Regional difference in microRNA regulation in the skull vault.

BACKGROUND: The murine calvaria has several membrane bones with different tissue origins (e.g., neural crest-derived frontal bone vs. mesoderm-derived parietal bone). Neural crest-derived frontal bone exhibits superior osteogenic activities and bone regeneration. MicroRNA (miRNA) has been emerged as a crucial regulator during organogenesis and is involved in a range of developmental processes. However, the underlying roles of miRNA regulation in frontal bone and parietal bone is unknown. RESULTS: Total of 83 significantly expressed known miRNAs were identified in frontal bones versus parietal bones. The significantly enriched gene ontology and KEGG pathway that were predicted by the enrichment miRNAs were involved in several biological processes (cell differentiation, cell adhesion, and transcription), and multiple osteogenic pathways (e.g., focal adhesion, MAPK, VEGF, Wnt, and insulin signaling pathway. Focal adhesion and insulin signaling pathway were selected for target verification and functional analysis, and several genes were predicted to be targets genes by the differentially expressed miRNAs, and these targets genes were tested with significant expressions. CONCLUSIONS: Our results revealed a novel pattern of miRNAs in murine calvaria with dual tissue origins, and explorations of these miRNAs will be valuable for the translational studies to enhance osteogenic potential and bone regeneration in the clinic.

The effect of experimental diabetes and glycaemic control on guided bone regeneration: histology and gene expression analyses.

OBJECTIVES: To investigate the effect of experimental diabetes and metabolic control on intramembranous bone healing following guided bone regeneration (GBR). MATERIAL AND METHODS: Ninety-three Wistar rats were allocated to three experimental groups, healthy (H), uncontrolled diabetes (D) and controlled diabetes (CD). Twenty one days following diabetes induction, a standardised 5-mm defect was created at the mid-portion of each parietal bone. In 75 animals (25H, 25D, 25CD), one defect was treated with an intracranial and extracranial membrane according to the GBR principle, and one defect was left empty (control); five animals per group were then randomly sacrificed at 3, 7, 15, 30 and 60 days and processed for decalcified histology. In 18 animals (6H, 6D, 6CD), both defects were treated according to the GBR principle; three animals from each group were then randomly sacrificed at 7 and 15 days of healing and employed for gene expression analysis. RESULTS: Application of the GBR therapeutic principle led to significant bone regeneration even in the D group. However, at 15 and 30 days, the osteogenesis process was impaired by uncontrolled diabetes, as shown by the significant reduction in terms of defect closure (38-42%) and newly formed bone (54-61%) compared to the healthy group. The comparison of the D vs. H group at 15 days of healing yielded the largest number of genes with significantly differential expression, among which various genes associated with the ossification process (bmp4, ltbp4, thra and cd276) were identified. CONCLUSIONS: Uncontrolled diabetes seems to affect early phases of the bone regeneration following GBR. A misregulation of genes and pathways related to cell division, energy production, inflammation and osteogenesis may account for the impaired regeneration process in D rats. Further studies are warranted to optimise the GBR process in this medically compromised patient population.

Physiological Signatures of Dual Embryonic Origins in Mouse Skull Vault.

BACKGROUND/AIMS: The mammalian skull vault is a highly regulated structure and consists of several membrane bones of different tissue origins (e.g. neural crest derived frontal bone and mesoderm derived parietal bone). Although membrane bones form through intramembranous ossification, neural crest derived frontal bone has superior osteoblast activity and bone regeneration ability, triggering a novel conception for craniofacial reconstruction and bone regeneration called endogenous calvarial regeneration. However, a comprehensive landscape of the genes and signaling pathways involved in this process is not clear. METHODS: Transcriptome analysis within the two bone elements is firstly performed to determine the physiological signatures of differential gene expressions in mouse skull vault. RESULTS: Frontal bone tissues and parietal bone tissues maintain tissue origin through special gene expression similar to neural crest vs mesoderm tissue, and physiological functions between these two tissues are also found in differences related to proliferation, differentiation and extracellular matrix production and clustered signaling pathways. CONCLUSION: Our data provide novel insights into the potential gene regulatory network in regulating the development of neural crest-derived frontal bone and mesoderm-derived parietal bone.

Calcium-containing scaffolds induce bone regeneration by regulating mesenchymal stem cell differentiation and migration.

BACKGROUND: Osteoinduction and subsequent bone formation rely on efficient mesenchymal stem cell (MSC) recruitment. It is also known that migration is induced by gradients of growth factors and cytokines. Degradation of Ca2+-containing biomaterials mimics the bone remodeling compartment producing a localized calcium-rich osteoinductive microenvironment. The aim of our study was to determine the effect of calcium sulfate (CaSO4) on MSC migration. In addition, to evaluate the influence of CaSO4 on MSC differentiation and the potential molecular mechanisms involved. METHODS: A circular calvarial bone defect (5 mm diameter) was created in the parietal bone of 35 Balb-C mice. We prepared and implanted a cell-free agarose/gelatin scaffold alone or in combination with different CaSO4 concentrations into the bone defects. After 7 weeks, we determined the new bone regenerated by micro-CT and histological analysis. In vitro, we evaluated the CaSO4 effects on MSC migration by both wound healing and agarose spot assays. Osteoblastic gene expression after BMP-2 and CaSO4 treatment was also evaluated by qPCR. RESULTS: CaSO4 increased MSC migration and bone formation in a concentration-dependent manner. Micro-CT analysis showed that the addition of CaSO4 significantly enhanced bone regeneration compared to the scaffold alone. The histological evaluation confirmed an increased number of endogenous cells recruited into the cell-free CaSO4-containing scaffolds. Furthermore, MSC migration in vitro and active AKT levels were attenuated when CaSO4 and BMP-2 were in combination. Addition of LY294002 and Wortmannin abrogated the CaSO4 effects on MSC migration. CONCLUSIONS: Specific CaSO4 concentrations induce bone regeneration of calvarial defects in part by acting on the host's undifferentiated MSCs and promoting their migration. Progenitor cell recruitment is followed by a gradual increment in osteoblast gene expression. Moreover, CaSO4 regulates BMP-2-induced MSC migration by differentially activating the PI3K/AKT pathway. Altogether, these results suggest that CaSO4 scaffolds could have potential applications for bone regeneration.

The Morphogenesis of Cranial Sutures in Zebrafish.

Using morphological, histological, and TEM analyses of the cranium, we provide a detailed description of bone and suture growth in zebrafish. Based on expression patterns and localization, we identified osteoblasts at different degrees of maturation. Our data confirm that, unlike in humans, zebrafish cranial sutures maintain lifelong patency to sustain skull growth. The cranial vault develops in a coordinated manner resulting in a structure that protects the brain. The zebrafish cranial roof parallels that of higher vertebrates and contains five major bones: one pair of frontal bones, one pair of parietal bones, and the supraoccipital bone. Parietal and frontal bones are formed by intramembranous ossification within a layer of mesenchyme positioned between the dermal mesenchyme and meninges surrounding the brain. The supraoccipital bone has an endochondral origin. Cranial bones are separated by connective tissue with a distinctive architecture of osteogenic cells and collagen fibrils. Here we show RNA in situ hybridization for col1a1a, col2a1a, col10a1, bglap/osteocalcin, fgfr1a, fgfr1b, fgfr2, fgfr3, foxq1, twist2, twist3, runx2a, runx2b, sp7/osterix, and spp1/ osteopontin, indicating that the expression of genes involved in suture development in mammals is preserved in zebrafish. We also present methods for examining the cranium and its sutures, which permit the study of the mechanisms involved in suture patency as well as their pathological obliteration. The model we develop has implications for the study of human disorders, including craniosynostosis, which affects 1 in 2,500 live births.

Calvarial Defect Healing Induced by Small Molecule Smoothened Agonist.

Hedgehog (Hh) signaling positively regulates both endochondral and intramembranous ossification. Use of small molecules for tissue engineering applications poses several advantages. In this study, we examined whether use of an acellular scaffold treated with the small molecule Smoothened agonist (SAG) could aid in critical-size mouse calvarial defect repair. First, we verified the pro-osteogenic effect of SAG in vitro, using primary neonatal mouse calvarial cells (NMCCs). Next, a 4 mm nonhealing defect was created in the mid-parietal bone of 10-week-old CD-1 mice. The scaffold consisted of a custom-fabricated poly(lactic-co-glycolic acid) disc with hydroxyapatite coating (measuring 4 mm diameter × 0.5 mm thickness). Treatment groups included dimethylsulfoxide control (n = 6), 0.5 mM SAG (n = 7) or 1.0 mM SAG (n = 7). Evaluation was performed at 4 and 8 weeks postoperative, by a combination of high-resolution microcomputed tomography, histology (H & E, Masson's Trichrome), histomorphometry, and immunohistochemistry (BSP, OCN, VEGF). In vivo results showed that SAG treatment induced a significant and dose-dependent increase in calvarial bone healing by all radiographic parameters. Histomorphometric analysis showed an increase in all parameters of bone formation with SAG treatment, but also an increase in blood vessel number and density. In summary, SAG is a pro-osteogenic, provasculogenic stimulus when applied locally in a bone defect environment.

Mice lacking the conserved transcription factor Grainyhead-like 3 (Grhl3) display increased apposition of the frontal and parietal bones during embryonic development.

BACKGROUND: Increased apposition of the frontal and parietal bones of the skull during embryogenesis may be a risk factor for the subsequent development of premature skull fusion, or craniosynostosis. Human craniosynostosis is a prevalent, and often serious embryological and neonatal pathology. Other than known mutations in a small number of contributing genes, the aetiology of craniosynostosis is largely unknown. Therefore, the identification of novel genes which contribute to normal skull patterning, morphology and premature suture apposition is imperative, in order to fully understand the genetic regulation of cranial development. RESULTS: Using advanced imaging techniques and quantitative measurement, we show that genetic deletion of the highly-conserved transcription factor Grainyhead-like 3 (Grhl3) in mice (Grhl3 -/- ) leads to decreased skull size, aberrant skull morphology and premature apposition of the coronal sutures during embryogenesis. Furthermore, Grhl3 -/- mice also present with premature collagen deposition and osteoblast alignment at the sutures, and the physical interaction between the developing skull, and outermost covering of the brain (the dura mater), as well as the overlying dermis and subcutaneous tissue, appears compromised in embryos lacking Grhl3. Although Grhl3 -/- mice die at birth, we investigated skull morphology and size in adult animals lacking one Grhl3 allele (heterozygous; Grhl3 +/- ), which are viable and fertile. We found that these adult mice also present with a smaller cranial cavity, suggestive of post-natal haploinsufficiency in the context of cranial development. CONCLUSIONS: Our findings show that our Grhl3 mice present with increased apposition of the frontal and parietal bones, suggesting that Grhl3 may be involved in the developmental pathogenesis of craniosynostosis.

Enhanced Activation of Canonical Wnt Signaling Confers Mesoderm-Derived Parietal Bone with Similar Osteogenic and Skeletal Healing Capacity to Neural Crest-Derived Frontal Bone.

Bone formation and skeletal repair are dynamic processes involving a fine-tuned balance between osteoblast proliferation and differentiation orchestrated by multiple signaling pathways. Canonical Wnt (cWnt) signaling is known to playing a key role in these processes. In the current study, using a transgenic mouse model with targeted disruption of axin2, a negative regulator of cWnt signaling, we investigated the impact of enhanced activation of cWnt signaling on the osteogenic capacity and skeletal repair. Specifically, we looked at two calvarial bones of different embryonic tissue origin: the neural crest-derived frontal bone and the mesoderm-derived parietal bone, and we investigated the proliferation and apoptotic activity of frontal and parietal bones and derived osteoblasts. We found dramatic differences in cell proliferation and apoptotic activity between Axin2-/- and wild type calvarial bones, with Axin2-/- showing increased proliferative activity and reduced levels of apoptosis. Furthermore, we compared osteoblast differentiation and bone regeneration in Axin2-/- and wild type neural crest-derived frontal and mesoderm-derived parietal bones, respectively. Our results demonstrate a significant increase either in osteoblast differentiation or bone regeneration in Axin2-/- mice as compared to wild type, with Axin2-/- parietal bone and derived osteoblasts displaying a "neural crest-derived frontal bone-like" profile, which is typically characterized by higher osteogenic capacity and skeletal repair than parietal bone. Taken together, our results strongly suggest that enhanced activation of cWnt signaling increases the skeletal potential of a calvarial bone of mesoderm origin, such as the parietial bone to a degree similar to that of a neural crest origin bone, like the frontal bone. Thus, providing further evidence for the central role played by the cWnt signaling in osteogenesis and skeletal-bone regeneration.

Accelerating bone generation and bone mineralization in the Interparietal sutures of rats using an rhBMP-2/ACS composite after rapid expansion.

This study aims to investigate the effects of rhBMP-2/ACS composite on bone regeneration and mineralization during expansion of the interparietal suture in rats. Forty 10-week-old Sprague-Dawley rats were divided into four groups (n=10). The first group (intact group) did not receive any intervention. The second group (expansion control group) received an expansion force of 60 g. The remaining two groups received an expansion force of 60 g and were implanted with an atelo-type I absorbable collagen sponge and rhBMP-2/ACS composite positioned on the suture beneath the periosteum. The relapse, relapse ratio, relevant bone remodelling, and calcium and osteocalcin contents were evaluated. Bone regeneration in the interparietal suture was estimated by the histological method. The osteocalcin content was measured by radioimmunoassay, and the calcium content was measured by atomic absorption spectrophotometry. Bone regeneration was more active in the suture after application of the expansion force compared with that of the suture without any intervention. Bone bridges formed in the rhBMP-2/collagen composite group. Both osteocalcin and calcium content were higher in the rhBMP-2/collagen composite group than in the other three groups (P<0.01). The relapse ratio in the rhBMP-2/collagen group was much lower than that in the other two expansion groups (P<0.01). RhBMP-2/ACS composite can promote bone regeneration and bone mineralization in the expanded suture and decrease the relapse ratio. Thus, the rhBMP-2/ACS composite may be therapeutically beneficial to the inhibition of relapse and shortening of the retention period during rapid expansion.

Negative pressure technology enhances bone regeneration in rabbit skull defects.

BACKGROUND: Bone is a slowly regenerating tissue influenced by various physiological processes, including proliferation, differentiation, and angiogenesis, under the control of growth factors. Shortening this healing time is an important and popular clinical research focus in orthopedics. Negative pressure can stimulate angiogenesis, improve blood circulation, promote granulation tissue growth and accelerate tissue wound healing. We sought to determine whether negative pressure could reduce bone healing time in a rabbit cranial defect model. METHODS: Four symmetrical holes (diameter, 3.5 mm) were drilled into the skulls of 42 New Zealand white rabbits, with two holes in each parietal bone. For each rabbit, the two sides were then randomly assigned into experimental and control groups. Using negative pressure suction tubes, experimental holes were treated with -50 kPa for 15 minutes, four times per day, whereas the control holes remained untreated. After 4 weeks, the negative pressure suction tubes were removed. At 2, 4, 6 and 8 weeks, three-dimensional (3D) reconstruction computed tomography (CT), X-ray radiopacity, and two-photon absorptiometry were used to evaluate new bone formation. Histological changes were determined by hematoxylin and eosin (H.E) staining. At weekly intervals until 6 weeks, the mRNA expression levels of vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP)-2 were evaluated by RT-PCR. A paired student's t-test was employed to compare X-ray radiopacity and bone density measurements between the experimental and control groups. RESULTS: 3D-reconstruction CT showed that new bone regeneration in the experimental group was greater than that in the control group at 4 and 6 weeks. At these time points, the experimental group presented with higher X-ray radiopacity and increased bone density (P < 0.05) as compared with the control group. Cartilage islands and new bone were observed by H.E staining at 2 weeks in the experimental group. By 6 weeks, the new bone had matured into lamellar bone in the experimental group. RT-PCR results showed that VEGF and BMP-2 were highly expressed in the experimental group as compared with control. CONCLUSIONS: Intermittent negative pressure can promote the regeneration of bone possibly by enhancing the expression of VEGF and BMP-2.

Effects of mesenchymal stem cells in critical size bone defect.

BACKGROUND AND OBJECTIVES: The aim of this study was to compare culture-expanded, bone marrow-derived mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) loaded to biphasic calcium phosphate (BCP) bone ceramic in the repair of rat calvarial bone. MATERIALS AND METHODS: Critical-size (7 mm dia.) calvarial defects were prepared in the frontal-parietal bones of 90 adult female Sprague-Dawley rats. Rats were randomly divided into 5 groups, according to defect filling, as follows: Group I (n = 21), BCP; Group II (n = 21), BCP+PRP; Group III (n = 21), BCP+MSC; Group IV (n = 21), BCP+PRP+MSC; Group V (n = 6) (control), no treatment. Animals were sacrificed at 2, 8 and 12 weeks postsurgery and bone regeneration was evaluated both histologically and immunohistochemically. RESULTS: Statistically significant differences were observed in bone osteoblastic activity in calvarial defects among the groups (p < 0.05). PRP and MSC used in combination with BCP as a defect filling resulted in greater osteoblastic bone formation activity when compared to the use of BCP alone. CONCLUSIONS: The combination of mesenchymal stem cells, platelet rich plasma and synthetic bone substitute was found to be more effective in inducing new bone formation (osteogenesis) than the use of platelet rich plasma combined with synthetic bone substitute and the use of synthetic bone substitute alone.

Microtomographic evaluation of the bone-cell interactions with a silorane-based composite.

The low-shrink Silorane-based composite could bond effectively to bone and showed the potential be used as a bone cement. Bone organ culture maintains the anatomical order, natural cell-to-cell and cell-to-matrix relationship. The purpose of this study was to evaluate the responses of bone cells to a Silorane-based composite which was compared with a representative polymethyl methacrylate (PMMA) bone cement. The critical size defects were created through the parietal bones from one litter of mice. The paired bones were divided into two groups: Silorane-based composite group and PMMA group. The prepared two groups of disks were put into the defects. The cultures were grown in vitro for 38 days and analyzed with microcomputed-tomography, dissecting-microscope, phase- contrast-microscope, scanning-electron-microscopy, and energy- dispersive-X-ray. At the 10th day, the Silorane disk was almost fully covered by a sheet of cells but the cells hardly attached to the disk surface. The edge of the PMMA disk was covered by a sheet of cells and the migrated individual cells attached to the whole surface of the disk. At the 38th day, some cells attached to the exposed disk area of the Silorane disk while the formed tissues covered the whole surface of the PMMA disk. The collagen fibers, globular deposits and bone formation were visible in both groups. The Silorane-based composite showed promise as a potential bone cement when compared with PMMA which is used in clinical orthopedics. However, the cell attachment to PMMA was evidently better than to Silorane-based composite.

Wear debris stimulates bone-resorbing factor expression in the fibroblasts and osteoblasts.

Wear debris is believed to cause periprosthetic osteolysis and loosening of total joint arthroplasties. We investigated the wear debris-mediated osteolysis in wild-type mice and macrophage-deficient Csf1op/Csf1op (op/op) mice using high density polyethylene (HDP) particles transplanted on the parietal bone surface. Four weeks after surgery, phagocytosis of the HDP particles by F4/80-positive macrophages and tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts was observed in the normal mice, but not in the macrophage-deficient op/op mice. These results suggest that macrophages are implicated in wear debris-dependent osteoclast formation. However, HDP particles were phagocytosed not only by macrophages but also by F4/80-negative cells in both genotypes of mice. Electron microscopic observation identified these cells as fibroblasts. Cell culture studies demonstrated that fibroblasts cultured with HDP-particles showed upregulation of interleukin-6 (IL-6) expression compared with non-treated fibroblasts. When we examined the gene expression of osteoblasts that belong to the mesenchymal cell lineage as fibroblasts, we found that the expression of not only IL-6 but also interleukin-1 beta (IL-1ß), tumor necrosis factor-alpha (TNF-α) and cyclooxygenase2 (Cox2) were up-regulated by HDP particle-stimulation. These findings suggest the possibility that fibroblasts and osteoblasts are involved in wear debris-mediated osteolysis within the tissue surrounding artificial joints through the production of bone resorbing factors IL-6, IL-1ß, TNF-α, and Cox2.

Differential expression of sclerostin in adult and juvenile mouse calvariae.

BACKGROUND: An understanding of the molecular mechanisms controlling bone formation is central to skeletal tissue engineering efforts. The observation that immature animals are able to heal calvarial defects while adult animals are not has proven to be a useful tool for examining these mechanisms. Thus, the authors compared expression of sclerostin, a bone inhibitor, between the calvariae of juvenile and adult mice. METHODS: Parietal bone was harvested from juvenile (6-day-old; n = 20) and adult (60-day-old; n = 20) mice. Sclerostin transcript and protein levels were compared between the parietal bone of juvenile and adult mice using polymerase chain reaction, Western blotting, and immunohistochemistry. Finally, osteoblasts from the parietal bone of juvenile and adult mice were harvested and cultured under osteogenic differentiation conditions with and without recombinant sclerostin (200 ng/ml). Terminal osteogenic differentiation was assessed at 21 days with alizarin red staining. RESULTS: Polymerase chain reaction, Western blot analysis, and immunohistochemistry all confirmed greater expression of sclerostin in the parietal bone of adult mice when compared with that of juvenile mice. Osteoblasts, whether from juvenile or adult parietal bones, demonstrated reduced capacity for osteogenic differentiation when exposed to recombinant sclerostin. CONCLUSIONS: Given the role of sclerostin in inhibiting bone formation, the authors' findings suggest that differences in expression levels of sclerostin may play a role in the differential regenerative capacity of calvariae from juvenile and adult animals. These findings suggest it as a potential target to abrogate in future tissue engineering studies.

Immunohistochemical analysis of osteoconductivity of beta-tricalcium phosphate and carbonate apatite applied in femoral and parietal bone defects in rats.

The feature of osteoconductivity, and expression of inductive BMP and transcription factors (Runx2 and Osterix) for osteoblast differentiation, which was related to conductive bone formation, were observed in experimentally created defects in rat femoral and parietal bones filled with beta-tricalcium phosphate (beta-TCP) or carbonate apatite (CAP). Femoral cortical bone defects were repaired by conductive bone formed by osteoblasts differentiated around beta-TCP and CAP, and immunohistochemical observation revealed that the osteoblasts expressed BMPs, Runx2, and Osterix. However, the repair in parietal bone defects was incomplete despite the beta-TCP and CAP filling. Only cells, which differentiated around beta-TCP or CAP, and formed conductive bone expressed BMPs, Runx2, and Osterix. These findings revealed that the osteoconductivity of calcium phosphate materials required the expression of BMPs as the prerequisite for Runx2 and Osterix expression. Therefore, it is suggested that when calcium phosphate ceramics are used as bone substitute materials, BMPs are essential for osteoconductivity.

Differential FGF ligands and FGF receptors expression pattern in frontal and parietal calvarial bones.

The mammalian skull vault consists mainly of 5 flat bones, the paired frontals and parietals, and the unpaired interparietal. All of these bones are formed by intramembranous ossification within a layer of mesenchyme, the skeletogenic membrane, located between the dermal mesenchyme and the meninges surrounding the brain. While the frontal bones are of neural crest in origin, the parietal bones arise from mesoderm. The present study is a characterization of frontal and parietal bones at their molecular level, aiming to highlight distinct differences between the neural crest-derived frontal and mesodermal-derived parietal bone. We performed a detailed comparative gene expression profile of FGF ligands and their receptors known to play crucial role in skeletogenesis. This analysis revealed that a differential expression pattern of the major FGF osteogenic molecules and their receptors exists between the neural crest-derived frontal bone and the paraxial mesoderm-derived parietal bone. Particularly, the expression of ligands such as Fgf-2, Fgf-9 and Fgf-18 was upregulated in frontal bone on embryonic day 17.5, postnatal day 1 and postnatal day 60 mice. Frontal bone also elaborated higher levels of Fgf receptor 1, 2 and 3 transcripts versus parietal bone. Taken together, these data suggest that the frontal bone is a domain with higher FGF-signaling competence than parietal bone.

EphA4 as an effector of Twist1 in the guidance of osteogenic precursor cells during calvarial bone growth and in craniosynostosis.

Heterozygous loss of Twist1 function causes coronal synostosis in both mice and humans. We showed previously that in mice this phenotype is associated with a defect in the neural crest-mesoderm boundary within the coronal suture, as well as with a reduction in the expression of ephrin A2 (Efna2), ephrin A4 (Efna4) and EphA4 in the coronal suture. We also demonstrated that mutations in human EFNA4 are a cause of non-syndromic coronal synostosis. Here we investigate the cellular mechanisms by which Twist1, acting through Eph-ephrin signaling, regulates coronal suture development. We show that EphA4 mutant mice exhibit defects in the coronal suture and neural crest-mesoderm boundary that phenocopy those of Twist1(+/-) mice. Further, we demonstrate that Twist1 and EphA4 interact genetically: EphA4 expression in the coronal suture is reduced in Twist1 mutants, and compound Twist1-EphA4 heterozygotes have suture defects of greater severity than those of individual heterozygotes. Thus, EphA4 is a Twist1 effector in coronal suture development. Finally, by DiI labeling of migratory osteogenic precursor cells that contribute to the frontal and parietal bones, we show that Twist1 and EphA4 are required for the exclusion of such cells from the coronal suture. We suggest that the failure of this process in Twist1 and EphA4 mutants is the cause of craniosynostosis.

Differential effects of TGF-beta1 and TGF-beta3 on chondrogenesis in posterofrontal cranial suture-derived mesenchymal cells in vitro.

BACKGROUND: Transforming growth factor (TGF)-beta1 has been associated with cranial suture fusion, whereas TGF-beta3 has been associated with suture patency. The mouse posterofrontal suture, analogous to the human metopic suture, fuses through endochondral ossification. METHODS: TGF-beta1 and TGF-beta3 expression in the posterofrontal suture was examined by immunohistochemistry. Next, the authors established cultures of suture-derived mesenchymal cells from the posterofrontal suture and examined the cellular responses to TGF-beta1 and TGF-beta3. Proliferation in response to TGF-beta isoforms was examined by bromodeoxyuridine incorporation. High-density micromass culture of posterofrontal mesenchymal cells was used to study the effect of TGF-beta1 and TGF-beta3 on chondrogenic differentiation. RESULTS: TGF-beta1 but not TGF-beta3 protein was highly expressed in chondrocytes within the posterofrontal suture. Significant increases in posterofrontal cell proliferation were observed with TGF-beta3 but not TGF-beta1. TGF-beta1 led to significant increases in chondrogenic-specific gene expression (including Sox9, Col II, Aggrecan, and Col X) as compared with moderate effects of TGF-beta3. TGF-beta1 increased cellular adhesion molecule expression (N-cadherin and fibronectin) and promoted cellular condensation, whereas TGF-beta3 increased cellular proliferation (PCNA expression). Finally, TGF-beta1 and, to a lesser extent, TGF-beta3 induced the expression of fibroblast growth factors (FGF-2 and FGF-18). CONCLUSIONS: TGF-beta1 and TGF-beta3 exhibit marked differences in their effects on chondrogenesis in posterfrontal suture-derived mesenchymal cells, influencing different stages of chondrogenic differentiation. TGF-beta3 significantly increased cellular proliferation, whereas TGF-beta1 induced precartilage condensation, promoting chondrocyte differentiation.

Effect of solely applied platelet-rich plasma on osseous regeneration compared to Bio-Oss: a morphometric and densitometric study on rabbit calvaria.

BACKGROUND: The use of platelet-rich plasma (PRP) in bone augmentation procedures is well documented; however, the exact benefit of this material is not yet established. PURPOSE: This study aimed to evaluate the benefits of using PRP, when only used, and compare it to Bio-Oss (Geistlich Biomaterials, Wolhusen, Switzerland) in vertical bone augmentation capacity. MATERIALS AND METHODS: The study was performed in calvaria of eight adult female New Zealand rabbits using titanium bone conduction cylinder. Two titanium cylinders were fixed into perforated slits made on the parietal bone of each rabbit. On each rabbit, one chamber was grafted with Bio-Oss, and the contralateral was filled with PRP. Animals were sacrificed 4 weeks after intervention and biopsies were taken. Densitometric, histological, and histomorphometric analyses were performed to evaluate bone mineral density, vertical bone augmentation, and remaining graft volume, respectively. Statistical analyses were performed with Mann-Whitney test, using a significance level of p < .05. RESULTS: Densitometric and histomorphometric data analysis revealed that mean bone mineral densities and bone augmentation were significantly lower in the cylinders treated with PRP (p < .0001) 4 weeks after implantation. CONCLUSION: This study showed no beneficial effect of using PRP on osseous regeneration. In addition, it was emphasized that Bio-Oss presents good osteoconductive properties by achieving suitable bone volume values.