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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 β1, 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.

The role of angiogenesis during mechanically induced bone formation is incompletely understood. The relationship between the mechanical environment, angiogenesis, and bone formation was determined in a rat distraction osteogenesis model. Disruption of either the mechanical environment or endothelial cell proliferation blocked angiogenesis and bone formation. This study further defines the role of the mechanical environment and angiogenesis during distraction osteogenesis. Introduction: Whereas successful fracture repair requires a coordinated and complex transcriptional program that integrates mechanotransductive signaling, angiogenesis, and osteogenesis, the interdependence of these processes is not fully understood. In this study, we use a system of bony regeneration known as mandibular distraction osteogenesis (DO) in which a controlled mechanical stimulus promotes bone induction after an osteotomy and gradual separation of the osteotomy edges to examine the relationship between the mechanical environment, angiogenesis, and osteogenesis. Materials and Methods: Adult Sprague-Dawley rats were treated with gradual distraction, gradual distraction plus the angiogenic inhibitor TNP-470, or acute distraction (a model of failed bony regeneration). Animals were killed at the end of distraction (day 13) or at the end of consolidation (day 41) and examined with μCT, histology, and immunohistochemistry for angiogenesis and bone formation (n = 4 per time-point per group). An additional group of animals (n = 6 per time-point per group) was processed for microarray analysis at days 5, 9, 13, 21, and 41. Results and Conclusions: Either TNP-470 administration or disruption of the mechanical environment prevented normal osteogenesis and resulted in a fibrous nonunion. Subsequent analysis of the regenerate showed an absence of angiogenesis by gross histology and immunohistochemical localization of platelet endothelial cell adhesion molecule in the groups that failed to heal. Microarray analysis revealed distinct patterns of expression of genes associated with osteogenesis, angiogenesis, and hypoxia in each of the three groups. Our findings confirm the interdependence of the mechanical environment, angiogenesis, and osteogenesis during DO, and suggest that induction of proangiogenic genes and the proper mechanical environment are both necessary to support new vasculature for bone induction in DO.

Previous studies have documented the differences in expression of various genes associated with the process of osteogenesis in fusing and nonfusing cranial sutures, including growth factors, growth factor receptors, and extracellular matrix molecules. Most of these studies were performed in rats, and although the biology regulating cranial suture fusion in mice and rats is presumed to be similar, studies are needed to verify these expression patterns as mice become increasingly utilized for scientific inquiry into the molecular biology of suture fusion and patency. The purpose of this study was to determine the differences in expression of several genes known to be critical to osteoblast biology. Posterior frontal and sagittal suture complexes (including the associated dura mater, suture mesenchyme, and osteogenic fronts) were isolated from 5-, 15-, 25-, 35-, and 45-day-old male CD-1 mice (n = 8 per age; n = 40 total). Total cellular RNA was extracted and converted to cDNA. Quantitative real-time reverse transcriptase polymerase chain reaction was then performed for the following genes: transforming growth factor beta1 and beta3, fibroblast growth factor receptor 1, Runx2,Osteopontin, and Osteocalcin. Expression of all genes examined was increased significantly in the posterior frontal suture as compared with the sagittal suture. Peak expression for all genes was observed on day 25. These data demonstrate that the expression of osteogenic growth factors, growth factor receptors, transcription factors, and extracellular matrix molecules is increased in the fusing posterior frontal suture in mice.

Resume La mise au point de nouveaux tissus exige une approche qui associe la biologie du developpement, la biologie cellulaire et moleculaire a la medecine clinique, a la biochimie, a l’immunologie, a l’ingenierie, et aux sciences des materiaux. Tandis que de nombreux chercheurs essaient de reproduire des structures endogenes pour creer de nouveaux tissus, de nombreuses barrieres doivent etre surmontees pour creer des tissus complexes, vascularises, specifiques du patient pour une utilisation de remplacement et de reparation. Bien que la mise au point de tissus a composants multiples exige la mise en œuvre de nombreuses idees, dans un souci de clarte nous limiterons cette revue aux developpements recents de l'application des structures bioresorbables naturelles et synthetiques. Ici, nous mettons en valeur des biomateriaux potentiellement utiles aux chirurgiens plasticiens qui sont actuellement utilises ou ont ete developpes pour etre utilises pour des remplacements et des reparations de la peau et des os.

Young children are capable of healing large calvarial defects, whereas adults lack this endogenous osseous tissue-engineering capacity. Despite the important clinical implications, little is known about the molecular and cell biology underlying this differential ability. Traditionally, guinea pig, rabbit, and rat models have been used to study the orchestration of calvarial healing. To harness the research potential of knockout and transgenic mice, the authors developed a mouse model for calvarial healing. Nonsuture-associated parietal defects 3, 4, and 5 mm in diameter were made in both juvenile (6-day-old, n = 15) and adult (60-day-old, n = 15) mice. Calvariae were harvested after 8 weeks and analyzed radiographically and histologically. Percentage of healing was quantified using Scion Image software analysis of calvarial radiographs. A significant difference in the ability to heal calvarial defects was seen between 6-day-old and 60-day-old mice when 3-, 4-, or 5-mm defects were created. The authors' analysis revealed that juvenile mice healed a significantly greater percentage of their calvarial defects than adult mice (juvenile mean percentage of healing: 3-mm defects, 59 percent; 4-mm defects, 65 percent; 5-mm defects, 44 percent; adult mean percentage of healing

Craniosynostosis, the premature fusion of cranial sutures, is one of the most common craniofacial anomalies, with a reported incidence of up to one in 2500 live births. Despite its prevalence, the cause of craniosynostosis remains unknown. Previously, apoptosis has been postulated to be a contributing factor in the pathogenesis of craniosynostosis, although the role of programmed cell death in cranial sutures is poorly understood. To address this problem, the authors used an established rodent model of posterior-frontal suture fusion and sagittal suture patency to globally examine apoptosis in cranial sutures. Apoptosis was evaluated by systemically coinjecting Sprague-Dawley rats with both fluorescent and technetium-99m-labeled annexin V at time points before, during, and after the period of predicted posterior-frontal suture fusion to determine the magnitude and time course of overall apoptotic activity in both fusing and patent sutures. Using these novel in situ imaging techniques, the authors observed a significant increase in the overall levels of apoptosis in both the posterior-frontal and sagittal suture complexes during the period of predicted posterior-frontal suture fusion. To further explore this increase in apoptotic activity, they used microarray technology to study apoptosis-related genes within the suture complex. Interestingly, there was activation of distinct apoptotic pathways in the posterior-frontal and sagittal sutures during the period of predicted posterior-frontal suture fusion. Whereas increased transcription of genes associated with the mitochondria-mediated apoptotic pathway occurred in the posterior-frontal suture during fusion, activation of genes associated with the death receptor-mediated apoptotic pathway predominated in the patent sagittal suture during the same time period. These data suggest that although overall apoptotic activity in rat patent and fusing sutures is similar, the pathways mediating apoptosis within each suture are distinct.

In CD-1 mice, the posterior frontal suture (analogous to the human metopic suture) fuses while all other cranial sutures remain patent. In an in vitro organ culture model, the authors previously demonstrated that posterior frontal sutures explanted immediately before the onset of suture fusion (at 25 days old) mimic in vivo physiologic fusion. In the first portion of this study, the authors defined how early in development the posterior frontal suture fuses in their tension-free, serum-free organ culture system by serially analyzing posterior frontal suture fusion from calvariae explanted at different stages of postnatal development. Their results revealed a divergence of suture fate leading to abnormal patency or physiologic fusion between the first and second weeks of life, respectively, despite viability and continued growth of the calvarial explants in vitro. From these data, the authors postulated that the gene expression patterns present in the suture complex at the time of explant may determine whether the posterior frontal suture fuses or remains patent in organ culture. Therefore, to elucidate potentially important differences in gene expression within this "window of opportunity," they performed a cDNA microarray analysis on 5-day-old and 15-day-old posterior frontal and sagittal whole suture complexes corresponding to the age ranges for unsuccessful (1 to 7 days old) and successful (14 to 21 days old) in vitro posterior frontal suture fusion. Overall, their microarray results reveal interesting differential expression patterns of candidate genes in different categories, including angiogenic cytokines and mechanosensitive genes potentially important in cranial suture biology.

Retinoic acid has been shown to adversely affect craniofacial development. Cleft palate and craniosynostosis are two examples of craniofacial defects associated with prenatal exposure to this agent. Although the effects of retinoic acid on cephalic neural crest-derived tissues have previously been studied, the specific effects of retinoic acid on the cellular biology of osteoblasts remain unclear. The purpose of this study was to analyze in detail the effects of pharmacologic doses of retinoic acid on the differentiation and proliferation of osteoblasts derived from an intramembranous source. Primary rat calvarial osteoblasts were established in culture and treated with 1 or 10 μM all-trans-retinoic acid. Retinoic acid treatment markedly increased expression of osteopontin up to 48 h after stimulation. Consistent with this early stage of differentiation, both mRNA and protein analysis of FGF receptor isoforms demonstrated a switch in predominance from fibroblast growth factor receptor 2 (fgfr2) to fgfr1. Analysis of PCNA protein confirmed inhibition of proliferation by retinoic acid. To determine whether these alterations in osteoblast biology would lead to increased differentiation, we examined short term [alkaline phosphatase (AP) activity] and long term (von Kossa staining) surrogates of bone formation in vitro. These assays confirmed that retinoic acid increased osteogenesis, with a 4-fold increase in bone nodule formation in cells treated with 10 μM retinoic acid after 28 days. Overall, our results demonstrated that pharmacologic doses of all-trans-retinoic acid decreased osteoblast proliferation and increased differentiation, suggesting that retinoic acid may effect craniofacial development by pathologically enhancing osteogenesis. © 2005 Wiley-Liss, Inc.

Appropriately timed closure of the cranial sutures is a critical factor in normal postnatal morphogenesis of the cranial vault. Suture patency is necessary to permit rapid neonatal expansion of the cerebral hemispheres, and later ossification is important for bony protection of the cerebrum. Premature suture ossification (craniosynostosis) leads to myriad adverse functional and developmental consequences. Several murine studies have implicated dura-derived fibroblast growth factor-2 (FGF-2) paracrine signaling as a critical factor promoting physiologic posterior frontal suture fusion. In this study, the authors used real-time reverse transcription polymerase chain reaction (RT-PCR) to study an in vitro system that models the in vivo stimulation of suture calvarial osteoblasts by dura-derived FGF-2. The authors advocate real-time RT-PCR as a powerful and rapid technique that offers advantages in the highly sensitive, specific, and reproducible analyses of nine genes known to be important in cranial suture biology. The genes studied were growth factors [FGF-2, transforming growth factor (TGF)-beta 1, TGF-beta 2, and TGF-beta 3], growth factor receptors (FGF-R1, FGF-R2, TGF-beta RI, and TGF-beta RII), and a marker of osteoblast differentiation (Co1-I alpha I). These analyses provide a "snapshot" of several important genes involved in suture fusion that is more inclusive and quantitative than that which has been previously reported.

Mechanical tensile strain is believed to play an important role in regulating calvarial morphogenesis. To better understand the effects of mechanical strain on pathologic calvarial growth, we applied 10% constant equibiaxial tensile strain to neonatal rat calvarial osteoblast cultures and examined cellular proliferation, cytokine production, and extracellular matrix molecule expression. Mechanical strain markedly increased osteoblast proliferation as demonstrated by increased proliferating cell nuclear antigen (PCNA) protein. In addition, both transforming growth factor-beta1 (TGF-beta1) mRNA expression and fibroblast growth factor-2 (FGF-2) protein production were increased with exposure to strain. Moreover, mechanical strain induced expression of the extracellular matrix molecule collagen IalphaI. To further explore the relationship between mechanotransduction, osteogenesis, and angiogenesis, we examined the effect of mechanical strain on calvarial osteoblast expression of vascular endothelial growth factor (VEGF). Interestingly, we found that mechanical strain induced a rapid (within 3 hrs) increase in osteoblast VEGF expression. These data suggest that constant equibiaxial tensile strain-induced mechanotransduction can influence osteoblasts to assume an "osteogenic" and "angiogenic" phenotype, and these findings may have important implications for understanding the mechanisms of pathologic strain-induced calvarial growth.

Craniofacial anomalies can severely affect the appearance, function, and psychosocial well being of patients; thus, tissue engineers are developing new techniques to functionally and aesthetically rebuild craniofacial structures. In the past decade, there have been tremendous advances in the field of tissue engineering that will substantially alter how surgeons approach craniofacial reconstruction. In this brief review, we highlight some of the preclinical recombinant protein, gene transfer, and cell-based strategies currently being developed to augment endogenous tissue repair or create structures for replacement. In addition, we discuss the importance of studying endogenous models of tissue induction and present some of the current in vitro and in vivo approaches to growing complex tissues/organs for craniofacial reconstruction.

Fabricating new tissues requires an interdisciplinary approach that combines developmental, cellular, and molecular biology with clinical medicine, biochemistry, immunology, engineering, and the material sciences. While many researchers are attempting to replicate endogenous structures to create new tissues, numerous barriers must be overcome to create complex, vascularized, patient-specific tissue constructs for replacement and repair. Although multi-step, multi-component tissue fabrication requires an amalgamation of ideas, for clarity we will limit this review to recent developments in the application of natural and synthetic bioabsorbable scaffolds. Herein, we highlight biomaterials potentially useful to plastic and reconstructive surgeons that are currently being used or developed for the replacement and repair of skin and bone.

Adhesion formation after flexor tendon repair remains a clinical problem. Early postoperative motion after tendon repair has been demonstrated to reduce adhesion formation while increasing tendon strength. The authors hypothesized that during mobilization, tendon cells experience mechanical shear forces that alter their biology in a fashion that reduces scar formation but also activates key genes involved in tendon healing.To test this hypothesis, primary intrinsic tenocyte cultures were established from flexor tendons of 20 Sprague-Dawley rats and sheared at 50 rpm (0.41 Pa) using a cone viscometer for 6 and 12 hours. Total RNA was harvested and compared with time-matched unsheared controls using cDNA microarrays and Northern blot analysis.Microarray analysis demonstrated that mechanical shear stress induced an overall "antifibrotic" expression pattern with decreased transcription of collagen type I and collagen type III. Shear stress down-regulated profibrotic molecules in the platelet-derived growth factor, insulin-like growth factor, and fibroblast growth factor signaling pathways. In addition, shear stress induced an overall decrease in transforming growth factor (TGF)-beta signaling pathway molecules with down-regulation of TGF-beta2, TGF-beta3, TGF-RI, and TGF-RII expression. Moreover, sheared tendon cells increased expression of matrix metalloproteinases and decreased expression of tissue inhibitors of metalloproteinase, an expression pattern consistent with an antifibrotic increase in extracellular matrix degradation. However, the authors also found up-regulation of genes implicated in tendon healing, specifically, vascular endothelial growth factor-A and several bone morphogenetic proteins. Interestingly, the known mechanoresponsive gene, TGF-beta1, also implicated in tendon healing, was differentially up-regulated by shear stress. Northern blot validation of our results for TGF-beta1, TGF-beta2, TGF-beta3, and collagen type I demonstrated direct correlation with the authors' microarray data.The authors demonstrate an overall antifibrotic expression pattern in response to shear stress in tendon cells that may provide insight into the mechanisms by which early mobilization decreases adhesion formation without impaired tendon healing.

Background: It has widely been observed that young children are capable of reossi-fying large calvarial defects, while adults lack this endogenous tissue-engineering capacity. The ability of juvenile animals to regenerate calvarial defects has been investigated in multiple animal models, including mice. In this study, the authors used cDNA microarrays to investigate the expression of osteogenesis-associated genes upstream and downstream of Runx2 in juvenile and adult mouse calvaria. Methods: Nonsuture-associated parietal bone discs were harvested from 6-day-old (n = 50) and 60-day-old (n = 35) male CD-1 mice. After separation of the underlying dura mater and overlying pericranium, the calvarial discs were snap-frozen and RNA was extracted from pooled samples of calvaria for microarray analysis. Genes analyzed included cytokines, receptors, and cell-surface and matrix proteins both upstream and downstream of Runx2. Results: Genes associated with the Runx2 pathway had notably higher levels in the juvenile versus adult calvaria. All genes except for osteocalcin were expressed at least twofold higher in the juvenile calvaria. This pattern was validated with quantitative real-time polymerase chain reaction. In addition, mRNA for potent osteoinductive growth factors was present at higher levels in the juvenile compared with the adult calvaria. Conclusions: These findings reflect a genomic environment of active osteoblast differentiation and ossification in the juvenile calvaria compared with the adult quiescent calvarial tissue. These data suggest that a decreased osteogenic potential of adult calvarial osteoblasts may, in part, explain the inability of adult animals to heal calvarial defects.

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.

Accumulating clinical genetic data support the hypothesis that alterations in osteoblast differentiation are closely associated with craniosynostoses. Gain-of-function mutations in FGFR1, FGFR2, FGFR3, and Msx2 and loss-of-function mutations in Twist are examples of such alterations. Several studies have examined how these mutations alter the expression patterns for transcription factors such as Runx2 and noncollagenous extracellular matrix molecules such as osteopontin and osteocalcin. One limitation of such studies is that they examine samples derived from craniosynostotic patients with sutures that have already fused, thus missing the dynamic osteogenic process of suture fusion. In this study, in situ hybridization was used to localize Runx2, osteopontin, and osteocalcin expression in the sagittal and posterior frontal sutures in mice (n = 20), before (day 13), during (days 23, 33, and 43), and after (day 53) the period of physiological posterior frontal suture fusion. The data demonstrated similar patterns of expression in fusing (posterior frontal) and nonfusing (sagittal) sutures. The expression of all three genes was primarily concentrated in the osteogenic fronts of both sutures and decreased with time. Notably, none of the three genes was expressed in the mesenchyme of either fusing or nonfusing sutures. The data suggest that the molecular signals leading to bone formation along the osteogenic fronts in fusing and nonfusing sutures are similar, raising the possibility that other factors, such as antagonists of osteogenesis, might have a role in maintaining suture patency.

Introduction: Similarities between skeletal formation and repair have led to the theory that adult fracture repair may parallel development. We hypothesize that since the mandible is derived from cranial neural crest (CNC) and forms via intramembranous ossification, non-stabilized mandibular fractures will heal in a similar fashion. Methods: Non-stabilized osteotomies were created in the right hemi-mandible of adult male mice. Mandibles were harvested at 7, 14, 21, and 28 days and processed for histology. Ossification was assessed and the presence of cartilage determined using histology and in situ hybridization (ISH). To determine the embryologic origins of the regenerate, transgenic mice were generated where CNC cells express GFP constitutively under the Wnt-1 promoter. Fractures were created as described above and processed for frozen section. CNC cells were localized using immunohistochemistry (IHC) for GFP. Closed, non-stabilized tibial fractures were also performed to determine the presence, if any, of CNC-derived cells in appendicular skeletal fractures. Results: In contrast to non-stabilized fractures of the appendicular skeleton, mandibular fractures healed predominantly via intramembranous ossification, with minimal and delayed cartilage production seen via histology and collagen2 ISH. IHC for GFP revealed that the mandibular fracture callous and regenerate was comprised primarily of GFP+, CNC-derived cells. In contrast, the tibial fracture callous was devoid of any CNC-derived cells. Conclusions: These data suggest that mandibular fractures heal by recapitulating their developmental program. These findings have important implications for designing appropriate cell-based approaches to craniofacial regeneration.

Introduction: Data indicates that TGF-betas may play an important role in craniosynostosis. In a murine organ culture model, we previously demonstrated that blocking TGF-beta signaling leads to pathologic suture patency. In this study, we investigated both the osteogenic properties of TGF-beta1 and determined whether adenoviral overexpression of TGF-beta1 in a normally patent sagittal (SAG) suture would lead to pathologic suture fusion. Methods: Primary calvarial rat osteoblasts were treated with TGF-beta1 adenovirus or vehicle and cultured for four weeks. Expression of TGF-beta1 was confirmed using quantitative real-time PCR (QRT-PCR) and bone nodule formation assessed with von Kossa staining. The dura mater of 22 day CD-1 mouse calvarial explants were then transfected with adenovirus overexpressing TGF-beta1 or lacZ. SAG sutures were harvested weekly for four weeks and analyzed with H&E as well as beta-galactosidase staining. Results: QRT-PCR data confirmed the marked up regulation of TGF-beta1 mRNA in osteoblasts transfected with the TGF-beta1 overexpression adenovirus. TGF-beta1 adenoviral overexpression led to a 5-fold increase in bone nodule formation compared to untreated osteoblasts. Interestingly TGF-beta1 adenoviral transfection in the underlying dura mater did not lead to pathologic SAG suture fusion in any specimens. Conclusions: TGF-beta1 has potent osteogenic capabilities as demonstrated by a significant increase in bone nodule formation. Although blocking TGF-beta signaling prevents physiologic suture fusion, overexpression of TGF-beta in a normally patent cranial suture does not lead to pathologic craniosynostosis. TGF-beta is potentially necessary but not sufficient in the mechanism of programmed cranial suture fusion.

Introduction: Guided tissue regeneration (GTR) utilizes barrier materials to create environments that favor tissue regeneration. In the current study, we applied the principle of GTR to our rat mandibular distraction osteogenesis (DO) model in an attempt to enhance bone regeneration. Methods: Osteoconductive Gore Resolut XT membranes (a co-polymer of polylactic acid and polyglycolic acid) were used in this study. Twelve (n = 6 for each group) adult Sprague-Dawley rats underwent routine gradual distraction (GD, 5-day latency, 8-day distraction for a total of 4mm followed by 4-weeks of consolidation) and acute lengthening (AL, immediate lengthening to 4mm followed by 6-weeks of consolidation). An additional 10 animals underwent AL followed by application of Gore membranes (AL + GTR). Membranes were wrapped around the distraction gap in its entirety. All animals were sacrificed at 6 and 8 weeks post-operatively. Mandibles were analyzed with a Faxitron x-ray system, and routine H&E histology. Quantitative histomorphometric analyses were performed to compare relative bone formation between all three groups. Results: The GD group achieved complete bony union by 6 weeks. The AL group had only 28.27 ± 4.8% bone formation, while the AL + GTR group achieved 57.43 ± 16.5% bony union (∗p Conclusions: Our data demonstrates that GTR can significantly enhance osteogenesis. It may therefore have a role in accelerating bone regeneration in DO clinically.

Introduction: Although a wealth of data demonstrates that regionally patterned dura mater regulates suture fusion or patency, the onset and extent of this patterning remains unknown. In this study we used microarray analyses as a starting point to investigate the differences in suture-specific dura mater from the mouse posterior frontal (PF) and sagittal (SAG) suture. Methods: Regional dura mater was harvested from the PF and SAsutures of 15 and 25 day male CD-1 mice (n = 40 each) and RNA isolated. Microarray analyses were performed using 42,000 element cDNA arrays. Slug was also evaluated in non-suture dura from young (5-day-old) and adult (60-day-old) animals. Investigation into regulation of novel genes, such as the transcription factor Slug, were carried out using rat calvarial osteoblasts stimulated with FGF-2, TGF-beta 1, and BMP2 and 4. Results: Microarray analyses demonstrated differential regulation of over 220 genes in the PF suture at d15, and over 280 genes at d25. Although the number of genes down-regulated was similar, there was a dramatic increase in the number of genes up-regulated in the PF suture at d25 vs. d15 (137 vs. 43). Distinct patterns of expression were seen for several groups of genes, including cytokines (FGF and FGF receptors) and extra-cellular matrix molecules (Col-1, osteopontin, osteocalcin). Several novel genes were identified, including the transcription factor Slug. QRT-PCR verified a > 4-fold increase in Slug mRNA in PF vs. SAG dura, and a > 3-fold increase in young vs. adult dura mater. Functional studies demonstrated a selective inhibition of Slug mRNA in osteoblasts by FGF-2 and TGF-beta 1, but not the BMPs, up to 24 hrs post stimulation (see figure). Conclusions: Our data demonstrate genomic patterning of regional dura mater as early as ten days prior to PF suture fusion. In addition we show that the transcription factor Slug, an evolutionarily conserved gene involved in epithelial-to-mesenchymal cell transitions, is regulated by two growth factors known to be expressed in dura mater.

Between 1960 and 1994, the population of those 85 years and older in the United States grew 274%. 1 Similarly, the fastest-growing sector of surgical patients older than 65 years is those older than 85 years. 2 These figures are critical because elderly persons have the highest mortality in the adult surgical population (5.8%-6.2% in those >80 years in 500 consecutive patients requiring general or regional anesthesia and 8.4% in those >90 years in 795 in-house operations). 3-5 Why do elderly persons face such high surgical mortality rates? In addition to a higher incidence and prevalence of disease, elderly persons experience baseline physiological changes associated with senescence. 6 it is vital for the modern surgeon to be aware of the physiological changes associated with aging to minimize morbidity and mortality in the aging surgical population.