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Background: Transforming growth factor (TGF)-beta1 and fibroblast growth factor (FGF)-2 have both been shown to have significant roles in the regulation of murine calvarial suture fusion. Methods to decrease gene expression of these cytokines and their respective receptors have been established, but because of side effects, clinical applications are limited. In this study, the authors examined the effect of TGF-beta1-specific small interfering RNA (siRNA) on the messenger RNA (mRNA) expression of TGF-beta1, its TGF-betaR1 and TGF-betaR2 receptors, and FGF-2 and its R1 receptor in murine dura cells., Methods: A primary dura cell line was established from CD-1 mice. Transfection efficiency using Lipofectamine was determined using BLOCKiT. Dura cells were transfected with serial concentrations of TGF-beta1 siRNA to determine the optimal dose. In subsequent experiments, cells were transfected with 16 nM TGF-beta1 siRNA and harvested on posttransfection days 4, 7, 10, and 14 for RNA isolation and quantitative polymerase chain reaction., Results: Optimal inhibition of TGF-beta1 mRNA expression was achieved at 16 nM siRNA. On posttransfection day 4, TGF-beta1 mRNA levels were significantly decreased but returned to baseline by day 14. TGF-betaR1 mRNA expression remained unaffected by transfection throughout the time course. However, TGF-betaR2, FGF-2, and FGF-R1 demonstrated significant inhibition of mRNA expression on posttransfection day 4., Conclusions: These results indicate that TGF-beta1 siRNA has the potential to alter the murine dura cytokines responsible for suture fusion in vitro. Manipulating underlying cranial suture biology with siRNA technology may ultimately allow control over suture fusion. This intervention may ultimately function as an effective adjunct to surgical intervention for craniosynostosis.

Background: Evidence from animal studies has associated transforming growth factor (TGF)-beta signaling with both normal and premature cranial suture fusion. However, the mechanisms whereby this pleiotropic cytokine mediates suture fusion remain uncertain. The authors established cultures of suture-derived mesenchymal cells from normally fusing (posterofrontal) and patent (sagittal) sutures and examined the in vitro effects of TGF-beta1 on these distinct cell populations., Methods: Skulls were harvested from 80 5-day-old mice. Posterofrontal and sagittal sutures were dissected, and cultures of suture-derived mesenchymal cells were established. The mitogenic, osteogenic, and chondrogenic effects of recombinant TGF-beta1 were then assessed on posterofrontal and sagittal suture-derived mesenchymal cells (1 to 10 ng/ml). Quantitative real-time polymerase chain reaction was used to examine the effects of TGF-beta1 on gene expression., Results: TGF-beta1 significantly decreased proliferation of both posterofrontal and sagittal suture-derived mesenchymal cells, by bromodeoxyuridine incorporation assays (n = 6). TGF-beta1 also inhibited osteogenesis in both suture-derived mesenchymal cells determined by alkaline phosphatase activity and mineralization (n = 3 for all assays). During chondrogenic differentiation, TGF-beta1 markedly increased expression of chondrocyte-specific gene markers in posterofrontal suture-derived mesenchymal cells (Sox9, Col II, Aggrecan, and Col X) (p

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.

Background: Craniosynostosis, the premature fusion of cranial sutures, affects one in 2500 children. In the mouse, the posterofrontal suture is programed to fuse postnatally, but the adjacent sagittal suture remains patent throughout life. To study the cellular process of suture fusion, the authors isolated and studied suture-derived mesenchymal cells., Methods: Skulls were harvested from 80 mice (2 to 5 days old), and posterofrontal and sagittal sutures were dissected meticulously. Suture mesenchymal tissue was separated from the underlying dura mater and overlying pericranium and cultured in growth media. After the cells migrated from the explant tissues, the morphologies of the two cell populations were studied carefully, and quantitative real-time polymerase chain reaction was performed to evaluate gene expression., Results: Both posterofrontal and sagittal cells exhibited highly heterogeneous morphologies, and the posterofrontal cells migrated faster than the sagittal cells. Accordingly, growth factors such as transforming growth factor-beta1 and fibroblast growth factor (FGF)-2 were expressed significantly more highly in posterofrontal compared with sagittal suture mesenchymal cells. In contrast, FGF receptor 2 and FGF-18 were expressed significantly more in sagittal than in posterofrontal suture cells. Importantly, bone morphogenic protein-3, the only osteogenic inhibitor in the bone morphogenic protein family, and noggin, a bone morphogenic protein antagonist, were expressed significantly more in sagittal than in posterofrontal suture cells, suggesting a possible mechanism of suture patency., Conclusions: To the authors' knowledge, this is the first analysis of mouse suture-derived mesenchymal cells. The authors conclude that isolation of suture-derived mesenchymal cells will provide a useful in vitro system with which to study the mechanisms underlying suture biology.

Background: Clinical genetics data and investigative studies have contributed greatly to our understanding of the role of numerous genes in craniosynostosis. Recent studies have introduced antagonists of osteogenesis as potential key regulators of suture fusion and patency. The authors investigated the expression pattern of the bone morphogenetic protein antagonist BMP3 in rat cranial sutures and the factors regulating its expression in vitro., Methods: Microarray analysis was performed on rat posterior frontal and sagittal cranial sutures at 5, 10, 15, 20, and 30 days of life (n = 30 per group). Gene expression was confirmed using quantitative real-time reverse transcriptase polymerase chain reaction. Regulation of BMP3 expression was determined using primary rat calvarial osteoblasts stimulated with recombinant human fibroblast growth factor 2 or recombinant human transforming growth factor beta1, or cultured with primary rat nonsuture dura mater. Gene expression was quantified with quantitative real-time reverse transcriptase polymerase chain reaction., Results: BMP3 expression in the posterior frontal suture decreased over the time course analyzed, whereas it increased in the sagittal suture. Notably, BMP3 expression was higher in the patent sagittal suture during the window of posterior frontal suture fusion. Stimulation of osteoblasts with recombinant human fibroblast growth factor 2 led to a rapid and sustained suppression of BMP3 expression (85 percent, p < 0.01) when compared with controls. Co-culture with dural cells decreased BMP3 mRNA by 50 percent compared with controls (p < 0.01)., Conclusions: BMP3 is expressed in rat cranial sutures in a temporal pattern suggesting involvement in cranial suture patency and fusion. Furthermore, BMP3 is regulated in calvarial osteoblasts by recombinant human fibroblast growth factor 2 and by paracrine signaling from dura mater. These data add to our knowledge of the role of osteogenic antagonists in cranial suture biology.

Using a physiologic model of mouse cranial suture fusion, the authors' laboratory has previously demonstrated that transforming growth factor (TGF)-betas appear to be more abundantly expressed in the suture complex of the fusing posterior frontal compared with the patent sagittal suture. Furthermore, the authors have shown that by blocking TGF-beta signaling with a replication-deficient adenovirus encoding a defective, dominant negative type II TGF-beta receptor (AdDN-TbetaRII), posterior frontal suture fusion was inhibited. In this study, the authors attempt to further elucidate the role of TGF-beta in cranial suture fusion by investigating possible mechanisms of AdDN-TbetaRII-mediated cranial suture patency using both an established organ culture model and a novel in vitro co-culture system that recapitulates the in vivo anatomic dura mater/cranial suture relationship. In this article, the authors demonstrate that blocking TGF-beta signaling with the AdDN-TbetaRII construct led to inhibition of cellular proliferation in the suture mesenchyme and subjacent dura mater during the early period of predicted posterior frontal suture fusion. Interestingly, co-culture experiments revealed that transfecting osteoblasts with AdDN-TbetaRII led to alterations in the gene expression levels of two important bone-related molecules (Msx2 and osteopontin). Inhibiting TGF-beta signaling prevented time-dependent suppression of Msx2 and prevented induction of osteopontin, thereby retarding osteoblast differentiation. Furthermore, the authors demonstrated that the AdDN-TbetaRII construct was capable of blocking TGF-beta -mediated up-regulation of collagen IalphaI, an extracellular matrix molecule important for bone formation. Collectively, these data strongly suggest that AdDN-TbetaRII maintains posterior frontal patency, in part by altering early events in de novo bone formation, including cellular proliferation and early extracellular matrix production.

The human brain grows rapidly during the first 2 years of life. This growth generates tensile strain in the overlying dura mater and neurocranium. Interestingly, it is largely during this 2-year growth period that infants are able to reossify calvarial defects. This clinical observation is important because it suggests that calvarial healing is most robust during the period of active intracranial volume expansion. With a rat model, it was previously demonstrated that immature dura mater proliferates more rapidly and produces more osteogenic cytokines and markers of osteoblast differentiation than does mature dura mater. It was therefore hypothesized that mechanical strain generated by the growing brain induces immature dura mater proliferation and increases osteogenic cytokine expression necessary for growth and healing of the overlying calvaria. Human and rat (n = 40) intracranial volume expansion was calculated as a function of age. These calculations demonstrated that 83 percent of human intracranial volume expansion is complete by 2 years of age and 90 percent of Sprague-Dawley rat intracranial volume expansion is achieved by 2 months of age. Next, the maximal daily circumferential tensile strains that could be generated in immature rat dura mater were calculated, and the corresponding daily biaxial tensile strains in the dura mater during this 2-month period were determined. With the use of a three-parameter monomolecular growth curve, it was calculated that rat dura mater experiences daily equibiaxial strains of at most 9.7 percent and 0.1 percent at birth (day 0) and 60 days of age, respectively. Because it was noted that immature dural cells may experience tensile strains as high as approximately 10 percent, neonatal rat dural cells were subjected to 10 percent equibiaxial strain in vitro, and dural cell proliferation and gene expression profiles were analyzed. When exposed to mechanical strain, immature dural cells rapidly proliferated (5.8-fold increase in proliferating cell nuclear antigen expression at 24 hours). Moreover, mechanical strain induced marked up-regulation of dural cell osteogenic cytokine production; transforming growth factor-beta1 messenger RNA levels increased 3.4-fold at 3 hours and fibroblast growth factor-2 protein levels increased 4.5-fold at 24 hours and 5.6-fold at 48 hours. Finally, mechanical strain increased dural cell expression of markers of osteoblast differentiation (2.8-fold increase in osteopontin levels at 3 hours). These findings suggest that mechanical strain can induce changes in dura mater biological processes and gene expression that may play important roles in coordinating the growth and healing of the neonatal calvaria.

For decades surgeons have exploited the ability of infants to reossify large calvarial defects. To demonstrate the role of dura mater-osteoblast communication during the process of calvarial reossification, the authors used a novel in vitro system that recapitulates the in vivo anatomic relationship of these cell populations. Primary cultures of osteoblast cells from 2-day-old Sprague-Dawley rat pups were grown on six-well plates, and cultures of immature, non-suture-associated dura mater cells from 6-day-old Sprague-Dawley rat pups were grown on Transwell inserts. When the osteoblast and dura mater cell cultures reached confluence, they were combined. This Transwell co-culture system permitted the two cell populations to grow together in the same well, but it prevented direct cell-to-cell contact. Therefore, the authors were able to determine, for the first time, whether paracrine signaling from immature, non-suture-associated dura mater could influence the biologic activity of osteoblasts. Osteoblasts co-cultured with dural cells proliferated significantly faster after 2 days (2.1 x 10(5) +/- 2.4 x 10(4) versus 1.4 x 10(5) +/- 2.2 x 10(4), p < or = 0.05) and 4 days (3.1 x 10(5) +/- 5 x 10(4) versus 2.2 x 10(5) +/- 4.0 x 10(4), p < or = 0.01) than did osteoblasts cultured alone. After 20 days, co-cultured osteoblasts expressed greater amounts of mRNA for several markers of osteoblast differentiation, including collagen I alpha I (4-fold), alkaline phosphatase (2.5-fold), osteopontin (3-fold), and osteocalcin (4-fold), than did osteoblasts cultured alone. After 30 days, co-cultured osteoblasts produced bone nodules that were significantly greater both in number (324 +/- 29 nodules versus 252 +/- 29 nodules per well, p , < or = 0.04) and total area of nodules (65 +/- 11 mm(2) versus 24 +/- 1.6 mm(2), p < or = 0.003) than osteoblasts cultured alone. To begin to understand how dural cells effect changes in osteoblast gene expression, the authors compared the expression of candidate genes, transforming growth factor beta 1 and fibroblast growth factor 2, in dural cells and osteoblasts before and after 5 days of culture. Interestingly, the dura mater produced marked amounts of these osteogenic cytokines compared with osteoblasts.The described co-culture system demonstrated that co-cultured osteoblasts proliferated more rapidly and experienced an increased rate and degree of cellular maturation than did osteoblasts cultured alone. The authors hypothesize that this effect was due to paracrine signaling (e.g., transforming growth factor beta 1 and fibroblast growth factor 2) from the dura mater, and they are investigating those mechanisms in ongoing experiments. Collectively these data verify that immature, non-suture-associated dura mater can influence the biologic activity of osteoblasts. Moreover, the production of cytokines derived from the dura mater (e.g., transforming growth factor beta 1 and fibroblast growth factor 2), and they may begin to explain why immature animals and infants with intact dura mater can reossify large calvarial defects.

The dura mater, the outermost layer of the meninges, is thought to be essential for calvarial morphogenesis, postnatal suture fusion, and osseous repair of calvarial defects. Despite numerous studies illustrating the fundamental role of the dura mater, there is little information about the autocrine and paracrine mechanisms regulating dural cell biology during calvarial ossification. Previous work conducted in the authors' laboratory demonstrated that non-suture-associated dural cells from 6-day-old rat pups expressed high levels of fibroblast growth factor 2 (FGF-2), whereas dural cells from 60-day-old adult rats expressed very little FGF-2. Because young mammals can successfully heal large calvarial defects, the authors sought to investigate the autocrine and/or paracrine effects of FGF-2 on the proliferation, gene expression, and alkaline phosphatase production of dural cells. Cultures of non-suture-associated dural cells were established from 6-day-old Sprague-Dawley rat pups and then stimulated with recombinant human FGF-2 (rhFGF-2; 10 ng/ml). Dural cells stimulated with rhFGF-2 proliferated significantly faster than untreated dural cells at 24 hours (2.1 x 10(5) +/- 3.2 x 10(4) versus 1.1 x 10(5) +/- 1.8 x 10(4), p < or = 0.001) and 48 hours (2.3 x 10(5) +/- 4.2 x 10(4) versus 1.2 x 10(5) +/- 1.3 x 10(4), p < or = 0.001). Moreover, dural cells stimulated with rhFGF-2 expressed 7-fold more proliferating cell nuclear antigen than did control cultures. Treatment with rhFGF-2 increased dural cell expression of genes important for skeletal repair: FGF-2 (7-fold), transforming growth factor beta 1 (3-fold), transforming growth factor beta 3 (4-fold), and type I collagen (4-fold). Furthermore, rhFGF-2 increased dural cell expression of osteopontin (2-fold), a "late" marker of osteoblastic differentiation. Interestingly, dural cell alkaline phosphatase activity, an "earlier" marker of osteoblast differentiation, was significantly decreased by treatment with rhFGF-2 compared with control cultures at 24 hours (0.005 +/- 0.001 versus 0.01 +/- 0.003, p < or = 0.01) and 48 hours (0.004 +/- 0.0009 versus 0.01 +/- 0.0009). Together these data provide insight into the autocrine and paracrine effects of FGF-2 on the biology of the dura mater.

A significant body of literature supports a role for the dura mater underlying cranial sutures in the regulation of sutural fate. These studies have implicated regional differentiation of the dura mater based on association with fusing and patent rat cranial sutures. The purpose of these experiments was to isolate and characterize dural cells associated with fusing (posterior frontal) and patent (sagittal) rat cranial sutures. Six-day-old rats were killed, and the dura mater underlying the posterior frontal and sagittal sutures was harvested. Dural cells were briefly trypsinized and allowed to reach confluence. Two litters (10 animals per litter) were used for each set of experiments. Cells were harvested after the first and fifth passages for analysis of vimentin and desmoplakin expression (characteristic of human meningeal cells), cellular proliferation, density at confluence (a measure of cellular contact inhibition), and alkaline phosphatase production. In addition, bone nodule formation and collagen I production were analyzed in first passage cells. The results indicate that suture-derived dural cells can be established and that these cells coexpress vimentin and desmoplakin. In addition, it is demonstrated that first-passage sagittal suture-derived dural cells proliferate significantly faster and have decreased cellular contact inhibition than posterior frontal suture-derived cells (p < 0.01). Finally, it is shown that suture-derived dural cells have osteoblast-like properties, including alkaline phosphatase production, collagen I expression, and bone nodule formation in vitro. The possible mechanisms by which regional differentiation of suture-derived dural cells occur are discussed.