A coupled reaction-diffusion-strain model predicts cranial vault formation in development and disease.

How cells utilize instructions provided by genes and integrate mechanical forces generated by tissue growth to produce morphology is a fundamental question of biology. Dermal bones of the vertebrate cranial vault are formed through the direct differentiation of mesenchymal cells on the neural surface into osteoblasts through intramembranous ossification. Here we join a self-organizing Turing mechanism, computational biomechanics, and experimental data to produce a 3D representative model of the growing cerebral surface, cranial vault bones, and sutures. We show how changes in single parameters regulating signaling during osteoblast differentiation and bone formation may explain cranial vault shape variation in craniofacial disorders. A key result is that toggling a parameter in our model results in closure of a cranial vault suture, an event that occurred during evolution of the cranial vault and that occurs in craniofacial disorders. Our approach provides an initial and important step toward integrating biomechanics into the genotype phenotype map to explain the production of variation in head morphology by developmental mechanisms.

Expression of glutathione peroxidase 1 in the spheno-occipital synchondrosis and its role in ROS-induced apoptosis.

BACKGROUND/OBJECTIVE: Chondrogenesis is an integral part of endochondral bone formation, by which the midline cranial base is developed. Reactive oxygen species (ROS) are required in chondrogenic differentiation and antioxidant enzymes regulate their levels. The aim of this study was to localize the antioxidant enzyme glutathione peroxidase 1 (Gpx1) at the spheno-occipital synchondrosis, as well as its effect on ROS challenge and its expression pattern in the course of differentiation. MATERIALS AND METHODS: Gpx1 was semiquantified in immunohistochemically stained sections of spheno-occipital synchondroses of rats. The effect of Gpx1 on ROS-induced apoptosis was investigated by manipulating the expression of Gpx1 in ATDC5 cells. The temporal pattern of Gpx1 expression was determined during chondrocyte differentiation for 21 days in vitro. RESULTS: Proliferating chondrocytes exhibited the greatest Gpx1 immunoreactivity and hypertrophic ones the lowest (P = 0.02). Cells transfected with Gpx1-siRNA had the highest apoptotic rate, while cells overexpressing Gpx1 the lowest one (P < 0.001). Gpx1 was significantly increased on days 10 (P = 0.02) and 14 (P = 0.01). CONCLUSIONS: Hypertrophic chondrocytes have the lowest Gpx1 activity in the spheno-occipital synchondrosis. Gpx1 is implicated in the ROS-induced apoptosis in chondrocytes. Its expression was not constitutive during chondrogenic differentiation.

Down-regulation of ubiquitin ligase Cbl induced by twist haploinsufficiency in Saethre-Chotzen syndrome results in increased PI3K/Akt signaling and osteoblast proliferation.

Genetic mutations of Twist, a basic helix-loop-helix transcription factor, induce premature fusion of cranial sutures in Saethre-Chotzen syndrome (SCS). We report here a previously undescribed mechanism involved in the altered osteoblastogenesis in SCS. Cranial osteoblasts from an SCS patient with a Twist mutation causing basic helix-loop-helix deletion exhibited decreased expression of E3 ubiquitin ligase Cbl compared with wild-type osteoblasts. This was associated with decreased ubiquitin-mediated degradation of phosphatidyl inositol 3 kinase (PI3K) and increased PI3K expression and PI3K/Akt signaling. Increased PI3K immunoreactivity was also found in osteoblasts in histological sections of affected cranial sutures from SCS patients. Transfection with Twist or Cbl abolished the increased PI3K/Akt signaling in Twist mutant osteoblasts. Forced overexpression of Cbl did not correct the altered expression of osteoblast differentiation markers in Twist mutant cells. In contrast, pharmacological inhibition of PI3K/Akt, but not ERK signaling, corrected the increased cell growth in Twist mutant osteoblasts. The results show that Twist haploinsufficiency results in decreased Cbl-mediated PI3K degradation in osteoblasts, causing PI3K accumulation and activation of PI3K/Akt-dependent osteoblast growth. This provides genetic and biochemical evidence for a role for Cbl-mediated PI3K signaling in the altered osteoblast phenotype induced by Twist haploinsufficiency in SCS.

Erk1/2 signaling is required for Tgf-beta 2-induced suture closure.

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

Expression of matrix metalloproteinase genes in the rat intramembranous bone during postnatal growth and upon mechanical stresses.

A cranial suture consists of neural-crest derived cells and matrices between mineralized skull bones. Little is known regarding the involvement of matrix metalloproteinases (MMPs) in the degradation of extracellular matrix of cranial sutures. In the postnatal rat model, the posterior frontal suture (PFS) undergoes complete ossification between P12-P22, whereas the sagittal suture (SS) remains patent. The present study utilized reverse transcriptase-polymerase chain reaction (RT-PCR) to explore the expression of MMP-1 and MMP-2 genes in the PFS and SS in P8 and P32 rats, and also to determine whether these MMP genes are modulated by exogenous mechanical forces. RNA was isolated from P8 and P32 normal PFS and SS each by pooling sutural specimens from 14 to 20 rats. RT-PCR analysis and semi-quantitative luminosity demonstrated the expression of MMP-1 and MMP-2 genes in the patent P8 PFS, P8 SS, and P32 SS, but no apparent MMP-2 expression in the physiologically ossified P32 PFS. Exogenous cyclic forces applied to the maxilla at 1000 mN and 4 Hz elicited corresponding cyclic bone strain waveforms with peak strain of 134.14+/-38.15 muepsilon (mean+/-S.D.) for the PFS, and 28.35+/-10.86 muepsilon for the SS in P32 rats. These cyclic forces delivered for 20 min/d over 2 consecutive days induced the expression of MMP-2 gene in the physiologically fused P32 PFS that was not expressed without mechanical stresses. Taken together, these data suggest potentially important roles of MMP genes in the postnatal development of cranial sutures, and their susceptibility to mechanical stresses.

Immunolocalization of urokinase and its receptor in prematurely fused cranial sutures of infants.

In cranial sutural samples derived from five children with premature cranial suture fusion we have performed immunostaining for the urokinase plasminogen activator (uPA) and urokinase receptor (uPAR). We have found a strong reactivity for cell- or matrix-bound uPA and uPAR in the sutural connective tissue and associated with the osteoblasts and osteocytes lining the calvarial bone. The sutural tissue itself showed a banding with different intensity of urokinase and uPAR staining concerning connective tissue. It is proposed that the components of the plasminogen activating system are involved in tissue turnover of sutural tissue and in sutural growth.

Enzymatic activation associated with programmed fusion of the posterior interfrontal sutures in rats.

Fetal rat coronal sutures in culture undergo fusion in the absence of their dura mater. Coinciding with the period of fusion are marked cellular enzymatic changes. Alkaline phosphatase, a marker of osteoblastic activity, and tartrate-resistant acid phosphatase (TRAP), a marker of osteoclastic activity, both increase significantly within fusing sutures and indicate changes in the control of bone synthesis and breakdown. Other enzymes not specifically related to bone formation or degradation also show activation within these fusing sutures. These enzymes include tartrate-sensitive acid phosphatase (TSAP), a marker of lysosomal activity; hexokinase, a glycolytic enzyme; glucose 6-phosphate dehydrogenase (G6PD), an enzyme of the pentose monophosphate shunt; and glutathione reductase, an enzyme of the antioxidant pathway. In the present study, we compared the enzymatic changes previously seen ex vivo with those occurring in vivo during the programmed closure of the posterior interfrontal suture of the rat. This suture fuses between postnatal days 10 and 30 in the rat. The sagittal suture, which remains patent during this period, was used to establish baseline enzymatic activities in a comparable midline suture. Neonatal rats were killed at postnatal days 2, 4, 5, 8, 10, 12, 15, 20, and 30, and posterior interfrontal and sagittal sutures with bone plates on either side were removed. The suture regions of the samples were isolated, dura mater was removed, and suture regions were assayed by microanalytical techniques. Activities of alkaline phosphatase, TRAP, TSAP, hexokinase, G6PD, and glutathione reductase were measured. DNA content was also assayed, and enzyme activities were expressed per amount of DNA. Three pups were killed at each time point, and three to five assays were performed per suture (posterior interfrontal or sagittal) for each time point assayed. Alkaline phosphatase and TRAP activities showed marked increases in fusing sutures compared with nonfusing controls, similar to the increases demonstrated ex vivo. TSAP and hexokinase also showed elevations in the fusing posterior interfrontal sutures, with the greatest differences predominantly during the period of fusion, comparable to the changes seen ex vivo. However, G6PD and glutathione reductase, enzymes of the antioxidant pathway, did not demonstrate the same degree of activation seen ex vivo in fusing sutures. In fact, the levels were actually higher in the patent sagittal samples for the majority of time points examined. Alkaline phosphatase and TRAP activity elevations indicated both osteoblastic and osteoclastic activation during fusion, as seen in the ex vivo phenomenon. TSAP and hexokinase increases also reflected activation in lysosomes and in cellular metabolism during fusion, paralleling the ex vivo situation. However, a less clear pattern of activation in the antioxidant pathway, in contrast to the pattern seen ex vivo, was present. These differences may reflect the different environments of sutures in vivo and ex vivo. Alternatively, oxidative stress may play a more central role in the pathologic process of induced suture fusion ex vivo than in programmed suture fusion in vivo.

Magnetic resonance images and histology of the spheno-occipital synchondrosis in young monkeys (Macaca fuscata).

Magnetic resonance images and the histology of spheno-occipital synchondrosis were examined in young monkeys in order to compare the magnetic resonance images with their histologic observations. In serial magnetics resonance images of posterior cranial base, the spheno-occipital synchondrosis showed a low signal zone with unclear boundaries, running through the posterior cranial base perpendicularly to the clivus. The zone was always interposed between nonsignal zones. These observations were the same as those in young juvenile human beings. The histologic sections also revealed that the low signal zone was really the spheno-occipital synchondrosis, which consisted of hyaline cartilage and that the nonsignal zones were bone tissues. The chondroblasts in the spheno-occipital synchondrosis were arranged bipolarly. Intense alkaline phosphatase activity was located in the areas along the bone. Tetracycline labeling was also noticed in the bone formed in the endochondral ossification. These results suggest that magnetic resonance imaging enables us to observe the spheno-occipital synchondrosis in the posterior cranial base and also to elucidate its influences on the growth of maxilla and mandible in the future.

Iron-induced rat coronal suture fusion in vitro: the role of redox regulation.

The presumptive coronal sutures of rat fetuses at gestation days 19 and 20 have been shown to fuse prematurely when grown in the absence of dura mater in culture. In the present study, the representative enzymes of glucose metabolism and the antioxidative pathway were assayed during the process of suture fusion. The coronal sutures of fetal day 19.5 (F19) and neonatal day 1 rats were grown in the presence or absence of dura mater in serum-free culture. The enzymes assayed were hexokinase (HK) and pyruvate kinase (PK) of glycolysis, and glucose 6-phosphate dehydrogenase (G6PD) and glutathione reductase (GR) of the antioxidative pathway. F19 sutures cultured without dura mater, which fused, showed significant increases in enzyme activities over the preculture levels. HK increased by 200% to 300% of the preculture levels, G6PD by 400% to 500%, GR by 200%, and PK by 400% to 500%. The fetal sutures cultured with dura mater, which did not fuse, showed little alterations of HK, G6PD, and GR activities, but showed a significant 200% to 400% increase in PK activity. Neonatal sutures showed significant increases in enzyme activities during culture, but the presence of dura mater did not significantly affect enzyme activities. High activity levels of enzymes of the antioxidative pathway in F19 sutures coincided with the period of premature suture fusion. Treatment of fetal calvaria with prooxidant (induced by ferrous iron and ascorbic acid) produced suture fusion even in the presence of dura mater. Treatment with deferoxamine (an iron chelator and antioxidant) during the culture prevented suture fusion. The results suggest that fusing sutures experience increased biosynthetic demands and are placed under oxidative stress. When oxidative stress overwhelms the dural influence, the sutures undergo premature fusion.

Regeneration of cranial suture and bone plate lesions in rabbits. Implications for positioning of osteotomies.

The positioning of osteotomies in intramembranous cranial bone was studied by exploring the pattern of bone regeneration in growth areas (the sutural region) as compared to that of the bone plate proper. Trephine defects in the left coronal suture area and the right parietal bone were produced in fifty-nine young rabbits. A pilot study to refine operative and analytical methods comprised 22 animals. The experiments were terminated at one, three, and six weeks after surgery. The bone regenerative response was assessed by x-ray planimetry, plain microscopy, enzyme histochemistry, and fluorescent labelling. Only minor divergences in healing capacity between the two defects were found. No adverse effects on the growth process were indicated. As to clinical management, the findings suggest that osteotomies designed to traverse sutural areas will, under normal circumstances, regenerate in a similar manner and rate to adjoining bone plates.

Immunolocalization of collagenase and tissue inhibitor of metalloproteinases (TIMP) in mechanically deformed fibrous joints.

To investigate the effects of mechanical deformation on matrix degradation in fibrous joints, coronal suture explants from neonatal rabbits were stressed in vitro for 24 hours in an established tooth-movement model system. The metalloproteinase collagenase (CL) and its inhibitor, TIMP (tissue inhibitor of metalloproteinases), were immunolocalized in two ways by a two-step indirect technique: (1) extracellularly by immunoprecipitation at the site of secretion, and (2) intracellularly by incubation of the explants with the ionophore monensin. Immunoprecipitates of CL and TIMP were distributed throughout the sutural and periosteal tissues of nonstressed explants. In stressed explants, however, CL immunoprecipitates were predominantly associated with an area of rounded cells between the bone ends. In explants treated with monensin a significant increase in the number of CL-positive cells was observed in this cellular area; active enzyme was suggested by the demonstration of CL bound to collagen. Extracellular TIMP was not seen within the area of rounded cells of stressed explants, but intracellular TIMP was detectable; this suggests that insufficient TIMP was available to immunoprecipitate with anti-TIMP, probably because it had become irreversibly complexed with active CL. Since the area of rounded cells corresponds to the site of increased cell proliferation in this and other animal models of tooth movement, these data suggest that collagenase production and cell proliferation might be correlated. We speculate that matrix degradation is an essential prerequisite for cell proliferation as it creates room to accommodate an increase in cell population.

The morphologic and biochemical effects of tensile force application to the interparietal suture of the Sprague-Dawley rat.

The purpose of this study was to correlate the histologic and biochemical responses of the interparietal suture to a range of tensile forces. Stainless steel spring implants, calibrated to generate expansive forces from 50 to 250 g, were placed across the interparietal suture in 85 female Sprague-Dawley rats. After experimental periods from 2 hours to 14 days, the interparietal sutures were evaluated by radiography, histology, and biochemistry. An in vivo/in vitro system was used for the biochemical analysis; total protein, proline incorporated, percent collagen, and alkaline phosphatase activity were measured. The radiographs and histology showed that in vivo suture expansion was achievable with 50 to 70 g of force, but the heavier forces showed greater sutural opening, more cellular proliferation, and more bone formation. This increased biologic response by the heavier forces was substantiated by an increase in sutural protein and alkaline phosphatase activity but not in percent collagen. It was concluded that changes in the total protein content of the suture were not primarily caused by proliferation of osteogenic cells and fibroblasts but due to an influx of transudate. In contrast, the increase in incorporation of 3H-proline and alkaline phosphatase activity correlated with the observance of bone formation. This study indicated a positive correlation between the magnitude of tensile forces and osteogenic response.

The use of in vitro models for investigating the response of fibrous joints to tensile mechanical stress.

To clarify the role of mechanical deformation in the remodeling of fibrous joints, organ culture systems have been developed to apply mechanical stress to cranial sutures under controlled experimental conditions. Tensile mechanical stress applied to cranial sutures from newborn rabbits produces a two- to threefold increase in protein synthesis and a twofold increase in collagen synthesis that can be detected within 6 hours. There is also a threefold increase in the DNA content of the sutures after 48 hours. Under normal conditions sutural fibroblasts synthesize type I collagen but respond to tensile deformation by synthesizing significant amounts of type III collagen. This suggests that the biomechanical environment of a connective tissue cell is an important determinant of the collagen type synthesized. However, the effect is likely to be an indirect one by virtue of its influence on the metabolic activity of the cells. Mechanically activated cells do not preferentially synthesize structural proteins, since mechanical stress stimulates the synthesis not only of structural macromolecules but also of the enzymes responsible for their specific hydrolysis. This is not accompanied by increased degradation, however, perhaps because the metalloproteinase inhibitor TIMP synthesized by the tissues is also increased. Confluent rabbit and mouse periosteal fibroblasts synthesize and release into the culture medium factors that can inhibit bone cell proliferation and stimulate bone resorption in vitro. It seems likely that further investigation of the interaction between fibroblasts and osteoblasts at the bone--fibrous tissue interface will require a reassessment of current thinking concerning the mechanisms regulating sutural osteogenesis.