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Background: The purpose of this study was to assess the effects of systemically given krill oil (KO) on the development of new bone formation in the sutura palatina media following rapid maxillary expansion (RME)., Methods: 28 4-5 week-old male Wistar albino rats were randomly divided into 4 groups: Control (C), Only Expansion (OE) (no supplement but undergoing expansion and retention), KE (supplemented during both the expansion and retention phases), Krill Oil Nursery Group (KN) (supplemented during the 40-day nursery phase as well as during the expansion and retention phases). A 5-day RME was followed by a 12-day retention period. All rats were euthanized simultaneously. Micro-computerized tomography (Micro-CT), hemotoxylen-eosin (H&E) staining, and immunohistochemical analysis were conducted. Kruskal-Wallis and Dunn tests with Bonferonni corrrection were applied (p < 0.05)., Results: Expansion and KO supplementation did not cause a statistically significant change in bone mineral density (BMD), bone volume fraction (BV/TV), spesific bone surface (BS/BV) and trabecular thickness (Tb.Th). While the expansion prosedure increased the trabecular seperation (Tb.Sp), KO supplemantation mitigated this effect. The KE group exhibited a statistically significantly increase in trabecular number (Tb.N) compared to the OE group. Although receptor activator of nuclear factor-kappa-Β ligand (RANKL)/osteoprotegerin (OPG) ratios did not show significant differences between groups, the KE and OE groups demonstrated the lowest and highest value, respectively. KE showed a reduced amount of tartrate-resistant acid phosphatase (TRAP) compared to the OE., Conclusion: KO positively affected the architecture of the new bone formed in the mid-palatal suture. In this rat model of RME, results support the idea that administering of KO during the expansion period or beginning before the RME procedure may reduce relapse and enhance bone formation within the mid-palatal suture., (© 2024. The Author(s).)

Background: The potential of relapse of craniofacial disharmony after trans-sutural distraction osteogenesis is high due to the failure to produce a stable bone bridge in the suture gap. The aim of this study is to evaluate whether hydroxyapatite nanoparticles (nHAP) have the effect of promoting osteoblast differentiation of suture-derived stem cells (SuSCs) and bone formation in sagittal suture during expansion., Methods: SuSCs were isolated from sagittal sutures and exposed to various concentrations of nHAP (0, 25, 50, and 100 μg mL -1 ) to determine the optimal concentration of nHAP in osteoblast differentiation via performing Western Blotting and RT-qPCR. Twenty 4-week-old male Sprague-Dawley rats were randomly assigned into 4 groups: SHAM (sham-surgery), distraction, ACS (absorbable collagen sponge) and ACS+nHAP groups. In the ACS and ACS+nHAP groups, saline solution and nHAP suspended in a saline solution were delivered by ACS placed across the sagittal suture, respectively. In the latter three groups, the suture was expanded for 14 days by 50 g of constant force via a W shape expansion device. Suture gap area, bone volume fraction (BV/TV) and bone mineral density (BMD) of sagittal sutures were assessed via micro-CT, while the mechanical properties of sagittal sutures were evaluated via nanoindentation test. The efficacy of nHAP on bone formation in sagittal suture was also evaluated via BMP-2 immunohistochemistry staining., Results: The expression of osteoblast related genes and proteins induced by 25μg mL -1 nHAP were significantly higher than the other groups in vitro (p<0.05). Furthermore, treating with 25μg mL -1 nHAP in vivo, the suture gap area was significantly reduced when compared with the distraction group. Correspondingly, the BV/TV, BMD, hardness and modulus of sagittal sutures were significantly increased in the ACS+nHAP group (p<0.05)., Conclusion: The 25μg mL -1 dose of nHAP delivered by ACS can facilitate bone formation into the sagittal suture during expansion via inducing osteoblast differentiation of SuSCs., Competing Interests: The authors report no conflicts of interest for this work and declare no competing financial interests., (© 2021 Liang et al.)

Sclerostin antibody (Scl-Ab) is an anabolic bone agent that has been shown to increase bone mass in clinical trials of adult diseases of low bone mass, such as osteoporosis and osteogenesis imperfecta (OI). Its use to decrease bone fragility in pediatric OI has shown efficacy in several growing mouse models, suggesting translational potential to pediatric disorders of low bone mass. However, the effects of pharmacologic inhibition of sclerostin during periods of rapid growth and development have not yet been described with respect to the cranium, where lifelong deficiency of functioning sclerostin leads to patterns of excessive bone growth, cranial compression, and facial palsy. In the present study, we undertook dimensional and volumetric measurements in the skulls of growing Brtl/+ OI mice treated with Scl-Ab to examine whether therapy-induced phenotypic changes were similar to those observed clinically in patients with sclerosteosis or Van Buchem disorder. Mice treated between 3 and 14 weeks of age with high doses of Scl-Ab show significant calvarial thickening capable of rescuing OI-induced deficiencies in skull thickness. Other changes in cranial morphology, such as lengths and distances between anatomic landmarks, intracranial volume, and suture interdigitation, showed minimal effects of Scl-Ab when compared with growth-induced differences over the treatment duration. Treatment-induced narrowing of foramina was limited to sites of vascular but not neural passage, suggesting patterns of local regulation. Together, these findings reveal a site specificity of Scl-Ab action in the calvaria with no measurable cranial nerve impingement or brainstem compression. This differentiation from the observed outcomes of lifelong sclerostin deficiency complements reports of Scl-Ab treatment efficacy at other skeletal sites with the prospect of minimal cranial secondary complications. © 2019 American Society for Bone and Mineral Research. © 2019 American Society for Bone and Mineral Research., (© 2019 American Society for Bone and Mineral Research.)

Objectives: The aim of this study was to investigate the effects of continuous parathyroid hormone (cPTH) and intermittent parathyroid hormone (iPTH) on bone formation and bone resorption in midpalatal suture during maxillary expansion., Methods: Forty-eight male SD rats were randomly divided into four groups (n = 12 each), including the control, the expansion (E), the E + cPTH, and the E + iPTH. A thermosensitive controlled-release hydrogel was synthesized for cPTH administration. All animals were sacrificed after seven days. Microcomputed tomography, histochemical staining and real-time PCR were used to investigate the bone remodeling of midpalatal suture. Serum chemistry was adopted to evaluate the systemic condition of experimental animals., Results: The suture width was increased by the expansion, and further elevated by cPTH and iPTH administration. Both regimes improved bone volume fraction and trabecular thickness of suture bone region. Moreover, both cPTH and iPTH decreased SOST expression and enhanced the expression of β-catenin and Col-I. cPTH increased RANKL expression, inhibited OPG expression, and resulted in an increment of osteoclasts, while iPTH had no influence on osteoclastogenesis. The serum calcium concentration was enhanced by PTH administration., Conclusion: Both cPTH and iPTH promote midpalatal suture expansion by enhancing bone formation, probably via SOST downregulation and the resulting β-catenin activation. Our results demonstrated that PTH administration may have potential to be an adjunctive approach for maxillary expansion treatment., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Craniosynostosis (CS), the premature and pathological fusion of cranial sutures, is a relatively common developmental disorder. Elucidation of the pathways involved and thus therapeutically targeting it would be promising for the prevention of CS. In the present study, we examined the role of BMP pathway in the all-trans retinoic acid (atRA)-induced CS model and tried to target the pathway in vivo via PLGA-based control release. As expected, the posterior frontal suture was found to fuse prematurely in the atRA subcutaneous injection mouse model. Further mechanism study revealed that atRA could repress the proliferation while promote the osteogenic differentiation of suture-derived mesenchymal cells (SMCs). Moreover, BMP signal pathway was found to be activated by atRA, as seen from increased expression of BMPR-2 and pSMAD1/5/9. Recombinant mouse Noggin blocked the atRA-induced enhancement of osteogenesis of SMCs in vitro. In vivo, PLGA microsphere encapsulated with Noggin significantly prevented the atRA-induced suture fusion. Collectively, these data support the hypothesis that BMP signaling is involved in retinoic acid-induced premature fusion of cranial sutures, while PLGA microsphere-based control release of Noggin emerges as a promising strategy for prevention of atRA-induced suture fusion.

Objectives: An overexpression of Tgf-β2 leads to calvarial hyperostosis and suture fusion in individuals with craniosynostosis. Inhibition of Tgf-β2 may help rescue fusing sutures and restore normal growth. The present study was designed to test this hypothesis., Design: Twenty-eight New Zealand White rabbits with delayed-onset coronal synostosis had radiopaque markers placed on either side of the coronal sutures at 10 days of age. The rabbits were randomly assigned to: (1) sham control rabbits (n = 10), (2) rabbits with control IgG (100 μg/suture) delivered in a collagen vehicle (n = 9), and (3) rabbits with Tgf-β2 neutralizing antibody (100 μg/suture) delivered in a collagen vehicle (n = 9). Longitudinal growth data were collected at 10, 25, 42, and 84 days of age. Sutures were harvested at 84 days of age for histomorphometry., Results: Radiographic analysis showed significantly greater ( P < .05) coronal suture marker separation, craniofacial length, cranial vault length, height, shape indices, cranial base length, and more lordotic cranial base angles in rabbits treated with anti-Tgf-β2 antibody than in controls at 42 and 84 days of age. Histologically, rabbits treated with anti-Tgf-β2 antibody at 84 days of age had patent and significantly ( P < .05) wider coronal sutures and greater sutural area compared to controls., Conclusions: These data support our hypothesis that antagonism of Tgf-β2 may rescue fusing coronal sutures and facilitate craniofacial growth in this rabbit model. These findings also suggest that cytokine therapy may have clinical significance in infants with progressive postgestational craniosynostosis.

Objectives: Craniofacial sutures are important growth sites for skull development and are sensitive to mechanical stress. In order to determine the role of bone resorption in stress-mediated sutural bone growth, midpalatal suture expansion was performed in mice receiving alendronate, an anti-resorptive bisphosphonate., Materials and Methods: The midpalatal sutures of 8-week-old C57BL/6 mice were expanded by orthodontic wires over the period of 2 weeks. Mice with maxillary expansion without drug treatment as well as untreated animals served as controls. Skulls were analyzed with micro-computed tomography (micro-CT), immunohistochemistry and histology., Results: Maxillary expansion in mice without drug treatment resulted in an increase of TRAP-positive osteoclasts. In contrast, no increase in osteoclasts was observed in expanded sutures of mice with bisphosphonate treatment. Double calcein labeling demonstrated rapid bone formation on the oral edges of the expanded sutures in mice without bisphosphonate treatment. Less bone formation was observed in bisphosphonate-treated mice after expansion. Histology revealed that the sutural architecture was reestablished in expanded sutures of mice without bisphosphonate treatment. In contrast, the sutural architecture was disorganized and the cartilage had an irregular form, following expansion in bisphosphonate-treated mice. Finally, micro-CT imaging demonstrated that the total amount of maxillary expansion was significantly lower in mice with bisphosphonate treatment as compared to those of mice without drug treatment., Conclusions: In conclusion, our results indicate that osteoclast-mediated bone resorption is needed for maxillary suture expansion and reorganization of sutural architecture., Clinical Significance: Orthodontic palatal expansion can be complicated in patients with inherited or drug-induced diseases of osteoclast dysfunction.

Background: Saethre-Chotzen syndrome is an autosomal dominantly inherited disorder caused by mutations in the twist family basic helix-loop-helix transcription factor 1 (TWIST1) gene. Surgical procedures are frequently required to reduce morphological and functional defects in patients with Saethre-Chotzen syndrome. Therefore, the development of noninvasive procedures to treat Saethre-Chotzen syndrome is critical. We identified that periostin, which is an extracellular matrix protein that plays an important role in both bone and connective tissues, is downregulated in craniosynostosis patients., Methods: We aimed to verify the effects of different concentrations (0, 50, 100, and 200 μg/l) of recombinant mouse periostin in Twist1 +/- mice (a mouse model of Saethre-Chotzen syndrome) coronal suture cells in vitro and in vivo. Cell proliferation, migration, and osteogenic differentiation were observed and detected. Twist1 +/- mice were also injected with recombinant mouse periostin to verify the treatment effects., Results: Cell Counting Kit-8 results showed that recombinant mouse periostin inhibited the proliferation of suture-derived cells in a time- and concentration-dependent manner. Cell migration was also suppressed when treated with recombinant mouse periostin. Real-time quantitative PCR and Western blotting results suggested that messenger ribonucleic acid and protein expression of alkaline phosphatase, bone sialoprotein, collagen type I, and osteocalcin were all downregulated after treatment with recombinant mouse periostin. However, the expression of Wnt-3a, Wnt-1, and β-catenin were upregulated. The in vivo results demonstrated that periostin-treated Twist1 +/- mice showed patent coronal sutures in comparison with non-treated Twist1 +/- mice which have coronal craniosynostosis., Conclusion: Our results suggest that recombinant mouse periostin can inhibit coronal suture cell proliferation and migration and suppress osteogenic differentiation of suture-derived cells via Wnt canonical signaling, as well as ameliorate coronal suture fusion in Twist1 +/- mice.

Background and Objectives: Relaxin (RLN) is an insulin-like hormone associated with extracellular matrix degradation, osteoclastogenesis, and osteoblast differentiation. This study aimed to assess the effect of RLN during and after lateral expansion of murine calvarial sagittal sutures., Materials and Methods: RLN was injected topically using a nano-sized liposome carrier into the sagittal sutures of 8- to 10-week-old wild type mice just before lateral expansion. Suture morphology, bone mineral density (BMD), and bone volume were analysed by micro-computed tomography. Collagen deposition and osteoclast differentiation were observed by Verhoeff-Van Gieson (VVG) and tartrate-resistant acid phosphatase (TRAP) staining, respectively., Results: Less collagen staining and higher tissue-specific relaxin/insulin-like family peptide receptor (Rxfp)-1 and -2 expression were observed in the RLN-treated samples after 48 hours. Increased BMD and volume, and thick well-organised osteoid tissue, with multinucleated TRAP-positive cells, were observed in RLN-treated samples after 1 week. Increased Rxfp-1 expression was observed in the sagittal sutures in the mid-suture fibrous tissue following RLN treatment. Rxfp-2 was only expressed in the calvarial bone under tensile stimulation and RLN treatment further increased its expression., Limitations: RLN-liposomes were not detected at any instance under the current experimental conditions. This is a preliminary study and the sample number limits the power of its results. VVG staining cannot quantify collagen contents but can provide preliminary information on the presence of collagen fibres., Conclusions: RLN treatment may modify bone remodelling and collagen metabolism during and after suture expansion., (© The Author 2016. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com)

Human bone marrow-derived mesenchymal stem cells (hBMSCs) present promising opportunities for therapeutic medicine. Carbon derivatives showed only marginal enhancement in stem cell differentiation toward bone formation. Here we report that red-light absorbing carbon nitride (C 3 N 4 ) sheets lead to remarkable proliferation and osteogenic differentiation by runt-related transcription factor 2 (Runx2) activation, a key transcription factor associated with osteoblast differentiation. Accordingly, highly effective hBMSCs-driven mice bone regeneration under red light is achieved (91% recovery after 4 weeks compared to 36% recovery in the standard control group in phosphate-buffered saline without red light). This fast bone regeneration is attributed to the deep penetration strength of red light into cellular membranes via tissue and the resulting efficient cell stimulation by enhanced photocurrent upon two-photon excitation of C 3 N 4 sheets near cells. Given that the photoinduced charge transfer can increase cytosolic Ca 2+ accumulation, this increase would promote nucleotide synthesis and cellular proliferation/differentiation. The cell stimulation enhances hBMSC differentiation toward bone formation, demonstrating the therapeutic potential of near-infrared two-photon absorption of C 3 N 4 sheets in bone regeneration and fracture healing.

Maxillary expansion of the median palatal suture is a common procedure in orthodontics. Even after retention, there is a strong tendency to relapse in the expanded suture. The authors' objectives are to accelerate the bone formation process in the expanded suture and to reduce the required retention time by using an energy drink (ED). Twenty rats were divided into 2 groups (n = 20). The expansion-only group was defined as the control group (Group C). The other group was defined as the expansion-plus-energy drink group (Group ED). In Group ED, ED was administered systemically through oro-gastric tubes after the expansion period. After 5 days of expansion, the springs were removed and replaced with short lengths of rectangular retaining wire. Tooth separation was maintained for 12 days. Histomorphometric analysis showed significant differences between the 2 groups in terms of newly formed bone (P = 0.018) and the bone area (P = 0.007). For the parameters that were investigated, Group ED had better results than Group C. These results show that systemic administration of an ED during the early stages of the orthopedic expansion of the inter-maxillary suture areas can stimulate bone formation and decrease the time required for retention.

Large scale surveillance studies, case studies, as well as cohort studies have identified the influence of thyroid hormones on calvarial growth and development. Surveillance data suggests maternal thyroid disorders (hyperthyroidism, hypothyroidism with pharmacological replacement, and Maternal Graves Disease) are linked to as much as a 2.5 fold increased risk for craniosynostosis. Craniosynostosis is the premature fusion of one or more calvarial growth sites (sutures) prior to the completion of brain expansion. Thyroid hormones maintain proper bone mineral densities by interacting with growth hormone and aiding in the regulation of insulin like growth factors (IGFs). Disruption of this hormonal control of bone physiology may lead to altered bone dynamics thereby increasing the risk for craniosynostosis. In order to elucidate the effect of exogenous thyroxine exposure on cranial suture growth and morphology, wild type C57BL6 mouse litters were exposed to thyroxine in utero (control = no treatment; low ~167 ng per day; high ~667 ng per day). Thyroxine exposed mice demonstrated craniofacial dysmorphology (brachycranic). High dose exposed mice showed diminished area of the coronal and widening of the sagittal sutures indicative of premature fusion and compensatory growth. Presence of thyroid receptors was confirmed for the murine cranial suture and markers of proliferation and osteogenesis were increased in sutures from exposed mice. Increased Htra1 and Igf1 gene expression were found in sutures from high dose exposed individuals. Pathways related to the HTRA1/IGF axis, specifically Akt and Wnt, demonstrated evidence of increased activity. Overall our data suggest that maternal exogenous thyroxine exposure can drive calvarial growth alterations and altered suture morphology., Competing Interests: The authors have declared that no competing interests exist.

Objective: To determine whether systemically given stinging nettle (SN) has an effect on bone formation in response to expansion of the rat inter-premaxillary suture., Materials and Methods: A total of 28 male Wistar albino rats were randomly divided into 4 equal groups: control (C), only expansion (OE), SN extract given only during the expansion and retention periods (SN group; a total of 17days), and SN extract given during the nursery phase before expansion (a period of 40days) and during the expansion and retention periods (N+SN group; a total of 57days). After the 5-day expansion period was completed, the rats in the OE, SN, and N+SN groups underwent 12days of mechanical retention, after which they were sacrificed, and their premaxilla were dissected and fixed. A histologic evaluation was done to determine the number of osteoblasts, osteoclasts, and capillaries, as well as the number and intensity of inflammatory cells and new bone formation., Results: Statistically significant differences were found between the groups in all histologic parameters except the ratio of intensities of inflammatory cells. New bone formation and the number of capillaries were significantly higher in the SN groups than in the other groups. The statistical analysis also showed that the numbers of osteoblasts, osteoclasts, and capillaries were highest in the N+SN group., Conclusion: Systemic administration of SN may be effective in accelerating new bone formation and reducing inflammation in the maxillary expansion procedure. It may also be beneficial in preventing relapse after the expansion procedure., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: To evaluate the histomorphometric effects of different molecular weight hyaluronic acid on bone formation in rats after expansion of the interpremaxillary suture., Material and Methods: Twenty-four male Sprague Dawley rats were divided into three groups. Each group was subjected to expansion for 5 days and retention for 10 days. Group 1 received 50 μl of high molecular weight hyaluronic acid (HMWHA), group 2 received 50 μl of low molecular weight hyaluronic acid (LMWHA), and the control group received same amount of saline solution to the interpremaxillary suture. Ten days after injection, the rats were killed and their maxillas dissected. For the histomorphometric evaluation, blocks were serially sectioned at 10-μm intervals. Sections were stained with hematoxylin-eosin (HE) and evaluated with image analysis software. Bone area (μm²) (BA), bone perimeter of suture borders (μm) (BP), and ratio of osteoblast cells and capillary cells to BA and BP parameters were evaluated., Results: HMWHA showed a statistically higher ratio of osteoblast and capillary cell scores compared with the LMWHA and control groups (p < 0.05). There were no statistically significant differences in between LMWHA and control groups (p > 0.05)., Conclusions: Local injection of HMWHA in the interpremaxillary suture after rapid maxillary expansion stimulated new bone formation, which may shorten the retention period and may reduce the risk of relapse. LMWHA has no effect on bone formation in interpremaxillary suture., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Administration of onabotulinumtoxinA (BoNT-A) to peripheral tissues outside the calvaria reduces the number of days chronic migraine patients experience headache. Because the headache phase of a migraine attack, especially those preceded by aura, is thought to involve activation of meningeal nociceptors by endogenous stimuli such as changes in intracranial pressure (i.e. mechanical) or chemical irritants that appear in the meninges as a result of a yet-to-be-discovered sequence of molecular/cellular events triggered by the aura, we sought to determine whether extracranial injections of BoNT-A alter the chemosensitivity of meningeal nociceptors to stimulation of their intracranial receptive fields., Material and Methods: Using electrophysiological techniques, we identified 161 C- and 135 Aδ-meningeal nociceptors in rats and determined their mechanical response threshold and responsiveness to chemical stimulation of their dural receptive fields with TRPV1 and TRPA1 agonists seven days after BoNT-A administration to different extracranial sites. Two paradigms were compared: distribution of 5 U BoNT-A to the lambdoid and sagittal sutures alone, and 1.25 U to the sutures and 3.75 U to the temporalis and trapezius muscles., Results: Seven days after it was administered to tissues outside the calvaria, BoNT-A inhibited responses of C-type meningeal nociceptors to stimulation of their intracranial dural receptive fields with the TRPV1 agonist capsaicin and the TRPA1 agonist mustard oil. BoNT-A inhibition of responses to capsaicin was more effective when the entire dose was injected along the suture lines than when it was injected into muscles and sutures. As in our previous study, BoNT-A had no effect on non-noxious mechanosensitivity of C-fibers or on responsiveness of Aδ-fibers to mechanical and chemical stimulation., Discussion: This study demonstrates that extracranial administration of BoNT-A suppresses meningeal nociceptors' responses to stimulation of their intracranial dural receptive fields with capsaicin and mustard oil. The findings suggest that surface expression of TRPV1 and TRPA1 channels in dural nerve endings of meningeal nociceptors is reduced seven days after extracranial administration of BoNT-A. In the context of chronic migraine, reduced sensitivity to molecules that activate meningeal nociceptors through the TRPV1 and TRPA1 channels can be important for BoNT-A's ability to act as a prophylactic., (© International Headache Society 2016.)

Background/objective: The aim of this study was to investigate the effects of different concentrations of ozone (O3) therapy on bone regeneration in response to an expansion of the inter-premaxillary suture in rats., Materials and Methods: Forty-eight Wistar rats were randomly divided into four groups (n = 12). In groups I, II, and III, 1ml of O3 at 10, 25, and 40 µg/ml was injected at the premaxillary suture, respectively. In group IV (control group), 1ml of saline solution was injected at the same point during the expansion procedure for 5 days. Bone regeneration in the suture was evaluated histomorphometrically. The area of new bone and fibrotic area, the number of osteoblasts and osteoclasts, and the amount of vascularity were measured and compared. The density of the newly formed bone in the expansion area was measured by using cone beam computed tomography. Data were analyzed using the Kruskal-Wallis one-way analysis of variance and post hoc Student-Newman-Keuls tests., Results: New bone area, fibrotic area, osteoblast and osteoclast numbers, and the amount of vascularity were significantly higher in experimental groups compared with the control group (P < 0.001). The density of newly formed bone (P < 0.001), new bone formation (P = 0.009), number of capillaries (P < 0.001), number of osteoclasts (P = 0.016), and number of osteoblasts (P < 0.001) in the maxillary sutures were highest in the 25 μg/ml O3 group compared with the other experimental groups and control group., Conclusions/implications: The application of O3 therapy can stimulate bone regeneration in an orthopedically expanded inter-premaxillary suture during both the expansion and retention periods., (© The Author 2015. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The use of selective serotonin reuptake inhibitors (SSRIs) for the treatment of depression during pregnancy is suggested to increase the incidence of craniofacial abnormalities including craniosynostosis. Little is known about this mechanism, however based on previous data we propose a mechanism that affects cell cycle. Excessive proliferation, and reduction in apoptosis may lead to hyperplasia within the suture that may allow for differentiation, bony infiltration, and fusion. Here we utilized in vivo and in vitro analysis to investigate this proposed phenomenon. For in vivo analysis we used C57BL-6 wild-type breeders treated with a clinical dose of citalopram during the third trimester of pregnancy to produce litters exposed to the SSRI citalopram in utero. At post-natal day 15 sutures were harvested from resulting pups and subjected to histomorphometric analysis for proliferation (PCNA) and apoptosis (TUNEL). For in vitro studies, we used mouse calvarial pre-osteoblast cells (MC3T3-E1) to assess proliferation (MTS), apoptosis (Caspase 3/7-activity), and gene expression after exposure to titrated doses of citalopram. In vivo analysis for PCNA suggested segregation of effect by location, with the sagittal suture, showing a statistically significant increase in proliferative response. The coronal suture was not similarly affected, however there was a decrease in apoptotic activity at the dural edge as compared to the periosteal edge. No differences in apoptosis by suture or area due to SSRI exposure were observed. In vitro results suggest citalopram exposure increased proliferation and proliferative gene expression, and decreased apoptosis of the MC3T3-E1 cells. Decreased apoptosis was not confirmed in vivo however, an increase in proliferation without a concomitant increase in apoptosis is still defined as hyperplasia. Thus prenatal SSRI exposure may exert a negative effect on post-natal growth through a hyperplasia effect at the cranial growth sites perhaps leading to clinically significant craniofacial abnormalities.

Objective: To investigate the effects of systemically administered Capparis spinosa extract (CSE) on expanded sutures in rats via three dimensionally morphometric method (stereological method)., Materials and Methods: Thirty-two Wistar rats were used. Subjects were divided into four groups, each with eight rats. Orthopaedic expansion force was applied for 5 days to maxillary incisors by attaching springs. Control-1 and CSE-1 waited 1 week for consolidation, and Control-2 and CSE-2 waited 2 weeks for consolidation. After the consolidation period, the subjects were sacrificed. Stereological examination was performed to determine the volume and area of new bone, connective tissue, and capillaries., Results: New bone area, new bone volume, connective tissue space, and connective tissue volume were statistically different in CSE-1 compared to Control-1. But there were no statistically difference between CSE-2 and Control&#95;2. In terms of the volume of blood vessels and vascular area, there were no statistically significant differences when comparing Groups CSE-1 and Control-1 or CSE-2 and Control-2., Conclusion: Systemic use of CSE accelerated fastened osteoblastic activity in the early period., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background/objective: The aim of this study was to investigate the effect of systemic St John's wort (Hypericum perforatum) on bone formation in the expanded premaxillary suture in rats., Materials/methods: A total of 28 rats were randomly divided into four groups of equal numbers: control (C); only expansion (OE); St John's wort extract given only during the expansion and retention period (a total of 17 days; SJW group); and St John's wort extract given during the nursery phase before expansion (a period of 40 days), and during the expansion and retention periods (a total of 57 days; N + SJW group). After the 5 day expansion period was completed, the rats in the OE, SJW, and N + SJW groups underwent 12 days of mechanical retention, following which they were killed, and their premaxilla dissected and fixed. Histological examination was performed to determine the number of osteoclasts and capillaries, as well as the number of osteoblasts, inflammatory cell infiltration, and the amount of new bone formation., Results: Statistical analysis showed that the number of osteoclasts and capillaries, and the inflammatory cell infiltration, as well as new bone formation, were higher in the SJW and N + SJW groups than in the other groups. However, statistical analysis demonstrated that among these two groups, all parameters, with the exception of the number of capillaries, were higher in the N + SJW group than the SJW group., Conclusions/implications: Although more effective in long-term usage, systemic use of St John's wort hastens new bone regeneration at the premaxillary suture and may help prevent relapse after expansion., (© The Author 2014. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

During the first month of life, the murine posterior-frontal suture (PF) of the cranial vault closes through endochondral ossification, while other sutures remain patent. These processes are tightly regulated by canonical Wnt signaling. Low levels of active canonical Wnt signaling enable endochondral ossification and therefore PF-suture closure, whereas constitutive activation of canonical Wnt causes PF-suture patency. We therefore sought to test this concept with a knockout mouse model. PF-sutures of Axin2(-/-) mice, which resemble a state of constantly activated canonical Wnt signaling, were investigated during the physiological time course of PF-suture closure and compared in detail with wild type littermates. Histological analysis revealed that the architecture in Axin2(-/-) PF-sutures was significantly altered in comparison to wild type. The distance between the endocranial layers was dramatically increased and suture closure was significantly delayed. Moreover, physiological endochondral ossification did not occur, rather an ectopic cartilage appeared between the endocranial and ectocranial bone layers at P7 which eventually involutes at P13. Quantitative PCR analysis showed the lack of Col10α1 upregulation in Axin2(-/-) PF-suture. Immunohistochemistry and gene expression analysis also revealed high levels of type II collagen as compared to type I collagen and absence of Mmp-9 in the cartilage of Axin2(-/-) PF-suture. Moreover, TUNEL staining showed a high percentage of apoptotic chondrocytes in Axin2(-/-) PF-sutures at P9 and P11 as compared to wild type. These data indicated that Axin2(-/-) PF-sutures lack physiological endochondral ossification, contain ectopic cartilage and display delayed suture closure.

Introduction: Suture expansion stimulates bone growth to correct craniofacial deficiencies but has a high potential of treatment relapse. The objective of this study was to investigate whether there is a dose-dependent relationship between the recombinant human bone morphogenetic protein-2 (rhBMP-2) and bone formation during suture expansion., Methods: Fifty 6-week-old male New Zealand white rabbits were randomly assigned to 5 groups to receive 0 (control), 0.01, 0.025, 0.1, or 0.4 mg/mL of rhBMP-2 delivered by absorbable collagen sponge placed over the interfrontal suture. The suture was expanded for 33 days by 200 g of constant force via a spring anchored with 2 miniscrew implants. Distance of suture expansion, suture volume, and cross-sectional area after expansion were measured using radiographs with bone markers and microcomputed tomography. Suture widths and mineralization appositional rates were calculated based on the widths between bone labels under an epifluorescent microscope. Software (Multilevel Win 2.0; University of Bristol, Bristol, United Kingdom) was used to model distance of suture expansion over time as polynomials to compare group differences. Wilcoxon signed rank tests were performed to compare the suture volume and cross-sectional area, mineral apposition rate, and suture width between groups. The significance level was set at P = 0.05., Results: Whereas the sutures were expanded in all groups, sutures were expanded by significantly greater amounts in the control and the 0.01 mg/mL groups without fusing the sutures than in the 0.025, 0.1, and 0.4 mg/mL groups with fusing sutures. Compared with the controls, the 0.01 mg/mL group showed significantly lower suture volumes, cross-sectional areas, and suture widths after expansion. The mineral apposition rate was significantly higher in the 0.01 mg/mL group than in the controls from days 10 to 30., Conclusions: The 0.01 mg/mL dose of rhBMP-2 delivered by absorbable collagen sponge can stimulate bone formation at the bony edges of the suture during suture expansion; however, higher concentrations cause suture fusion. With an appropriate concentration, rhBMP-2 might facilitate suture expansion for clinical uses., (Copyright © 2013 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.)

Objective :  Studies described in this paper were designed to test the hypothesis that an increase in nonviral, plasmid-encoded Tgf-β3 production, localized to the rat posterior frontal suture, prevents programmed suture fusion. Design :  We developed a gene delivery system based on a dense collagen gel to deliver nonviral plasmids that encode for Tgf-β3. Studies were performed to test the ability of this system to rescue rat cranial suture fusion in vitro and in vivo. Immunohistochemical studies were conducted to characterize the possible mechanisms by which increased production and presence of Tgf-β3 protein interferes with suture fusion. Results :  Posterior frontal sutures in the Tgf-β3 plasmid-treated group exhibited 77% to 85% less bony bridging than the collagen control and untreated groups after 15 days in culture. In animals treated with Tgf-β3 plasmid or Tgf-β3 protein, there was a significant reduction in suture fusion in the middle region of the posterior frontal sutures when compared with control groups. In this region the Tgf-β3 plasmid-treated group revealed 70% to 75% less bony bridging than control groups in vivo. Conclusions :  Collagen gel can be formulated to provide release of nonviral plasmid DNA that results in cell transfection and elevated Tgf-β3 protein production. Tgf-β3 is an important regulator of suture fusion, and an increase in plasmid-encoded Tgf-β3 protein is effective in inhibiting programmed suture fusion in rats.

C-type natriuretic peptide (CNP) is a potent stimulator of long bone and vertebral development via endochondral ossification. In the present study, we investigated the effects of CNP on craniofacial skeletogenesis, which consists of both endochondral and membranous ossification. Morphometric analyses of crania from CNP knockout and transgenic mice revealed that CNP stimulates longitudinal growth along the cranial length, but does not regulate cranial width. CNP markedly increased the length of spheno-occipital synchondrosis in fetal murine organ cultures, and the thickness of cultured murine chondrocytes from the spheno-occipital synchondrosis or nasal septum, resulting in the stimulation of longitudinal cranial growth. Mandibular growth includes endochondral and membranous ossification; although CNP stimulated endochondral bone growth of condylar cartilage in cultured fetal murine mandibles, differences in the lengths of the lower jaw between CNP knockout or transgenic mice and wild-type mice were smaller than those observed for the lengths of the upper jaw. These results indicate that CNP primarily stimulates endochondral ossification in the craniofacial region and is crucial for midfacial skeletogenesis.

Objectives: Glycogen synthase kinase-3β (GSK-3β)/β-catenin signaling mediates osteogenesis in response to mechanical loading. We tested the hypothesis that local administration of a GSK-3β inhibitor could stimulate new bone formation in the expanding premaxillary suture., Materials and Methods: Thirty-five Sprague-Dawley rats were subjected to premaxillary suture expansion using a helix spring. The experimental rats were given one or two local injections of SB-415286, a small-molecule GSK-3β inhibitor. Animals were administered calcein and sacrificed on day 7 to quantify new bone formation. To evaluate the proliferation and differentiation of osteoblasts, rats were labeled with bromodeoxyuridine on day 1 and sacrificed on day 2 or 4. β-catenin expression was evaluated by immunohistochemical staining., Results: Two injections of SB-415286 led to an elevation of β-catenin expression and an increase in the number of proliferating osteoblasts in expanding sutures on day 2 and day 4. Consequently, new bone formation in the suture increased significantly on day 7., Conclusions: These results suggest that local delivery of a GSK-3β inhibitor could stimulate bone formation in the expanding premaxillary suture by eliciting β-catenin signaling. GSK-3β could be a pharmaceutical target for improving the effect of orthodontic treatments such as rapid palatal expansion., (© 2012 John Wiley & Sons A/S.)

Objective: Methionine is an essential amino acid and pivotal for normal growth and development. However, previous animal studies have shown that excessive maternal intake of methionine causes growth restrictions, organ damages, and abnormal growth of the mandible in newborn animals. However, the effect of excessive methionine on the development of the cranial growth plate is unknown. This study investigated histological alterations of the cranial growth plate induced by high methionine administration in newborn rats., Design: Twenty pregnant dams were divided into a control and an experimental group. The controls received a diet for rats and the experimental group was fed from the 18th gestational day with a special manufactured high methionine diet for rats. The high methionine diet was maintained until the end of the lactation phase (day 20). The offspring of both groups were killed at day 10 or 20 postnatally and their spheno-occipital synchondroses were collected for histological analysis., Results: The weight of the high-dose methionine treated experimental group was considerably reduced in comparison to the control group at day 10 and 20 postnatally. The cartilaginous area of the growth plate and the height of the proliferative zone were markedly reduced at postnatal day 10 in the experimental group., Conclusions: In summary, the diet-induced hypermethioninemia in rat dams resulted in growth retardations and histomorphological changes of the spheno-occipital synchondrosis, an important craniofacial growth centre in newborns. This finding may elucidate facial dysmorphoses reported in patients suffering from hypermethioninemia., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objectives: Craniosynostosis affects 1 in 2000 to 3000 live births and may result in craniofacial and neural growth disturbances. Histological data have shown that thick collagenous bundles are present in the sutural ligament, which may tether the osteogenic fronts, resulting in premature fusion. The hormone relaxin has been shown to disrupt collagen fiber organization, possibly preventing craniosynostosis by relaxing the sutural ligament and allowing osteogenic fronts to separate normally and stay patent. This study tested this hypothesis with a rabbit model of delayed-onset coronal suture synostosis., Methods: A total of 18 New Zealand White rabbits with craniosynostosis were randomly assigned to one of three groups: sham control, protein control (BSA), relaxin treatment. After initial diagnosis, sham surgery, BSA, or relaxin was delivered to the fusing coronal suture in a slow-release (56-day) collagen vehicle. Longitudinal radiographs and body weights were collected at 10, 25, 42, and 84 days of age, and sutures were harvested for histology., Results: Relaxin-treated animals had more disorganized intrasuture content than control groups. These specimens also appeared to have relatively wider sutures ectocranially. There were no significant differences in relaxin-treated animals for all craniofacial growth measures, or suture separation compared with controls., Conclusions: These data do not support our initial hypothesis that the use of relaxin may rescue sutures destined to undergo premature suture fusion. These findings suggest that collagen fiber arrangement may not be important for suture fusion. This protein therapy would not be clinically useful for craniosynostosis.

Craniofacial sutures are bone growth fronts that respond and adapt to biomechanical environments. Little is known of the role sutures play in regulating the skull biomechanical environment during patency and fusion conditions, especially how delayed or premature suture fusion will impact skull biomechanics. Tgf-β3 has been shown to prevent or delay suture fusion over the short term in rat skulls, yet the long-term patency or its consequences in treated sutures is not known. It was therefore hypothesized that Tgf-β3 had a long-term impact to prevent suture fusion and thus alter the skull biomechanics. In this study, collagen gels containing 3 ng Tgf-β3 were surgically placed superficial to the posterior interfrontal suture (IFS) and deep to the periosteum in postnatal day 9 (P9) rats. At P9, P24, and P70, biting forces and strains over left parietal bone, posterior IFS, and sagittal suture were measured with masticatory muscles bilaterally stimulated, after which the rats were sacrificed and suture patency analyzed histologically. Results demonstrated that Tgf-β3 treated sutures showed less fusion over time than control groups, and strain patterns in the skulls of the Tgf-β3-treated group were different from that of the control group. Although bite force increased with age, no alterations in bite force were attributable to Tgf-β3 treatment. These findings suggest that the continued presence of patent sutures can affect strain patterns, perhaps when higher bite forces are present as in adult animals., (Copyright © 2012 Wiley Periodicals, Inc.)

This investigation examined the effects of pharmacologically induced precocious puberty on cranial growth in Wistar rats. Forty-eight female newborn Wistar rats were divided into two groups: a control group (C) and an experimental group (E), with four subgroups of six animals each. The time interval from birth until sacrifice differed between the subgroups, and was set at 30, 60, 90, and 120 days. An intramuscular single dose (300 μg) of steroid hormone danazol was administered on day 5 after birth, as a means of inducing precocious puberty. Alizarin (2 mg/100 g) was administered to three animals in each subgroup three days prior to sacrifice. Body mass and dates corresponding to the beginning of the oestrous cycle were recorded. Craniometric measurements were undertaken. Histological analysis using light and fluorescence microscopy was then carried out to qualitatively and quantitatively evaluate the spheno-occipital synchondrosis and to visualize bone deposition patterns. The results were analysed with a Student's t-test and analysis of variance. Precocious puberty was effectively induced and differences between groups denoted an earlier maturation in the experimental rats. In qualitative analysis, a significant increase of total synchondrosis width was noted only in group E60, in comparison with C60, and an increase in the E90 subgroup cortical bone width compared with the C90 subgroup. Histomorphometrically, a statistical difference between total width values of subgroups E60 (434.3 μm) and C60 (323.5 μm) was detected. However, body mass and macroscopic measurements did not show statistically significant differences. An appropriate model for studying bone growth associated with precocious puberty in Wistar female rats was not achieved using steroid hormone danazol, when evaluated at 30 day intervals.

The aim of this study was to investigate the effects of systemically applied zoledronic acid (ZA) on osteoblastic bone formation and relapse in the rat sagittal suture after expansion. Eighteen 12-week-old male Wistar rats were divided into three groups. In groups 1 and 2, a saline solution was given subcutaneously after expansion and the retention period lasted for 14 and 7 days, respectively. In group 3, 0.1 mg of ZA was diluted with saline and given subcutaneously after expansion: the retention period lasted for 7 days. Computed tomography (CT) measurements were obtained at the start of the study (T1), after expansion (T2), after the retention period (T3), and after the follow-up period (T4). The amount of expansion and relapse and the density of the newly formed bone in the expansion area were measured. The mean bone density values in hounsfield unit (HU) of the newly formed bone were recorded using MX View Workstation. Data were analysed using the Kruskal-Wallis, Friedman, Wilcoxon, and Mann-Whitney U-tests. The results showed that there were significant differences between the groups in the density of newly formed bone after the retention period (P < 0.05). Statistically significant differences were observed when the relapse percentages were compared between the groups (P < 0.05). ZA stimulated bone formation and decreased the relapse ratio after expansion in the rat sagittal suture.

Objective: To identify the expressions of SOX9 and type II collagen in spheno-occipital synchondrosis in response to quercetin, using a mouse in vitro model., Materials and Methods: A total of 50 one-day-old male BALB/c mice were randomly assigned to the control and experimental groups. Each group was subdivided into five different time points, which were 6, 24, 48, 72, and 168 hours, and each subgroup contained 5 mice (n  =  5). In the experimental group, the spheno-occipital synchondrosis was immersed in the BGJb medium + quercetin dihydrate 1 µM. In the control group, the spheno-occipital synchondrosis was immersed in the BGJb medium. Tissue sections were subjected to immunohistochemical staining for SOX9 and type II collagen expressions., Results: Quantitative analysis revealed there was a statistically significant increase of 32.31% (P < .001) in the expression of SOX9 between experimental groups and control groups at 24 hours. Furthermore, there was a statistically significant increase of 22.99% (P < .001) in the expression of type II collagen between experimental groups and control groups at 72 hours., Conclusion: The expressions of SOX9 and type II collagen in the spheno-occipital synchondrosis can be increased by quercetin.

Excess exogenous retinoic acid (RA) has been well documented to have teratogenic effects in the limb and craniofacial skeleton. Malformations that have been observed in this context include craniosynostosis, a common developmental defect of the skull that occurs in 1 in 2500 individuals and results from premature fusion of the cranial sutures. Despite these observations, a physiological role for RA during suture formation has not been demonstrated. Here, we present evidence that genetically based alterations in RA signaling interfere with human development. We have identified human null and hypomorphic mutations in the gene encoding the RA-degrading enzyme CYP26B1 that lead to skeletal and craniofacial anomalies, including fusions of long bones, calvarial bone hypoplasia, and craniosynostosis. Analyses of murine embryos exposed to a chemical inhibitor of Cyp26 enzymes and zebrafish lines with mutations in cyp26b1 suggest that the endochondral bone fusions are due to unrestricted chondrogenesis at the presumptive sites of joint formation within cartilaginous templates, whereas craniosynostosis is induced by a defect in osteoblastic differentiation. Ultrastructural analysis, in situ expression studies, and in vitro quantitative RT-PCR experiments of cellular markers of osseous differentiation indicate that the most likely cause for these phenomena is aberrant osteoblast-osteocyte transitioning. This work reveals a physiological role for RA in partitioning skeletal elements and in the maintenance of cranial suture patency., (Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Objective: To examine whether periostin-like factor (PLF) stimulation of the expanded palatal suture would accelerate mineral formation of bone., Materials and Methods: Expanded palates of 6-week-old male C57BL/6 mice were maintained in an organ culture system, and tissue was either unstimulated or stimulated with PLF or BMP-7 for 8 days. Bone mineral formation was assessed by Raman microspectroscopy analysis and histological examination., Results: Raman microspectroscopy analysis demonstrated that unstimulated palates maintained their bone mineral concentration within the palatal suture over 8 days (%increase = 11.29 ± 2.34). In comparison, palates exposed to either PLF (%increase = 29.33 ± 1.61, p = 0.012) or BMP-7 (% increase = 25.49 ± 1.09, p = 0.016) formed significantly more bone at the osteogenic fronts of the palatal suture compared with unstimulated samples at day 8. Alkaline phosphatase activity along the bone edges was markedly greater in the PLF and BMP-7 groups compared with that in unstimulated groups at day 0 and day 8. The levels of osteocalcin proteins in the palatal suture tissues of the PLF and BMP-7 groups were significantly higher than those in the unstimulated group., Conclusion: PLF can increase bone mineral formation within the expanded palatal suture., (© 2011 John Wiley & Sons A/S.)

Aim: The aim of this experimental study was to histomorphometrically evaluate the effects of vitamin C administration on bone formation in response to expansion of the rat inter-premaxillary suture., Materials and Methods: A total of 30 50- to 60-day old Wistar male rats were divided into three groups of equal number. The inter-premaxillary suture in each animal was expanded with a 0.49 N force applied to the upper incisors. At 24 h after appliance placement, control animals received saline solution (group I) and two groups were given a single dose of vitamin C using two different methods [locally into the suture (group II) and systemically via intramuscular injection (group III)]. The area of new bone (µm(2)), the perimeter around the new bone (µm), Feret's diameter (µm), and the percentage of new bone to non-ossified tissue (%) were measured and compared. Kruskal-Wallis and Tukey tests were used for statistical evaluation at the p<0.05 level., Results: We observed significant differences among the groups in all histomorphometric parameters. New bone area, bone perimeter, Feret's diameter, and percentage of new bone dimensions were significantly higher in group III than the others (p<0.001). Histomorphometric measurements of bone architecture revealed that this improved in the group administered with vitamin C systemically, while local injection revealed significantly less bone growth than the control group., Conclusion: The systemic administration of vitamin C during the early stages of inter-premaxillary suture expansion may stimulate bone growth. However, local injection of this antioxidant into an orthopedically-expanded suture area has negative effects on bone formation.

Introduction: The aim of this experimental study was to evaluate the effects of systemically applied zoledronic acid on bone regeneration in response to expansion of the sagittal suture and relapse in rats., Methods: Thirty-six male Wistar rats were divided into 3 groups. In the first and second groups, saline solution was given subcutaneously after expansion, and the retention periods lasted 14 and 7 days, respectively. In the third group, 0.1 mg of zoledronic acid was diluted with saline solution and given subcutaneously after expansion; the retention period lasted for 7 days. Expansion and relapse amounts were measured by using computed tomography. After the retention period, 6 rats from each group were killed for histologic and immunohistochemical assessments. The other 6 rats from each group were used for observation of the relapse., Results: The histologic evaluation showed that, in groups 1 and 2, the numbers of osteoblasts were less than observed in group 3. When scores of staining intensity were compared, immunoreactivities were statistically significantly increased in group 3 compared with groups 2 and 1. Statistically significant differences were found when the relapse percentages were compared between the groups (P <0.05). The smallest relapse occurred in group 3., Conclusions: Zoledronic acid has positive effects on bone formation in the sagittal suture in response to expansion and decreases the relapse ratio after expansion in rats., (Copyright © 2011 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.)

Objective: The aim of this study was to evaluate the effects of local resveratrol (RSVL) administration on bone formation in response to expansion of the inter-premaxillary suture, in rats., Material and Methods: Twenty 50- to 60-day-old male Wistar rats were separated into two equal groups. Both groups were subjected to expansion, and 30 cN of force was applied to the maxillary incisors with helical-spring. Twenty-four hours after appliance placement, single-dose 10 μmol/kg RSVL in the dimethylsulfoxide (DMSO) was injected to the inter-premaxillary suture in the experimental group. In the control group, the same amount of DMSO was injected to the suture of rats. Bone formation in the suture was evaluated histomorphometrically. The area of new bone (μm(2)), the perimeter around the new bone (μm), Feret's diameter (μm), the percentage of new bone to non-ossified tissue (%), and the number of osteoblast were measured and compared. Mann-Whitney U-test was used for statistical evaluation at p < 0.05 level., Results: Statistically significant differences were found between the groups for all histomorphometric parameters. New bone area (p < 0.001), bone perimeter (p < 0.001), Feret's diameter (p < 0.001), percentage of new bone (p < 0.001), and the number of osteoblast (p < 0.001) were significantly larger in the experimental group when compared with the control. Bone histomorphometric measurements revealed that bone architecture in the RSVL treated rats was improved., Conclusions: Local application of RSVL during the early stages to orthopedically expanded inter-premaxillary suture area may stimulate bone formation and shorten the retention period, in rats., (© 2011 John Wiley & Sons A/S.)

Background: Craniosynostosis is defined as the premature fusion of one or more cranial sutures. Bone morphogenetic proteins (BMPs), regulators of ossification, have been implicated in premature suture fusion. Noggin, an extracellular BMP inhibitor, has been shown experimentally to inhibit resynostosis following surgery. The present study was designed to test the hypothesis that BMP inhibition using noggin therapy may rescue sutures destined to fuse by inhibiting initial ossification., Methods: Twenty-six, 10-day old rabbits with familial, delayed-onset, coronal suture synostosis were randomly divided into three groups: (1) the sham surgical control group, (2) the bovine serum albumin-treated group [10 μg/suture (protein/vehicle controls)], and (3) the noggin therapy group (10 μg/suture; experimental group). Sutural growth was monitored by radiopaque markers implanted at 10 days of age. At 25 days, the bovine serum albumin or noggin was combined with a slow-resorbing collagen vehicle and injected subperiosteally above the coronal suture. Somatic and sutural growth data were collected at 10, 25, 42, and 84 days of age. Coronal sutures were harvested at 84 days to histologically assess fusion., Results: Results showed no significant (p > 0.05) differences in suture separation at any age. Suture fusion assessed by histomorphology did not differ among the three groups. Although previous data showed noggin to inhibit postoperative resynostosis in this craniosynostotic rabbit model, here there was no effect on initial suture fusion., Conclusion: These results suggest that in this rabbit model of craniosynostosis, BMPs do not play a role in the pathogenesis of craniosynostosis and only play a role in postoperative bony wound healing.

Research has implicated the faulty regulation of transforming growth factor beta signaling as one mechanism for premature calvaria suture fusion. Androgens have been shown to increase the expression and activity of the transforming growth factor beta, resulting in increased osteoblast proliferation and differentiation and possibly premature suture fusion. The present study was designed to test the hypothesis that flutamide, an androgen receptor-blocking agent, would "rescue" a coronal suture destined to fuse and improve craniofacial growth in a familial rabbit model of craniosynostosis. Thirty rabbits with delayed-onset, coronal suture synostosis were examined via longitudinal cephalometry. The rabbits were divided into 4 groups: (1) sham surgical controls (n = 10), (2) bovine serum albumin (500 ng) protein controls (n = 6), (3) flutamide diluent controls (n = 6), and (4) flutamide (15 mg dissolved in ethanol) experimental group (n = 8). At 10 days of age, radiopaque amalgam markers were implanted in all rabbits on either side of the coronal suture to monitor sutural growth. At 25 days of age, the bovine serum albumin, ethanol, and flutamide were combined with a slow-resorbing collagen vehicle and injected subperiosteally above the coronal suture into the respective groups. Although results revealed a slight but significant increase in coronal suture marker separation in flutamide-treated rabbits compared with controls at 42 days of age, few significant differences were noted for craniofacial growth and intracranial volume among groups. Results suggest that androgen receptor-blocking using flutamide may only provide a transient rescue to suture fusion in this model. Further research is needed to investigate the effects of hormones on suture development and maintenance.

Suture expansion osteogenesis, which induces active bone formation within the distracted area by mechanical stress, provides an alternative form of treatment of some bone deficiency problems in craniofacial field, such as craniosynostosis, palate cleft, or narrow maxilla. However, how to stimulate new bone formation within the distracted area remains a great challenge. In this study, the effect of recombinant human bone morphogenetic protein (rhBMP-4) on bone formation induced by mechanical stimuli was investigated. The expanded midsagittal sutures of the rat calvaria were maintained in an organ culture system in the absence (control group) or presence (experimental group) of rhBMP-4 (25 or 50 ng/mL). Proliferating cell nuclear antigen, alkaline phosphatase (ALP) activity, and messenger RNA levels of core-binding factor alpha 1 (Cbfa1) and ALP were detected to determine the cell proliferation and differentiation. Bone formation within the suture was evaluated by histologic and fluorescent examination. No difference in the number of proliferating cell nuclear antigen-positive cells was found between the control and the experimental groups, whereas ALPase activity and messenger RNA levels of Cbfa1 and ALP in the experimental group were higher than that in the control group. Bone formation was accelerated in the rhBMP-4-treated group when compared with the control group. In addition, the amounts of bone formation and cell differentiation in response to 25 ng/mL of rhBMP-4 were almost equal to those induced by 50 ng/mL of rhBMP-4. Our results suggest that application of exogenous rhBMP-4 could enhance new bone formation within the rapid expanded sutures by stimulating osteoblast differentiation.

Osteogenic supplements are a requirement for osteoblastic cell differentiation during in vitro culture of human calvarial suture-derived cell populations. We investigated the ability of ascorbic acid and beta-glycerophosphate with and without the addition of dexamethasone to stimulate in vivo-like osteoblastic differentiation. Cells were isolated from unfused and prematurely fused suture tissue from patients with syndromic and non-syndromic craniosynostosis and cultured in each osteogenic medium for varying lengths of time. The effect of media supplementation was investigated with respect to the ability of cells to form mineralised bone nodules and the expression of five osteodifferentiation marker genes (COL1A1, ALP, BSP, OC and RUNX2), and five genes that are differentially expressed during human premature suture fusion (GPC3, RBP4, C1QTNF3, WIF1 and FGF2). Cells from unfused sutures responded more slowly to osteogenic media but formed comparable bone nodules to fused suture-derived cells after 16 days of culture in either osteogenic media. However, gene expression differed between unfused and fused suture-derived cells, as did expression in each osteogenic medium. When compared to expression in the explant tissue of origin, neither medium induced a level or profile of gene expression similar to that seen in vivo. Overall, our results demonstrate that cells from the same suture that are isolated during different stages of morphogenesis in vivo, despite being de-differentiated to a similar level in vitro, respond uniquely and differently to each osteogenic medium. Further, we suggest that neither cell culture medium recapitulates differentiation via activation of the same genetic cascades as occurs in vivo., ((c) 2008 Wiley-Liss, Inc.)

Premature suture obliteration results in an inability of cranial and facial bones to grow, with craniofacial dysmorphology requiring surgical correction as a consequence. Understanding signaling pathways associated with suture morphogenesis might enable non-invasive treatment of patients with fused sutures. Tgf-beta 2 induces premature suture fusion associated with increased cell proliferation both in vitro and in vivo. Tgf-beta 2 and Egf signal transduction pathways use some signaling proteins in common to regulate proliferation and differentiation, leading to speculation that these two pathways converge to regulate normal suture development. It was therefore hypothesized that Egf could induce suture fusion, and that Tgf-beta 2-induced suture closure occurred via an Egf-dependent pathway. A well-established fetal calvarial organ culture system was used to expose developing E19.5 fetal rat coronal sutures to Egf, Tgf-beta 2 and SC-120, a blocker of Egf receptor activity. Co-culture experiments examined the effect of Egf on Tgf-beta 2-induced suture closure when Egf was given either prior to or after Tgf-beta 2 treatment. Histomorphometric measurement of suture width was done on sagittal sections through coronal sutures harvested after 5 days in culture. Western blotting using phospho-antibodies against Egf receptors was used to confirm Egf receptor activity. Suture width increased with increasing concentrations of Egf, demonstrating that Egf-induced cell activity alone was not sufficient to cause premature suture obliteration. Egf administered prior to Tgf-beta 2 treatment rescued sutures from Tgf-beta 2-induced suture obliteration, demonstrating that pre-exposure of cells to this powerful mitogen prevented their response to signals induced by Tgf-beta 2. However, Egf added after Tgf-beta 2 treatment had no effect on Tgf-beta 2-induced suture closure. Blocking Egf activity after Tgf-beta 2 treatment rescued sutures from Tgf-beta 2-induced obliteration, indicating that Tgf-beta 2 required Egf activity to induce suture obliteration. Appropriate timing of signal generation by Egf and Tgf-beta 2 is critical for normal suture development and maintenance of suture patency.

Premature fusion of calvarial sutures is the result of a long and complex reaction, and several growth factors including transforming growth factor beta and basic fibroblast growth factor have important role in this event. Several prostaglandins have important functions in local bone modeling and remodeling by autocrine and paracrine mechanisms. Although effects of prostaglandins on long bones were studied both experimentally and clinically, there are limited data about cranial bones and sutures. In this study, we investigated the effect of iloprost-a stable prostacyclin analogue, which is widely used for the treatment of pulmonary arterial hypertension even in early pregnancy, to rat calvarial sutures. In 2 study groups, iloprost was injected intraperitoneally 10 and 15 microg kg d, respectively. In the third group, dexamethasone 2 mg kg d + iloprost 15 microg kg d was injected intraperitoneally to antagonize the effects of iloprost. In every group, 4 rats were killed at the postoperative 15, 30, and 45 days, and specimens including the sagittal and frontal sutures were excised immediately. Routine histological and immunohistological staining were performed on the specimens. Morphological measurements were performed on the skulls, also. In histological evaluation, bone formation in the both frontal and sagittal suture area was increased and accelerated in iloprost groups. Dexamethasone inhibited the effects of iloprost on the third group. Expressions of transforming growth factor beta and basic fibroblast growth factor were also increased in immunohistological staining. In morphological measurements, statistically significant differences were found between control and study groups. Iloprost did not fused the rat calvarial sutures prematurely, but it narrowed the sagittal and frontal sutures especially after the second week of the study. This situation might effect the sutures of the babies of the pregnant patients with pulmonary arterial hypertension treated with iloprost. Cranial sutures, calvarial bones, and cranial shape of the babies of the pregnant patients who were treated with iloprost should be monitored to clarify the topic.

Objective: Overexpression of transforming growth factor-beta 2 has been associated with craniosynostosis and resynostosis following surgery. We examined the effects of localized transforming growth factor-beta 2 inhibition on craniofacial phenotype in rabbits with craniosynostosis., Design: Twenty-five New Zealand white rabbits with bilateral coronal craniosynostosis were divided into three treatment groups: (1) suturectomy control (n=8); (2) suturectomy with nonspecific, control immunoglobulin G antibody (n=6); and (3) suturectomy with anti-transforming growth factor-beta 2 antibody (n=11). At 10 days of age, a coronal suturectomy was performed on all rabbits. The sites in groups 2 and 3 were immediately filled with a slow-resorbing collagen gel mixed with either immunoglobulin G or anti-transforming growth factor-beta 2 antibody. Computed tomography scans of each rabbit were acquired at ages 10, 25, and 84 days. Craniofacial landmarks were collected from three-dimensional computed tomography reconstructions, and growth and form were compared among the three groups., Results: Rabbits treated with anti-transforming growth factor-beta 2 antibody differed in form at 84 days of age compared with suturectomy control rabbits, specifically in the snout and posterior neurocranium. Growth in some areas of the skull was greater in rabbits from the anti-transforming growth factor-beta 2 group than in suturectomy control rabbits, but not significantly greater than in IgG control rabbits., Conclusions: We find support for the hypothesis that transforming growth factor-beta 2 inhibition alters adult form, but these changes do not appear to be localized to the suturectomy region. Slight differences in form and growth between the two control groups suggest that the presence of the collagen vehicle itself may affect skull growth.

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: Postoperative resynostosis is a common clinical finding. It has been suggested that an overexpression of transforming growth factor (TGF)-beta2 may be related to craniosynostosis and may contribute to postoperative resynostosis. Interference with TGF-beta2 function with the use of neutralizing antibodies may inhibit resynostosis. The present study was designed to test this hypothesis., Methods: New Zealand White rabbits with bilateral coronal suture synostosis were used as suturectomy controls (group 1, n = 9) or given suturectomy with nonspecific, control immunoglobulin G antibody (group 2, n = 9) or suturectomy with anti-TGF-beta2 antibody (group 3, n = 11). At 10 days of age, a 3 x 15-mm coronal suturectomy was performed. The sites in groups 2 and 3 were immediately filled with 0.1 cc of a slowly resorbing collagen gel mixed with either immunoglobulin G (100 mug per suture) or anti-TGF-beta2 (100 mug per suture). Three-dimensional computed tomography scan reconstructions of the defects were obtained at 10, 25, 42, and 84 days of age, and the sutures were harvested for histomorphometric analysis., Results: Computed tomography scan data revealed that the suturectomy sites treated with anti-TGF-beta2 showed significantly (p < 0.05) greater areas through 84 days of age compared with controls. Histomorphometry also showed that suturectomy sites treated with anti-TGF-beta2 had patent suturectomy sites and more fibrous tissue in the defects compared with sites in control rabbits and had significantly (p < 0.001) less new bone area (by approximately 215 percent) in the suturectomy site., Conclusions: These data support the initial hypothesis that interference with TGF-beta2 function inhibited postoperative resynostosis in this rabbit model. They also suggest that this biologically based therapy may be a potential surgical adjunct to retard postoperative resynostosis in infants with craniosynostosis.

Objective: Crouzon syndrome is caused by mutations in fibroblast growth factor receptor 2 (FGFR2) leading to constitutive activation of receptors in the absence of ligand binding. The syndrome is characterized by premature fusion of the cranial sutures that leads to abnormal cranium shape, restricted brain growth, and increased intracranial pressure. Surgical remodeling of the cranial vault is currently used to treat affected infants. The purpose of this study was to develop a pharmacological strategy using tyrosine kinase inhibition as a novel treatment for craniosynostotic syndromes caused by constitutive FGFR activation., Methods: Characterization of cranial suture fusion in Fgfr2 mutant mice, which carry the most common Crouzon mutation, was performed using micro-computed tomographic analysis from embryogenesis through maturation. Whole calvarial cultures from wild-type and Fgfr2 mice were established and cultured for 2 weeks in the presence of dimethyl sulfoxide control or PD173074, an FGFR tyrosine kinase inhibitor. Paraffin sections were prepared to show suture morphology and calcium deposition., Results: In untreated Fgfr2 cultures, the coronal suture fused bilaterally with loss of overlap between the frontal bone and parietal bone. Calvaria treated with PD173074 (2 micromol/L) showed patency of the coronal suture and were without evidence of any synostosis., Conclusion: We report the successful use of PD173074 to prevent in vitro suture fusion in a model for Crouzon syndrome. Further studies are underway to develop an in vivo treatment protocol as a novel therapeutic modality for FGFR associated craniosynostotic syndromes.

Transforming growth factors beta (Tgf-betas) act by means of Smad signaling pathways and may also interact with the mitogen-activated protein kinase pathway. The hypothesis was tested that Erk1/2 signaling is required for Tgf-beta2-induced suture closure, by culturing embryonic mouse calvariae in the presence of Tgf-beta2 with or without Erk1/2 inhibitor PD98059 (PD). Suture widths were measured daily, and microdissected sutures and bones were homogenized and protein analyzed by Western blots. Tgf-beta2 induced narrowing of the sutures after 72 hr, an effect inhibited by treatment with PD. Erk1/2 and Egf but not Smad2/3 protein expression was up-regulated by Tgf-beta2 calvarial tissues at 72 hr. PD inhibited endogenous and Tgf-beta2-stimulated Erk1/2 protein as well as Tgf-beta2-stimulated Egf, but increased Smad2/3 protein expression. In tissues harvested 0, 15, and 30 min after exposure to Tgf-beta2, Erk1/2 phosphorylation was up-regulated after 15 min, an effect abrogated by the simultaneous addition of PD. In summary, Tgf-beta2 stimulated Erk1/2 phosphorylation and induced Egf and Erk1/2 expression, associated with suture closure after 72 hr. Blocking Erk1/2 activity with PD inhibited these effects but increased Smad2/3 expression. We postulate that Tgf-beta2 regulates suture closure directly by means of phosphorylation of Erk1/2 and indirectly by up-regulating Erk1/2, a substrate for Fgf receptor signaling required for Fgf induction of premature suture obliteration., ((c) 2005 Wiley-Liss, Inc.)

Maxillary bone deficiencies, such as cleft palate and underdeveloped maxilla that require bone graft or regeneration after orthopedic or surgical expansion, pose a significant biomedical burden. Nell-1 is a secreted molecule that possesses chordin-like domains and induces cranial suture bone growth and osteoblast differentiation. To accelerate bone formation in acutely distracted palatal sutures, rat organ cultures were stimulated with Nell-1 or BMP-7 for 8 days in vitro. We hypothesized that Nell-1 stimulation to the distracted palatal suture would accelerate bone formation. Distracted palates of 4-week-old male rats were maintained in an organ culture system, and tissue was either unstimulated or stimulated with Nell-1 or BMP-7 for 8 days. MicroCT was conducted to quantitate bone formation, while alcian blue staining was conducted for cartilage localization. Immunohistochemistry of Sox9 for chondrocyte proliferation, type X collagen for hypertrophic cartilage in endochondral bone formation, and bone sialoprotein for bone formation was conducted to characterize the cellular mechanism of newly developed tissues. Distracted palates cultured in the presence of Nell-1 or BMP-7 produced statistically significantly (P < 0.05) more bone and cartilage within the intermaxillary suture, relative to unstimulated control samples. While both BMP-7 and Nell-1 induced similar bone formation in the distracted suture, BMP-7 induced both chondrocyte proliferation and differentiation, while Nell-1 accelerated chondrocyte hypertrophy and endochondral bone formation. While both Nell-1 and BMP-7 are effective in forming bone in the distracted palatal suture, they are suggested to have distinctively different mechanisms. The ability of Nell-1 to accelerate bone formation within the palate suture demonstrates the versatility of Nell-1 within the craniofacial complex as well as an exciting advance in palate suture defect healing.

Craniosynostosis results in cranial deformities and increased intracranial pressure, which pose extensive and recurrent surgical management problems. Developmental studies in rodents have shown that low levels of transforming growth factor-beta 3 (Tgf-beta 3) are associated with normal fusion of the interfrontal (IF) suture, and that Tgf-beta 3 prevents IF suture fusion in a dose-dependent fashion. The present study was designed to test the hypothesis that Tgf-beta 3 can also prevent or "rescue" fusing sutures in a rabbit model with familial craniosynostosis. One hundred coronal sutures from 50 rabbits with delayed-onset, coronal suture synostosis were examined in the present study. The rabbits were divided into five groups of 10 rabbits each: 1) sham controls, 2) bovine serum albumin (BSA, 500 ng) low-dose protein controls, 3) low-dose Tgf-beta 3 (500 ng), 4) high-dose BSA (1,000 ng) controls, and 5) high-dose Tgf-beta 3 (1,000 ng). At 10 days of age, radiopaque amalgam markers were implanted in all of the rabbits on either side of the coronal suture to monitor sutural growth. At 25 days of age, the BSA or Tgf-beta 3 was combined with a slow-absorbing collagen vehicle and injected subperiosteally above the coronal suture. Radiographic results revealed that high-dose Tgf-beta 3 rabbits had significantly greater (P < 0.05) coronal suture marker separation than the other groups. Histomorphometric analysis revealed that high-dose Tgf-beta 3 rabbits also had patent coronal sutures and significantly (P < 0.01) greater sutural widths and areas than the other groups. The results suggest that there is a dose-dependent effect of TGF-beta 3 on suture morphology and area in these rabbits, and that the manipulation of such growth factors may have clinical applications in the treatment of craniosynostosis., (Copyright 2003 Wiley-Liss, Inc.)

Objective: To determine whether antibody perturbation of Tgf-beta, delivered in a collagen gel, could inhibit cranial suture fusion., Design: Attachment and proliferation of osteoblasts cultured on a collagen gel with or without anti-Tgf-beta2 antibody were determined by AlamarBlue dye assay and cell morphology by toluidine-blue staining. In rat calvarial organ culture, collagen gel with and without anti-Tgf-beta2 antibody was injected subperiosteally over the posterior frontal suture of postnatal day 15 rat calvariae. A quantitative analysis of suture fusion was used to measure suture bridging in histological serial sections at various time points., Results: Attachment and proliferation for cells cultured on collagen gel with anti-Tgf-beta2 antibody were similar to collagen gel controls. Although proliferation was lower than on tissue culture plastic, cells treated with anti-Tgf-beta2 antibody maintained an osteoblastic morphology. After 7, 10, and 15 days in organ culture, anti-Tgf-beta2 antibody treatment caused a reduction in the percent bridging of posterior frontal sutures, compared with controls. Sutures exposed to anti-Tgf-beta2 antibody and fibroblast growth factor-2 concurrently did not show an inhibition of bony bridging., Conclusions: These results support previous reports suggesting a role for Tgf-beta2 in cranial suture fusion. In cell culture the collagen gel, both with and without anti-Tgf-beta2 antibody, promoted similar osteoblast attachment, proliferation, and osteoblastic morphology. In organ culture anti-Tgf-beta2 antibody was delivered in a bioactive state via a collagen gel to inhibit cranial suture fusion. Also, the results suggest that the inductive effect of fibroblast growth factor-2 is not dependent on Tgf-beta2 activity. Together, these results provide further support for the role of Tgf-beta2 in cranial suture fusion.

Objective: To evaluate the effect of insulin-like growth factor 1 for the bone induction and the regulation for the fusion of the sagittal cranial sutures., Methods: The cells, derived from cranial sutures in the newborn SD rats and the sagittal suture from the mice, were cultured with a serum-free medium and treated with and without insulin-like growth factor 1. The osteoblast phetotypes (osteocalcin, alkaline, osteoponcin and type-1 collagen) were measured with the RT-PCR and ELISA, and the explanted sagittal sutures were then evaluated under light microscopy., Results: The cells, treated with the insulin-like growth factor 1, significantly produced more osteocalcin, alkaline, osteoponcin and type-1 collagen than those without insulin-like growth factor 1. The fusion of the sagittal suture explants will delay till to 30 days when it was not treated with IGF1. However, in the group with IGF1 the fusion was observed to start in 8 days, and a small amount of the sagittal suture fusion was found at the 20th day while a large amount was at the 30th day., Conclusion: The IGF1 has a direct effect on the fusion of cranial suture due to enhancing bone induction of cranial suture cell.