone and cartilage growth. Recent experimental evidence demonstrates that cyclic forces evoke greater anabolic responses of not only craniofacial sutures, but also cranial base cartilage. Mechanical forces are transmitted as tissue-borne and cell-borne mechanical strain that in turn regulates gene expression, cell proliferation, differentiation, maturation, and matrix synthesis, the totality of which is growth and development. Thus, hereditary and mechanical modulations of growth and development share a common pathway via genes. Combined approaches using genetics, bioengineering, and quantitative biology are expected to bring new insight into growth and development, and might lead to innovative therapies for craniofacial skeletal dysplasia including malocclusion, dentofacial deformities, and craniofacial anomalies such as cleft palate and craniosynostosis, as well as disorders associated with the temporomandibular joint.

Various activating mutations of FgfR2 have been linked to a number of craniosynostosis syndromes, suggesting that FGFR2-mediated signaling plays significant roles in intramembranous bone formation. To define (i) the roles of FGFR2-mediated signaling in osteogenesis and (ii) bone cell functions affected by abnormal signaling induced by craniosynostosis mutations, chicken calvarial osteoblasts were infected with replication competent avian sarcoma viruses expressing FgfR2 with dominant negative (DN), P253R (Apert), or C278F (Pfeiffer and Crouzon) mutation. Analyses of the infected osteoblasts revealed that attenuated FGF/FGFR signaling by DN-FgfR2 resulted in a decrease in cell proliferation and accelerated mineralization. In contrast, the C278F mutation, which causes ligand-independent activation of the receptor, significantly stimulated cell proliferation and inhibited mineralization. Interestingly, the P253R mutation, which does not cause ligand-independent activation of the receptor, showed a weaker mitogenic effect than the C278F mutation and did not inhibit mineralization. Gene expression analysis also revealed diverse effects of C278F and P253R mutations on expression of several osteogenic genes. Based on these results, we conclude that one of the major functions of FGFR2 is to mediate mitogenic signals in osteoblasts and that distinctively different cellular mechanisms underlie the pathogenesis of craniosynostosis phenotypes resulting from P253R and C278F mutations of the FGFR2 gene.

The mechanism(s) by which the cells within the calvaria tissue are restricted into the osteogenic versus the chondrogenic lineage during intramembranous bone formation were examined. Cells were obtained from 12-day chicken embryo calvariae after tissue condensation, but before extensive osteogenic differentiation, and from 17-day embryo calvariae when osteogenesis is well progressed. Only cell populations from the younger embryos showed chondrogenic differentiation as characterized by the expression of collagen type II. The chondrocytes underwent a temporal progression of maturation and endochondral development, demonstrated by the expression of collagen type II B transcript and expression of collagen type X mRNA. Cell populations from both ages of embryos showed progressive osteogenic differentiation, based on the expression of osteopontin, bone sialoprotein, and osteocalcin mRNAs. Analysis using lineage markers for either chondrocytes or osteoblasts demonstrated that when the younger embryonic cultures were grown in conditions that were permissive for chondrogenesis, the number of chondrogenic cells increased from approximately 15 to approximately 50% of the population, while the number of osteogenic cells remained almost constant at approximately 35-40%. Pulse labeling of the cultures with BrdU showed selective labeling of the chondrogenic cells in comparison with the osteogenic cells. These data indicate that the developmental restriction of skeletal cells of the calvaria is not a result of positive selection for osteogenic differentiation but a negative selection against the progressive growth of chondrogenic cells in the absence of a permissive or inductive environment. These results further demonstrate that while extrinsic environmental factors can modulate the lineage progression of skeletal cells within the calvariae, there is a progressive restriction during embryogenesis in the number of cells within the calvaria with a chondrogenic potential. Finally, these data suggest that the loss of cells with chondrogenic potential from the calvaria may be related to the progressive limitation of the reparative capacity of the cranial bones.