Coronal Suture Fusion Research Articles

Apert Syndrome with Combination of Synostosis: A Case Report

Apert syndrome is a rare condition affecting the skull, face, hands and feet due to premature closure of cranial sutures. This case report describes a 6-month-old boy with Apert syndrome presenting with an abnormal head shape. A CT scan revealed craniosynostosis (fused sutures) of both the coronal and lambdoid sutures. Surgery involved removing a 1-2 cm section of the fused sutures along the coronal suture and across the lambdoid suture. After 4 months, the boy showed improvement in both clinical appearance and imaging scans. While Apert syndrome is typically classified based on the suture involved, the specific timing of surgery depends on various factors. This case demonstrates that minimally invasive surgery can be effective with minimal complications. Even though unilateral coronal and lambdoid suture fusion in Apert syndrome is uncommon, it can still be successfully treated with surgery.

Targeting of C-ROS-1 Activity Using a Controlled Release Carrier to Treat Craniosynostosis in a Preclinical Model of Saethre-Chotzen Syndrome

Saethre-Chotzen syndrome (SCS) is one of the most prevalent craniosynostosis, caused by a loss-of-function mutation in the TWIST-1 gene, with current treatment options relying on major invasive transcranial surgery. TWIST-1 haploinsufficient osteogenic progenitor cells exhibit increased osteogenic differentiation potential due to an upregulation of the transmembrane tyrosine kinase receptor, C-ROS-1, a TWIST-1 target gene known to promote bone formation. The present study assessed the efficacy of suppressing C-ROS-1 activity using a known chemical inhibitor to C-ROS-1, crizotinib, to halt premature coronal suture fusion in a preclinical mouse model of SCS. Crizotinib (1 μM, 2 μM, or 4 μM) was administered locally over the calvaria of Twist‐1del/+ heterozygous mice prior to coronal suture fusion using either a nonresorbable collagen sponge (quick drug release) or a resorbable sodium carboxymethylcellulose microdisk (slow sustained release). Coronal suture fusion rates and bone parameters were determined by μCT imaging and histomorphometric analysis of calvaria postcoronal suture fusion. Results demonstrated a dose-dependent increase in the efficacy of crizotinib to maintain coronal suture patency, with no adverse effects to brain, kidney, liver, and spleen tissue, or blood cell parameters. Moreover, crizotinib delivered on microdisks resulted in a greater efficacy at a lower concentration to reduce bone formation at the coronal suture sites compared to sponges. However, the bone inhibitory effects were found to be diminished by over time following cessation of treatment. Our findings lay the foundation for the development of a pharmacological nonsurgical, targeted approach to temporarily maintain open coronal sutures in SCS patients. This study could potentially be used to develop similar therapeutic strategies to treat different syndromic craniosynostosis conditions caused by known genetic mutations.

Apical expansion of calvarial osteoblasts and suture patency is dependent on fibronectin cues.

The skull roof, or calvaria, is comprised of interlocking plates of bones that encase the brain. Separating these bones are fibrous sutures that permit growth. Currently, we do not understand the instructions for directional growth of the calvaria, a process which is error-prone and can lead to skeletal deficiencies or premature suture fusion (craniosynostosis, CS). Here, we identify graded expression of fibronectin (FN1) in the mouse embryonic cranial mesenchyme (CM) that precedes the apical expansion of calvaria. Conditional deletion of Fn1 or Wasl leads to diminished frontal bone expansion by altering cell shape and focal actin enrichment, respectively, suggesting defective migration of calvarial progenitors. Interestingly, Fn1 mutants have premature fusion of coronal sutures. Consistently, syndromic forms of CS in humans exhibit dysregulated FN1 expression, and we also find FN1 expression altered in a mouse CS model of Apert syndrome. These data support a model of FN1 as a directional substrate for calvarial osteoblast migration that may be a common mechanism underlying many cranial disorders of disparate genetic etiologies.

Orbital Foraminal Morphometrics in Nonsyndromic Unilateral Coronal Craniosynostosis.

Nonsyndromic unilateral coronal craniosynostosis (UCS) is a rare congenital disorder that results from premature fusion of either coronal suture. The result is growth restriction across the suture, between the ipsilateral frontal and parietal bones, leading to bony dysmorphogenesis affecting the calvarium, orbit, and skull base. Prior studies have reported associations between UCS and visual abnormalities. The present study utilizes a novel geometric morphometric analysis to compare dimensions of orbital foramina on synostotic versus nonsynostotic sides in patients with UCS. Computed tomography head scans of pediatric UCS patients were converted into 3-dimensional mesh models. Anatomical borders of left and right orbital structures were plotted by a single trained team member. Dimensions between synostotic and nonsynostotic sides were measured and compared. Medical records were examined to determine prevalence of visual abnormalities in this patient cohort. Visual abnormalities were reported in 22 of the 27 UCS patients (77.8%). Astigmatism (66.7%), anisometropic amblyopia (44.4%), and motor nerve palsies (33.3%) represented the 3 most prevalent ophthalmologic abnormalities. Orbits on synostotic sides were 11.3% narrower ( P < 0.001) with 21.2% less volume ( P = 0.028) than orbits on nonsynostotic sides. However, average widths, circumferences, and areas were similar between synostotic and nonsynostotic sides upon comparison of supraorbital foramina, infraorbital foramina, optic foramina, and foramina ovalia. Therefore, previously proposed compression or distortion of vital neurovascular structures within bony orbital foramina does not seem to be a likely etiology of visual abnormalities in UCS patients. Future studies will examine the role of ocular and/or neuro-ophthalmologic pathology in this disease process.

Craniofacial Dysmorphology in Infants With Non-Syndromic Unilateral Coronal Craniosynostosis.

Unilateral coronal craniosynostosis (UCS) is a congenital disorder resulting from the premature suture fusion, leading to complex primary and compensatory morphologic changes in the shape of not only the calvarium and but also into the skull base. This deformity typically requires surgery to correct the shape of the skull and prevent neurologic sequelae, including increased intracranial pressure, sensory deficits, and cognitive impairment. The present multicenter study sought to reverse-engineer the bone dysmorphogenesis seen in non-syndromic UCS using a geometric morphometric approach. Computed tomography scans for 26 non-syndromic UCS patients were converted to three-dimensional mesh models. Two hundred thirty-six unique anatomical landmarks and semi-landmarked curves were then plotted on each model, creating wireframe representations of the Patients' skulls. Generalized Procrustes superimposition, Principal Component Analysis, and heatmaps identified significant superior displacement of the ipsilateral orbit ("harlequin" eye deformity), anterior displacement of the ear ipsilateral to the fused coronal suture, acute deviation of midline skull base structures ipsilateral to the fused coronal suture and flattening of the parietal bone and associated failure to expand superiorly. The described technique illustrates the impact of premature coronal suture fusion on the development of the entire skull and proposes how bone dysmorphology contributes to the Patients' neurologic sequelae. By bridging novel basic science methodologies with clinical research, the present study quantitatively describes craniofacial development and bone dysmorphogenesis.

Quantitative Morphologic Analysis of Cranial Vault in Twist1+/- Mice: Implications in Craniosynostosis.

The haploinsufficiency in the TWIST1 gene encoding a basic helix-loop-helix transcription factor is a cause of one of the craniosynostosis syndromes, Saethre-Chotzen syndrome. Patients with craniosynostosis usually require operative release of affected sutures, which makes it difficult to observe the long-term consequence of suture fusion on craniofacial growth. In this study, we performed quantitative analysis of morphologic changes of the skull in Twist1 heterozygously-deleted mice (Twist1+/-) with micro-computed tomographic images. In Twist1+/- mice, fusion of the coronal suture began before postnatal day 14 and progressed until postnatal day 56, during which morphologic changes occurred. The growth of the skull was not achieved by a constant increase in the measured distances in wild type mice; some distances in the top-basal axis were decreased during the observation period. In the Twist1+/- mouse, growth in the top-basal axis was accelerated and that of the frontal cranium was reduced. In the unicoronal suture fusion mouse, the length of the zygomatic arch of affected side was shorter in the Twist1+/- mouse. In one postnatal day 56 Twist1+/- mouse with bilateral coronal suture fusion, asymmetric zygomatic arch length was identified. The authors'results suggest that measuring the length of the left and right zygomatic arches may be useful for early diagnosis of coronal suture fusion and for estimation of the timing of synostosis, and that more detailed study on the growth pattern of the normal and the synostosed skull could provide prediction of the risk of resynostosis. The data from this study can be useful to better understand the cranial growth pattern in patients with craniosynostosis.

Apert Syndrome

Apert syndrome is another genetically inherited syndrome characterized by craniosynostosis (premature fusion of coronal sutures) resulting skull and facial deformities and syndactyly. The syndrome was first described in 1906 by French physician Eugene Apert when he described nine people with similar facial and extremity characteristics. In 1906, Eugène Apert, a French physician, described nine people sharing similar attributes and characteristics.

Unicoronal Craniosynostosis: Is There a Lateral Difference in Retinal Morphology?

Craniosynostosis is the premature fusion of cranial sutures in pediatric patients, which may lead to elevated intracranial pressure due to cerebro-cephalic disproportion between a growing brain and constricted skull. It is unknown whether this increased pressure is distributed equally throughout the cranial vault, or whether certain areas of the brain experience greater pressure at these regions of premature osseous fusion. Optical coherence tomography (OCT) is a noninvasive modality for detecting elevated intracranial pressure. Optical coherence tomography was utilized to measure the peripapillary retinal nerve fiber layer (RNFL) thickness in patients undergoing surgical correction of craniosynostosis. Retinal nerve fiber layer in the eye ipsilateral to the unicoronal suture fusion was compared to the RNFL in the eye contralateral to the unicoronal suture fusion. During the study interval, 21 patients met inclusion criteria. Median age at operative intervention was 8.0 months, and 28.6% patients presented with left-sided unicoronal craniosynostosis, whereas 71.4% of patients presented with right-sided unicoronal craniosynostosis. Rather than universal increase on the affected side of coronal suture fusion, retinal nerve fiber layer thickness parameters showed a rotation phenomenon, such that the patterns of elevation had a 45° circumferential rotation in the direction of intorsion. The explanation for these results remains elusive, but they likely indicate either intracranial changes transmitted differentially to the peripapillary retina, or differing retinal morphology, between the ipsilateral and contralateral eyes in unicoronal craniosynostosis.

Intracranial Volume Measured and Correlated to Cephalic Index in Syndromic and Nonsyndromic Anterior Brachycephaly.

Premature fusion of both coronal sutures (anterior brachycephaly) alters skull shape and potentially affects intracranial volume (ICV). Currently little is known about preoperative ICV in anterior brachycephaly. Aim is to measure preoperative ICV and compare this with normative data. Additionally, ICV will be correlated to most used clinical method of quantification: cephalic index (CI). Preoperative patients with anterior brachycephaly (age, ≤12 months) were included and categorized by syndrome (when present). Computed tomography scans were used for ICV measurement by manual segmentation (OsiriX (Fondation OsiriX, Geneva, Switzerland)). Intracranial volume of each subgroup was compared with Lichtenberg normative cranial volume growth curves for controls. Cephalic index was calculated and correlated to ICV using Pearson correlation coefficient. Thirty-four patients with both syndromic and nonsyndromic anterior brachycephaly were included: 17 with Apert syndrome, 6 with Muenke syndrome, 5 with Saethre Chotzen syndrome, 3 with Crouzon, 1 with craniofrontonasal dysplasia, and 2 nonsyndromal. Mean age at preoperative computed tomography scan was 4 months (1-10 months). Mean ICV was 847.31 cm3 (473.91-1459.22 cm3). Nineteen of 34 patients had skull volumes between ±2 SD curves of Lichtenberg, none of the patients had an ICV smaller than -2 SD and 15 of 34 had an ICV larger than +2 SD. Mean ICV in Apert syndrome was 829.85 cm3 (473.91-1061.53 cm3), in Muenke syndrome 942.06 cm3 (768.02-1136.75 cm3), in Saethre Chotzen syndrome 779.72 cm3 (609.21-1002.95 cm3), in Crouzon syndrome 700.57 cm3 (652.31-784.32 cm3), in craniofrontonasal dysplasia 738.97 cm3, and in the nonsyndromal group 1154.64 cm3 (850.07-1459.22 cm3). Apert had a mean greater than +2SD above the mean, the other subgroups had a mean within normal ranges (±2 SD). Correlation between severity of brachycephaly and overall ICV was low (r = 0.42). Mean preoperative ICV in both syndromic and nonsyndromic anterior brachycephaly was 847.31 cm3. Intracranial volume in anterior brachycephaly is in 55.9% between normal ranges (±2 SD). In 44.1% ICV was greater than +2 SD, especially in Apert syndrome (11/16 Apert patients). None of the included patients had a deviant small ICV of less than -2 SD. Additionally, low correlation between ICV and CI (r = 0.42) was found and therefore CI is not suitable for estimating ICV in anterior brachycephaly.

Study of Pattern of Fusion of Coronal Suture Using Skull Radiography and its Association with Documented Age in Males

Study of Pattern of Fusion of Coronal Suture Using Skull Radiography and its Association with Documented Age in Males

Genetic background dependent modifiers of craniosynostosis severity

Craniosynostosis severity varies in patients with identical genetic mutations. To understand causes of this phenotypic variation, we backcrossed the FGFR2+/C342Y mouse model of Crouzon syndrome onto congenic C57BL/6 and BALB/c backgrounds. Coronal suture fusion was observed in C57BL/6 (88% incidence, p < .001 between genotypes) but not in BALB/c FGFR2+/C342Y mutant mice at 3 weeks after birth, establishing that that the two models differ in phenotype severity. To begin identifying pre-existing modifiers of craniosynostosis severity, we compared transcriptome signatures of cranial tissues from C57BL/6 vs. BALB/c FGFR2+/+ mice. We separately analyzed frontal bone with coronal suture tissue from parietal bone with sagittal suture tissues because the coronal suture but not the sagittal suture fuses in FGFR2+/C342Y mice. The craniosynostosis associated Twist and En1 transcription factors were down-regulated, while Runx2 was up-regulated, in C57BL/6 compared to BALB/c tissues, which could predispose to craniosynostosis. Transcriptome analyses under the GO term MAPK cascade revealed that genes associated with calcium ion channels, angiogenesis, protein quality control and cell stress response were central to transcriptome differences associated with genetic background. FGFR2 and HSPA2 protein levels plus ERK1/2 activity were higher in cells isolated from C57BL/6 than BALB/c cranial tissues. Notably, the HSPA2 protein chaperone is central to craniofacial genetic epistasis, and we find that FGFR2 protein is abnormally processed in primary cells from FGFR2+/C342Y but not FGFR2+/+ mice. Therefore, we propose that differences in protein quality control responses may contribute to genetic background influences on craniosynostosis phenotype severity.

Mesenchymal stem cells in tissue homeostasis and regeneration

The human skull is composed of twenty‐two bones connected by sutures, which are fibrous joints that contain skeletal progenitor cells. The calvarial sutures allow compression of the skull during childbirth and dramatic postnatal growth. Craniosynostosis is a common congenital defect characterized by premature suture fusion, which can cause severe outcomes including abnormal growth of the skull, increased intracranial pressure, retarded brain development and impaired neurocongnitive function. Currently, the only treatment option for infants with craniosynostosis is surgery that involves cutting the calvaria into pieces, manually reshaping them, and fixing them in place mechanically, necessitating blood transfusion and complex post‐surgical care. In many cases, the calvarial bones fuse again, which requires repeated operations to relieve the constriction on the brain during the sensitive period of development in order to improve physiological function. Clearly, there is an immense need for a better approach to the treatment of craniosynostosis and prevention of re‐occurrence.Recent studies have shown that Gli1+ cells are an indispensable mesenchymal stem cell (MSC) source within the cranial sutures. Ablation of Gli1+ cells leads to fusion of all craniofacial sutures in adult mice; we have also demonstrated there is a premature loss of Gli1+ cells prior to coronal suture fusion in Twist1+/− mice, which provide an important, clinically relevant craniosynostosis model of Saethre‐Chotzen syndrome. Here we took advantage of the Twist1+/− mouse model, developed an innovative scaffold combined with Gli1+ MSCs and were able to regenerate a coronal suture and improved the morphology and function of Twist1+/− mice. This discovery provides a potential new approach for treating patients with craniosynostosis.Support or Funding InformationSupported by the NIDCR, NIH R01 DE026339; U01 DE 026914

Radiographic Study of Fusion of Coronal Sutures in Females for Age Estimation

INTRODUCTION: Age estimation of individuals is a usual case faced by Forensic Practitioners. Establishment of the individuality of a person can be absolute or partial. Every age has medico-legal importance in this era. Age estimation as seen in the courts of law can be for age confirmation as in pension, benefits of senior citizen or in dementia cases. Cranial sutures with other criteria are helpful for age estimation.

PIN1 Attenuation Improves Midface Hypoplasia in a Mouse Model of Apert Syndrome

Premature fusion of the cranial suture and midface hypoplasia are common features of syndromic craniosynostosis caused by mutations in the FGFR2 gene. The only treatment for this condition involves a series of risky surgical procedures designed to correct defects in the craniofacial bones, which must be performed until brain growth has been completed. Several pharmacologic interventions directed at FGFR2 downstream signaling have been tested as potential treatments for premature coronal suture fusion in a mouse model of Apert syndrome. However, there are no published studies that have targeted for the pharmacologic treatment of midface hypoplasia. We used Fgfr2S252W/+ knock-in mice as a model of Apert syndrome and morphometric analyses to identify causal hypoplastic sites in the midface region. Three-dimensional geometric and linear analyses of Fgfr2S252W/+ mice at postnatal day 0 demonstrated distinct morphologic variance. The premature fusion of anterior facial bones, such as the maxilla, nasal, and frontal bones, rather than the cranium or cranial base, is the main contributing factor toward the anterior-posterior skull length shortening. The cranial base of the mouse model had a noticeable downward slant around the intersphenoid synchondrosis, which is related to distortion of the airway. Within a skull, the facial shape variance was highly correlated with the cranial base angle change along Fgfr2 S252W mutation–induced craniofacial anomalies. The inhibition of an FGFR2 downstream signaling enzyme, PIN1, via genetic knockdown or use of a PIN1 inhibitor, juglone, attenuated the aforementioned deformities in a mouse model of Apert syndrome. Overall, these results indicate that FGFR2 signaling is a key contributor toward abnormal anterior-posterior dimensional growth in the midface region. Our study suggests a novel therapeutic option for the prevention of craniofacial malformations induced by mutations in the FGFR2 gene.

Craniosynostosis and metabolic bone disorder. A review

IntroductionSome metabolic bone disorders may result in the premature closure of one or more calvarial sutures during childhood, potentially leading to a cranioencephalic disproportion. The aim of this paper is to review the characteristics and consequences of craniosynostosis associated with metabolic disorder. Material and methodsA review of the literature on metabolic forms of craniosynostosis was performed. ResultsThe most common forms of craniosynostosis associated with metabolic bone disorder were isolated sagittal suture fusion with or without scaphocephaly, and sagittal suture fusion associated with coronal suture fusion (oxycephaly) or also with lambdoid suture fusion (pansynostosis). Synostosis may be well-tolerated, but in some subjects results in neurodevelopmental and functional impairment that is sometimes severe. ConclusionThe impact of metabolic synostosis is very variable, depending on the specific underlying metabolic disease, with a large spectrum of morphological and functional consequences. Diagnosis should be early and management should be carried out by a multidisciplinary team with expertise in both rare skeletal disorders and craniosynostosis. The impact of emergent medical therapies recently developed for some of these diseases will be assessed by systematic coherent follow-up of international registries.

S7A-14 SESSION 7A: SAGITTAL SYNOSTOSIS 10 YEARS REVIEW OF « H CRANIECTOMY » TECHNIQUE FOR SAGITTAL SYNOSTOSIS IN 436 PATIENTS,EVALUATION OF SURGICAL ASPECTS, AESTHETIC AND COGNITIVE OUTCOME

Introduction: “H craniectomy” – open approach with partial craniectomy- represents one of the principal surgical techniques for scaphocephaly treatment. Several variations of this technique and mini invasive approaches have been proposed in the last years to improve the outcomes and reduce the complications. Methods: Between 2008 and 2018, 567 patients underwent H craniectomy for scaphocephaly correction in a single center (Necker Hospital, Paris). 476 patients had sufficient data available and were included. The following parameters were assessed: removal of coronal sutures, cranial index (pre-operative and late post-operative), associated closure of metopic suture during surgery, late postoperative fusion of coronal sutures and presence of copper-beaten skull. The aesthetic outcome was evaluated by one single surgeon using the Whitaker scale. Cognitiveissues were recorded (attention, memory, language, school performance) as well as the incidence of headaches. Results: 64% of patients were operated on beforetheage 6 months;30.5% were operated on between age 6 months and 1 yearof age; 5.5% were operatedonafter 1 yearof age. Mean follow-uptimewas 5.6 years (0.2–10.4). Mean cranial index was 70.5 preoperatively and 72.9 postoperatively. 17.3% of patients presented with copper-beaten skullon late follow-up. 58.4% presented with a radiologic fusion of the coronal sutures at late follow-up. 19% presented with an insufficient aesthetic result (Whitaker III or IV). 2.1% had papillary edema at some point during their follow-up. 27.7% reported headaches at some point of their follow-up. As to the cognitive outcomes, 75% had no reported problems at alland5.4% presented with cognitive or language impairment requiring professional assistance. 7 (1.5%) patients neededsurgical revision.The comparison between the classic technique and the surgical variant where coronal sutures were removed did not show any statistically significant differences in cognitive (p=0.633), aesthetic (p=0.782) or radiologic (p=0.461) outcomes. Conclusion: This is the largest long-term retrospective outcome study aiming to evaluate the H craniectomy technique: H craniectomy is a safe and effective procedure. Removal of coronal sutures did not change the incidence of secondary coronal fusion, aestheticand functional outcomes. Further investigations based on multivariate models are in process in order to screen for parameters with predictive values for various aspects the outcome of H craniectomy.

S4A-01SESSION 4A: GENERALITIES IN CRANIOSYNOSTOSIS – PART I CLINICAL AND GENETIC CHARACTERIZATION OF CRANIOSYNOSTOSIS; A RETROSPECTIVE COHORT STUDY AT KING SAUD UNIVERSITY MEDICAL CITY

Introduction: Craniosynostosis (CS) is defined as premature fusion of one or more of the cranial sutures and is classified to either simple or complex.CS can present as a part of a syndrome or as isolated finding.The aim of this study was to evaluate the clinical and genetic pattern of CS among Saudi population. Methods: A retrospective cohort study has been conducted from February 2015 till February 2019 at King Saud University Medical City among patients diagnosed with CS. Results: A total of 31 patients with CS has been identified in our center, of which 26 patients were operated, 4 patients refused surgery, and 1 was operated outside. 58 % (18 cases) of patients were male and 42% (13 cases) were female. Mean age at surgery among the 26 patients was 16 months. Simple CS was observed in 19 cases and complex CS in 12 cases. In our population, the most common simple skull suture fusion was unilateral coronal (plageocephaly) followed by metopic (trigonocephaly). Raised intracranial pressure was detected in 35.4% of the patients especially among syndromic cases and the most common grade of Papilledema were grade I and grade II preoperatively. The Most common procedure done was cranial vault reconstruction with fronto-orbital advancement, followed by posterior vault distraction. Post-operatively, Papilledema resolved in all but one patient who had improvement and is still being followed up. Furthermore, we found that 77.4% of patients had normal development and 22.6% presented with developmental delay. Developmental delay was observed commonly among complex suture fusion patients followed by metopic suture fusion. Genetics etiology was identified either by whole exome sequencing, single gene testing or microarray. Testing revealed; FGFR2 (5 cases), TWIST1 (4 cases), ALPL (2 cases), FREM1 (1 case), TGFBR2 (1 case), TCF12 (1 case), MAN2B1 (1 case), SETD2 (1 case), Interstitial deletion of the short arm of chromosome 7 in one case, and dual genetic diagnoses in one patient with ALDOB and ADAT3 gene mutations. Genetic testing for the remaining 9 cases is still not finalized. Parental consanguinity presented in 25.8% of the patients. Conclusion: Worldwide, Sagittal suture is the most commonly affected suture in simple craniosynostosis, but in our population coronal suture fusion was observed to be affected the most. This is the first study conducted about this topic in Saudi Arabia and further studies needed to investigate the characteristics of CS in our population.

S14-09 SESSION 14: FACIOCRANIOSYNOSTOSIS – PART III MITIGATION OF SHP2 ACTIVATION PREVENTS FGFR2 SIGNALING INDUCED CRANIOSYNOSTOSIS

Introduction: Activating-dominant mutations in the fibroblast growth factor receptor IIIc splice variant gene (Fgfr2c) cause premature cranial suture fusion (craniosynostosis), which can restrict growth of the craniofacial complex. Fgfr2c signaling is mediated via the docking protein Frs2a, which interacts with both Shp2, a tyrosine phosphatase, and Grb2, an adapter protein, to induce ERK-MAPK activation. The role of both Shp2 and Grb2 in craniosynostosis and craniofacial growth remains unknown; however, it is known that gain-of-function Fgfr2c mutant mice demonstrate hyperactivation of the ERK-MAPK signaling cascade and osteoblast activity. The objective of the current study is to determine the role Shp2 and Grb2 may play in Fgfr2-induced craniosynostosis and craniofacial growth. Methods: Gain-of-function Fgfr2c mice were crossed with mice deficient in two Shp2- or four Grb2-specific tyrosine phosphorylation sites within Frs2a. Skull morphology and sutures were analyzed grossly, histologically, and by micro-Computed Tomography. Coronal sutures were analyzed for ERK protein activation and whole calvaria were cultured and analyzed for alkaline phosphatase activity. Fluorescence-lifetime imaging microscopy – Fluorescence Resonance Energy Transfer (FLIM-FRET) was performed to study the interactions between FRS2a, Shp2, and Grb2 within WT or gain-of-function Fgfr2c mutant HEK293T cells. Analysis of Variance was used to compare three or more groups. An observed P value ≤ 0.05 was considered statistically significant. Results: Reduction of Shp2 activation prevents coronal suture fusion and restores normal craniofacial growth (P < 0.05), ERK-MAPK signaling (P < 0.05), and osteoblast activity (P < 0.05) in a mouse model of Fgfr2c-induced craniosynostosis via an Frs2a-dependent mechanism. Complete uncoupling of Grb2 from Frs2a resulted in only partial rescue of normal suture formation and growth. FLIM-FRET analysis revealed a complex interplay between FRS2a, Shp2, and Grb2 in the setting of dysregulated Fgfr2c signaling. Conclusion: This study provides mechanistic insight into craniofacial development and identifies Shp2 as a key regulator of craniofacial growth and development and highlights the therapeutic potential of Shp2 modulation for the treatment of Fgfr2c-related disorders.

Recombinant mouse periostin ameliorates coronal sutures fusion in Twist1+/\u2212 mice

BackgroundSaethre–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.MethodsWe 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.ResultsCell 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.ConclusionOur 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.

Depletion of Cranial Suture Stem Cells by Pharmacological Exposures May Precipitate Premature Suture Fusion

The CDC National Birth Defects Prevention Study has published data suggesting that “environmental” exposures including maternal thyroid diseases, use of selective serotonin reuptake inhibitors (SSRIs), and maternal nicotine use may increase the incidence and or severity of craniofacial anomalies including craniosynostosis. Craniosynostosis is defined as the premature fusion of the suture(s) of the skull occurring in 1:1800–2500 births. A proposed mechanism of craniosynostosis is the disruption of proliferation and differentiation of stem cells in the perisutural area. Here, we hypothesize that the aforementioned teratogenic exposures can cause a depletion of stem cells within the suture resulting in premature suture fusion. To determine if in utero pharmacological exposures deplete stem cells within the calvarial sutures, we exposed pregnant wild‐type mice to levothyroxine, citalopram (SSRI), and nicotine and investigated suture fusion and the presence of stem cell markers ex vivo. Additionally, we exposed primary calvarial suture derived cells to clinically relevant doses levothyroxine, citalopram, and nicotine in vitro and assessed the presence of stem markers via flow cytometry. Micro‐CT assessment of coronal and posterior interfrontal suture fusion revealed that in utero exposure to nicotine and citalopram increased the risk of premature posterior interfrontal suture fusion and exposure to citalopram resulted in an increased likelihood for coronal suture fusion at post‐natal day 15. Further, we confirmed a reduction in Gli1+ cells ex vivo in correlation with in utero teratogen exposure. Our in vitro analysis also indicates a depletion of stem cell populations with teratogen exposure via flow cytometry. Teratogenic exposures including maternal thyroid disorder, maternal use of SSRIs and maternal nicotine use may target calvarial stem cell populations for depletion precipitating an increased risk for craniosynostosis. Investigating the newly defined stem cell niche of the calvarial sutures and its relationship to suture maintenance will provide insight into future manipulation of these cells for therapeutic benefit.Support or Funding InformationNational Institute of Dental and Craniofacial Research [F31DE026684 to ELD; R03DE023350A and R03DE02619‐01A to JC; 5T32DE017551 to MUSC]Cleft Palate Foundation Cleft/Craniofacial Anomalies Grant Award to JCPlastic Surgery Education Foundation Pilot Award to JCNational Institute on Aging (NIA) 1P01AG036675 (Augusta University)National Institutes of Health National Institute of General Medicine [P30GM103331]South Carolina Clinical and Translational Research Institute (NIH/NCATS NIH/NCATS UL1TR000062)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.