Hydroxyapatite Nanoparticles Facilitate Osteoblast Differentiation and Bone Formation Within Sagittal Suture During Expansion in Rats.

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.

Sclerostin Antibody-Induced Changes in Bone Mass Are Site Specific in Developing Crania.

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.

Effects of continuous and intermittent parathyroid hormone administration on midpalatal suture expansion in rats.

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 ß-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 ß-catenin activation. Our results demonstrated that PTH administration may have potential to be an adjunctive approach for maxillary expansion treatment.

PLGA-based control release of Noggin blocks the premature fusion of cranial sutures caused by retinoic acid.

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.

Recombinant mouse periostin ameliorates coronal sutures fusion in Twist1+/- mice.

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 ß-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.

Inhibition of bone resorption by bisphosphonates interferes with orthodontically induced midpalatal suture expansion in mice.

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.

Rescue of Premature Coronal Suture Fusion with TGF-ß2 Neutralizing Antibody in Rabbits with Delayed-Onset Synostosis.

OBJECTIVES: An overexpression of Tgf-ß2 leads to calvarial hyperostosis and suture fusion in individuals with craniosynostosis. Inhibition of Tgf-ß2 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-ß2 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-ß2 antibody than in controls at 42 and 84 days of age. Histologically, rabbits treated with anti-Tgf-ß2 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-ß2 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.

Accelerated Bone Regeneration by Two-Photon Photoactivated Carbon Nitride Nanosheets.

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 (C3N4) 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 C3N4 sheets near cells. Given that the photoinduced charge transfer can increase cytosolic Ca2+ 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 C3N4 sheets in bone regeneration and fracture healing.

Effect of Energy Drink on Bone Formation in the Expanded Inter-Premaxillary Suture.

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.

A preliminary investigation of the effect of relaxin on bone remodelling in suture expansion.

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.

Effects of In Utero Thyroxine Exposure on Murine Cranial Suture Growth.

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.

Effect of hyaluronic acid on bone formation in the expanded interpremaxillary suture in rats.

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.

Does stinging nettle (Urtica dioica) have an effect on bone formation in the expanded inter-premaxillary suture?

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.

Extracranial injections of botulinum neurotoxin type A inhibit intracranial meningeal nociceptors' responses to stimulation of TRPV1 and TRPA1 channels: Are we getting closer to solving this puzzle?

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.

The effect of different concentrations of topical ozone administration on bone formation in orthopedically expanded suture in rats.

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.

Effects of Citalopram on Sutural and Calvarial Cell Processes.

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.

Effect of Capparis spinosa extract on sutural ossification: A stereological study.

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_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.

Effect of St John's wort on bone formation in the orthopaedically expanded premaxillary suture in rats: a histological study.

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.

Recombinant human bone morphogenetic protein-2 stimulates bone formation during interfrontal suture expansion in rabbits.

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.

Absence of endochondral ossification and craniosynostosis in posterior frontal cranial sutures of Axin2(-/-) mice.

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.