Analysis of TGF-beta production by fusing and nonfusing mouse cranial sutures in vitro.

The role of transforming growth factor beta (TGF-beta) in the regulation of cranial suture fusion has been studied by various qualitative techniques such as in situ hybridization and immunohistochemistry. Although the relative expression of TGF-beta isoforms has been assessed in these studies, increased expression of TGF-beta has not been demonstrated in a quantitative fashion. Therefore, the purpose of this study was to quantify TGF-beta production by fusing (posterofrontal [PF]) and nonfusing (sagittal) mouse sutures using two different quantitative TGF-beta assays. The PF and sagittal sutures of 25-day-old mice were harvested and cultured separately in vitro. Culture media conditioned for 48 hours were collected after 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 days of culture, and total TGF-beta production was assessed using a TGF-beta bioassay. For a quantitative TGF-beta1 immunoassay, media conditioned for 48 hours were collected after 3, 5, 7, 9, 14, 22, and 28 days of culture. The TGF-beta bioassay revealed large amounts of total TGF-beta activity in both PF and sagittal sutures during the first week of culture, with decreasing amounts thereafter. Absolute TGF-beta activity in conditioned media collected from PF sutures at several early time points was higher than those obtained from sagittal sutures; however, these differences were not statistically significant. The results of the TGF-beta1 immunoassay (enzyme-linked immunosorbent assay) were similar to the bioassay in that the highest TGF-beta1 levels were noted during the first week of culture period and decreased thereafter. Analysis of variance of these samples, however, revealed significantly more TGF-beta1 protein in samples collected from the PF suture compared with the sagittal suture on days 3 and 5 of culture (p < 0.05). TGF-beta1 levels in the conditioned media obtained from PF sutures remained elevated compared with the sagittal suture on days 7 and 9; however, these differences were not statistically significant. Increased production of TGF-beta in the conditioned media of fusing PF sutures is the first such quantitative demonstration of growth factor upregulation during suture fusion and supports the hypothesis that TGF-beta expression may be important in cranial suture fusion.     

Expression of high-affinity receptors for TGF-beta during rat cranial suture fusion.

The etiology of craniosynostosis is unknown. The elucidation of the biological pathways responsible for this disorder has been hampered by an inability to evaluate cranial sutures before, during, and after cranial suture fusion. The programmed fusion of the rat posterofrontal (PF) suture postnatally provides an excellent model to study the molecular events that occur during cranial suture fusion. Previous experiments have implicated transforming growth factor beta (TGF-beta) growth factors in the regulation of PF suture fusion. The purpose of these experiments was to localize the expression of high-affinity receptors for these growth factors during cranial suture fusion. Four rats were sacrificed on postnatal days 8, 12, 17, and 40 (N = 16). The PF and sagittal sutures were harvested and prepared for immunohistochemical localization of TGF-beta receptor 1 and receptor 2 (Tbeta-RI, Tbeta-RII) protein. Results indicate that immunostaining for Tbeta-RI and Tbeta-RII is markedly increased in the dura mater and osteoblasts of the sutural margin of the PF suture during active suture fusion (on postnatal days 12, 17, and 40) compared with the osteoblasts and dura mater underlying the patent sagittal suture. These results, in combination with the authors' previous findings as well as studies supporting a role for TGF-beta molecules in the regulation of osteogenesis, implicate TGF-beta signaling in the regulation of suture fusion. The possible mechanisms of ligand-receptor interaction are discussed.     

Cranial deformation in craniosynostosis. A new explanation.

Skull growth after premature fusion of a single suture was described by Virchow in 1851. He observed that growth was restricted in a plane perpendicular to a fused suture. However, he failed to predict the compensatory growth patterns that produce many of the deformities recognized as features of individual craniosynostosis syndromes. The deformities resulting from premature closure of a coronal, sagittal, metopic, or lambdoid suture can be predicted by the following observations: (1) cranial vault bones that are prematurely fused act as a single bone plate with decreased growth potential; (2) asymmetrical bone deposition occurs mainly at perimeter sutures, with increased bone deposition directed away from the bone plate; (3) sutures adjacent to the stenotic suture compensate in growth more than those sutures not contiguous with the closed suture; and (4) enhanced bone deposition occurs along both sides of a nonperimeter suture that is a continuation of the prematurely closed suture. These four rules were derived by critically examining the clinical deformities observed with each form of craniosynostosis. These rules assume that cranial sutures have the capacity to compensate by depositing bone asymmetrically along their edges. Unequal growth patterns have been demonstrated in the frontonasal suture of rabbits by Selman and Sarnat. In addition, unequal bone deposition has also been demonstrated along the parieto-interparietal suture in albino rats by Baer. Human studies to determine if asymmetrical bone deposition actively occurs along cranial vault sutures in response to a stenotic suture have not been performed, however. It is also unclear whether these four guidelines apply to cranial base abnormalities observed with craniosynostosis. As new radiologic techniques develop to define the configuration of the skull in intricate detail, a skull pattern of growth explaining the pathogenesis of all deformities created by premature fusion of a cranial vault suture may become apparent.     

胰岛素生长因子1在颅缝闭合中调节作用的实验研究

目的：研究胰岛素生长因子1对大鼠颅缝细胞的骨诱导作用和体外调节小鼠矢状缝闭合的作用。方法：获取新出生的SD大鼠的矢状缝细胞进行培养和出生8d的CD1小鼠矢状缝进行体外无血清器官培养基培养，加入胰岛素生长因子1（insulin lilce growth factor 1 IGF1），浓度分别为10ng/ml和40ng/ml，并设立不加IGF1者为对照组，应用RT－PCR检测颅缝细胞的成骨细胞表型碱性磷酸酶、骨钙素和骨桥蛋白mRNA表达，ELISA法检测培养液I型胶原的分泌，光镜观察小鼠矢状缝闭合的情况。结果：加入IGF1的矢状缝细胞的成骨细胞表型碱性磷酸酶、骨钙素和骨桥蛋白mRNA表达以及培养液I型胶原分泌量较对照组明显增高。有IGF1干预的矢状缝移植体培养8d时，颅缝内侧面骨板开始接近，培养20d时，颅缝小部分闭合，培养30d时颅缝大部分闭合，无IGF1干预的对照组培养30d，颅缝未发生闭合。结论：胰岛素生长因子1通过增强颅缝细胞的骨诱导促进颅缝的闭合。     

An analysis of the growth of the cranial vault in rabbits by vital staining with lead acetate

The mode and the rate of formation of individual bones in the cranium were studied by vital staining with lead acetate in 13 New Zealand white rabbits beginning at 35 days of age. These evaluations were compared with⁴⁵Ca uptake at bilaterally comparable sites.The growth patterns differed from one suture to another. Elongation of the cranial vault in the anterior direction in the snout area was rapid. In the sagittal suture complex, active growth was observed on the intrasutural surfaces of the internasal and metopic sutures, while much less growth was taking place in the sagittal suture. Active growth also was taking place at the intrasutural surface of the nasal bone at the naso-premaxillary suture while resorption was found on the surface of the parietal bone in the squamosal suture.Increase in thickness of the cranial vault appeared to be the result of accretion on the ectocranial and to a lesser extent on the endocranial surfaces of the frontal and parietal bones.Good correlations between the linear measurement with the lead lines and⁴⁵Ca uptake were observed in the bones of the cranial vault, especially when⁴⁵Ca was administered 3 or 6 days before sacrifice.Fellowship F05-TW-1029.     

Regional differentiation of cranial suture-associated dura mater in vivo and in vitro: implications for suture fusion and patency.

Despite its prevalence, the etiopathogenesis of craniosynostosis is poorly understood. To better understand the biomolecular events that occur when normal craniofacial growth development goes awry, we must first investigate the mechanisms of normal suture fusion. Murine models in which the posterior frontal (PF) suture undergoes programmed sutural fusion shortly after birth provide an ideal model to study these mechanisms. In previous studies, our group and others have shown that sutural fate (i.e., fusion vs. patency) is regulated by the dura mater (DM) directly underlying a cranial suture. These studies have led to the hypothesis that calvarial DM is regionally differentiated and that this differentiation guides the development of the overlying suture. To test this hypothesis, we evaluated the messenger RNA (mRNA) expression of osteogenic cytokines (transforming growth factor beta1 [TGF-beta1] and TGF-beta3) and bone-associated extracellular matrix (ECM) molecules (collagen I, collagen III, osteocalcin, and alkaline phosphatase) in freshly isolated, rat dural tissues associated with the PF (programmed to fuse) or sagittal (SAG; remains patent) sutures before histological evidence of sutural fusion (postnatal day 6 [N6]). In addition, osteocalcin protein expression and cellular proliferation were localized using immunohistochemical staining and 5-bromo-2'deoxyuridine (BrdU) incorporation, respectively. We showed that the expression of osteogenic cytokines and bone-associated ECM molecules is potently up-regulated in the DM associated with the PF suture. In addition, we showed that cellular proliferation in the DM associated with the fusing PF suture is significantly less than that found in the patent SAG suture just before the initiation of sutural fusion N6. Interestingly, no differences in cellular proliferation rates were noted in younger animals (embryonic day 18 [E18] and N2). To further analyze regional differentiation of cranial suture-associated dural cells, we established dural cell cultures from fusing and patent rat cranial sutures in N6 rats and evaluated the expression of osteogenic cytokines (TGF-beta1 and fibroblast growth factor 2 [FGF-2]) and collagen I. In addition, we analyzed cellular production of proliferating cell nuclear antigen (PCNA). These studies confirmed our in vivo findings and showed that dural cell cultures derived from the fusing PF suture expressed significantly greater amounts of TGF-beta1, FGF-2, and collagen I. In addition, similar to our in vivo findings, we showed that PF suture-derived dural cells produced significantly less PCNA than SAG suture-derived dural cells. Finally, coculture of dural cells with fetal rat calvarial osteoblastic cells (FRCs) revealed a statistically significant increase in proliferation (*p < 0.001) in FRCs cocultured with SAG suture-derived dural cells as compared with FRCs cocultured alone or with PF suture-derived dural cells. Taken together, these data strongly support the hypothesis that the calvarial DM is regionally differentiated resulting in the up-regulation of osteogenic cytokines and bone ECM molecules in the dural tissues underlying fusing but not patent cranial sutures. Alterations in cytokine expression may govern osteoblastic differentiation and ECM molecule deposition, thus regulating sutural fate. Elucidation of the biomolecular events that occur before normal cranial suture fusion in the rat may increase our understanding of the events that lead to premature cranial suture fusion.     

Cranial vault growth in craniosynostosis

Skull growth after single suture closure was described in 1851 by Virchow, who noted that growth in the plane perpendicular to a fused suture was restricted. However, this observation failed to predict compensatory growth patterns that produce many of the deformities recognized as features of individual syndromes. The deformities resulting from premature closure of a coronal, sagittal, metopic, or lambdoid suture can be predicted on the basis of the following observations: 1) cranial vault bones that are prematurely fused secondary to single suture closure act as a single bone plate with decreased growth potential; 2) asymmetrical bone deposition occurs mainly at perimeter sutures, with increased bone deposition directed away from the bone plate; 3) sutures adjacent to the prematurely fused suture compensate in growth more than those sutures not contiguous with the closed suture; and 4) enhanced symmetrical bone deposition occurs along both sides of a non-perimeter suture that is a continuation of the prematurely closed suture. These observations regarding growth in craniosynostosis are illustrated with clinical material in this report.     

Impaired posterior frontal sutural fusion in the biglycan/decorin double deficient mice

Biglycan (Bgn) and decorin (Dcn) are highly expressed in numerous tissues in the craniofacial complex. However, their expression and function in the cranial sutures are unknown. In order to study this, we first examined the expression of biglycan and decorin in the posterior frontal suture (PFS), which predictably fuses between 21 and 45 days post-natal and in the non-fusing sagittal (S) suture from wild-type (Wt) mice. Our data showed that Bgn and Dcn were expressed in both cranial sutures. We then characterized the cranial suture phenotype in Bgn deficient, Dcn deficient, Bgn/Dcn double deficient, and Wt mice. At embryonic day 18.5, alizarin red/alcian blue staining showed that the Bgn/Dcn double deficient mice had hypomineralization of the frontal and parietal craniofacial bones. Histological analysis of adult mice (45-60 days post-natal) showed that the Bgn or Dcn deficient mice had no cranial suture abnormalities and immunohistochemistry staining showed increased production of Dcn in the PFS from Bgn deficient mice. To test possible compensation of Dcn in the Bgn deficient sutures, we examined the Bgn/Dcn double deficient mice and found that they had impaired fusion of the PFS. Semi-quantitative RT-PCR analysis of RNA from 35 day-old mice revealed increased expression of Bmp-4 and Dlx-5 in the PFS compared to their non-fusing S suture in Wt tissues and decreased expression of Dlx-5 in both PF and S sutures in the Bgn/Dcn double deficient mice compared to the Wt mice. Failure of PFS fusion and hypomineralization of the calvaria in the Bgn/Dcn double deficient mice demonstrates that these extracellular matrix proteoglycans could have a role in controlling the formation and growth of the cranial vault.     

Interrelationship of Cranial Suture Fusion, Basicranial Development, and Resynostosis Following Suturectomy in Twist1+/? Mice, a Murine Model of Saethre-Chotzen Syndrome

The interrelationships among suture fusion, basicranial development, and subsequent resynostosis in syndromic craniosynostosis have yet to be examined. The objectives of this study were to determine the potential relationship between suture fusion and cranial base development in a model of syndromic craniosynostosis and to assess the effects of the syndrome on resynostosis following suturectomy. To do this, posterior frontal and coronal suture fusion, postnatal development of sphenooccipital synchondrosis, and resynostosis in Twist1(+/+) (WT) and Twist1(+/-) litter-matched mice (a model for Saethre-Chotzen syndrome) were quantified by evaluating μCT images with advanced image-processing algorithms. The coronal suture in Twist(+/-) mice developed, fused, and mineralized at a faster rate than that in normal littermates at postnatal days 6-30. Moreover, premature fusion of the coronal suture in Twist1(+/-) mice preceded alterations in cranial base development. Analysis of synchondrosis showed faster mineralization in Twist(+/-) mice at postnatal days 25-30. In a rapid resynostosis model, there was an inability to fuse both the midline posterior frontal suture and craniotomy defects in 21-day-old Twist(+/-) mice, despite having accelerated mineralization in the posterior frontal suture and defects. This study showed that dissimilarities between Twist1(+/+) and Twist1(+/-) mice are not limited to a fused coronal suture but include differences in fusion of other sutures, the regenerative capacity of the cranial vault, and the development of the cranial base.     

A Pro253Arg mutation in fibroblast growth factor receptor 2 (Fgfr2) causes skeleton malformation mimicking human Apert syndrome by affecting both chondrogenesis and osteogenesis

Apert syndrome is one of the most severe craniosynostosis that is mainly caused by either a Ser252Trp(S252W) or Pro253Arg(P253R) mutation in fibroblast growth factor receptor 2 (FGFR2). As an autosomal dominant disorder, Apert syndrome is mainly characterized by skull malformation resulting from premature fusion of craniofacial sutures, as well as syndactyly, etc. A P253R mutation of FGFR2 results in nearly one-thirds of the cases of Apert syndrome. The pathogenesis of Apert syndrome resulting from P253R mutation of FGFR2 is still not fully understood. Here we reported a knock-in mouse model carrying P253R mutation in Fgfr2. The mutant mice exhibit smaller body size and brachycephaly. Analysis of the mutant skulls and long bones revealed premature fusion of coronal suture, shortened cranial base and growth plates of long bones. In vitro organ culture studies further revealed that, compared with wild-type littermates, the mutant mice have prematurely fused coronal sutures and retarded long bone growth. Treatment of the cultured calvaria and femur with PD98059, an Erk1/2 inhibitor, resulted in partially alleviated coronal suture fusion and growth retardation of femur respectively. Our data indicated that the P253R mutation in Fgfr2 directly affect intramembranous and endochondral ossification, which resulted in the premature closure of coronal sutures and growth retardation of long bones and cranial base. And the Erk1/2 signaling pathway partially mediated the effects of P253R mutation of Fgfr2 on cranial sutures and long bones.     

A case of pycnodysostosis

Pycnodysostosis is a term introduced by Maroteaux and Lamy in 1962 to describe characteristic features such as dwarfism, separated cranial sutures, open fontanelles, hypoplasia and absence of mandibular angle, partial aplasia of the terminal phalanges of the fingers and toes, generalized condensation of bone shadow, bone fragility, and autosomal recessive inheritance. We reported a such case in a 13-years old boy associated with an epidural hematoma. Four weeks after removal of the hematoma, lumbar puncture was performed with the initial pressure of 200mm H2O in an awake state and the plateau waves were observed several times during sleep. Such increased intracranial pressure may have caused the open fontanelle and cranial sutures in addition to dysostosis. Thus such patients require careful attention in order to prevent major bleeding from the superior sagittal sinus directly beneath the separated sagittal suture.     

兔颅骨双侧冠状缝早闭后颅面骨的生长变化

为从骨生长量和生长方向两个方面探讨兔双侧冠状缝早闭后对颅骨骨生长发育的影响，用牙科釉质和剂固定２周龄幼兔双侧冠状缝，于冠状缝，鼻额缝和人字缝前后置入金属标记物，观察双侧冠状缝早闭后各骨缝及颅面长度，颅穹窿高度，长度和鼻骨长度变化情况，结果表明兔颅骨缝扩张性生长高峰期在２～８周龄，双侧冠状缝固定后，骨缝生长停止，鼻额缝补偿生长加快，４周解除固定带后，冠状缝在８周前出现补偿性生长高峰期，８周解除固定带     

颅面多骨缝固定对颅颌形态影响的实验研究

目的通过固定幼兔颅骨缝，模拟颅骨多骨缝早闭，观察固定后颅颌面生长方式的变化及相互影响的关系。方法采用牙科釉质粘合剂固定２周龄幼兔冠状缝、矢状缝和额间缝，模拟颅骨多骨缝早闭。术后不同时期测量颅穹窿长度、高度、面中部高度、上颌骨长度、面角、腭角和颅底角。结果多骨缝早闭后，颅穹窿高度和长度明显减少，使颅骨变短且扁平。面中部高度和上颌骨长度增大，整个面中骨结构有整体向上移动趋势。面、腭和颅底角增大，这与颅穹窿变浅，面中部高度增大相对应。结论兔出生后１０周前是颅骨缝扩张性生长高峰期，在此期间，某些原因影响颅骨正常生长，必然导致颅面畸形。实验结果与临床颅骨骨缝早闭后表现类似，和主张早期手术矫正畸形观点一致     

兔颅骨双侧冠状缝早闭后颅面骨的生长变化

为从骨生长量和生长方向两个方面探讨免双侧冠状缝早闭后对颅面骨生长发育的影响,用牙科釉质粘和剂固定2周龄幼免双侧冠状缝,于冠状缝、鼻额缝和人字缝前后置入金属标记物,观察双侧冠状缝早闭后各骨缝及颅面长度、颅穹窿高度、长度和鼻骨长度变化情况。结果表明兔颅骨缝扩张性生长高峰期在2～8周龄。双侧冠状缝固定后,骨缝生长停止,鼻额缝补偿性生长加快。4周解除固定带后,冠状缝在8周前出现补偿性生长高峰期,8周解除固定带无补偿性生长高峰出现。另外鼻骨长度增加并向下移位,颅穹隆长度和颅面长度缩短。提示临床手术矫正颅骨畸形时,除考虑到手术年龄的安全性外,必须强调早期手术的必要性。     

兔颅骨双侧冠状缝早闭后颅面骨的生长变化

为从骨生长量和生长方向两个方面探讨兔双侧冠状缝早闭后对颅面骨生长发育的影响，用牙科釉质粘和剂固定２周龄幼兔双侧冠状缝，于冠状缝、鼻额缝和人字缝前后置入金属标记物，观察双侧冠状缝早闭后各骨缝及颅面长度、颅穹窿高度、长度和鼻骨长度变化情况。结果表明兔颅骨缝扩张性生长高峰期在２～８周龄。双侧冠状缝固定后，骨缝生长停止，鼻额缝补偿性生长加快。４周解除固定带后，冠状缝在８周前出现补偿性生长高峰期，８周解除固定带无补偿性生长高峰出现。另外鼻骨长度增加并向下移位，颅穹隆长度和颅面长度缩短。提示临床手术矫正颅骨畸形时，除考虑到手术年龄的安全性外，必须强调早期手术的必要性。     

Craniofacial growth following experimental craniosynostosis and craniectomy in rabbits.

Premature fusion of the coronal suture was produced in 9-day-old rabbits by immobilization of the suture area bilaterally with methyl-cyanoacrylate adhesive. The effects of suture fusion and its surgical release on suture growth and on skull morphology were evaluated by radiographic cephalometry. Immobilization resulted in significant changes in the angular dimensions in the vault toward an anteroposterior shortening. No permanent deformity was observed in the angular relationship between the cranial base and the facial skeleton. Craniectomy at 30 days, when a skull deformity had been established, resulted in rapid separation of the bones at the suture site which returned the deformed skull to a normal configuration by 90 days of age. Surgical removal of a normal suture in a control group also resulted in accelerated separation of the bones at the excised suture site, but it was less than after removal of an immobilized suture. The experimental data indicate that premature fusion of rapidly growing sutures results in consistent skull deformity. Early release of the fusion, when this is the primary abnormality, will result in spontaneous correction of the deformity.     

Slit-ventricle syndrome secondary to shunt-induced suture ossification

OBJECTIVE: To report five children with slit-ventricle syndrome who were found to have increased intracranial pressure despite functioning cerebrospinal fluid shunts. METHODS: Computed tomographic scans demonstrated erosion of the inner table of the cranium and sclerosis of the cranial sutures, particularly the coronal suture. Magnetic resonance imaging scans demonstrated no cerebrospinal fluid over the convexities. The patients were treated with cranial expansion operations that included removal of the sclerotic sutures, which were examined histologically. RESULTS: Postoperatively, symptoms resolved for all children. Sutures were abnormal and contained foci of cartilage and bone within abnormally arranged fibrous tissue. CONCLUSION: We postulate that chronic overdrainage of cerebrospinal fluid via shunts dampens the normal cerebral pressure waves; growth of the calvarium is thus understimulated, and this leads to ossification of the sutures, which become unable to expand to allow normal brain growth. Shunt-induced craniostenosis should be considered for children with symptoms of slit-ventricle syndrome for whom shunts are functional but intracranial pressure is increased. Cranial expansion operations may be more appropriate treatments than subtemporal decompressions for such children, given the diffuseness of the suture pathological features.     

Craniosynostosis

Craniosynostosis is a premature pathologic fusion of one or more cranial vault sutures that leads to abnormal shape of the skull. The fused sutures lead to restricted growth in some areas and compensatory bossing in other areas. The head may assume different shapes depending upon the site and timing of the abnormally fused suture. The exact cause of this suture pathology is still unknown, but the local dura, cranial base and the fibroblast growth factors seem to influence this. The diagnosis rests on clinical examination and confirmation is generally on the computed tomography scan. The need for surgery is both for cosmetic and functional reasons. Many cases may be associated with raised intracranial pressure with its attendant deleterious effects on vision and brain. The aim of treatment is to increase the cranial volume and reshape the skull. The surgery can be safely undertaken around 9-12 months in most of the cases. The conventional management is through an open surgical approach; although, some centres have claimed impressive results with limited endoscopic techniques in selected cases. The review article deals with the aetiopathogenesis, clinical presentations and management of the common varieties of craniosynostoses seen in the Indian scenario.KEY WORDS: Abnormal skull shapes, cosmetic and functional issues, cranioplasty, fronto-orbital advancement, premature suture fusion     

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.