Sutural mineralization of rat calvaria characterized by atomic-force microscopy and transmission electron microscopy

The application of transmission electron microscopy (TEM) and atomic-force microscopy (AFM) aid the acquisition of detailed structural information on the process of hard tissue formation. The sutural mineralization of rat calvaria is taken as a model for a collagen-related mineralization system. After cryofixation or chemical fixation an anhydrous tissue preparation technique with no staining procedures is used. The atomic-force microscope and the transmission electron microscope are used for structural analysis of the mineralizing region of the sutural tissue. With the application of AFM the collagen macroperiod is shown to be well represented in the unmineralized sutural tissue. At the mineralization front the collagen fibrils are found to be thickened and to change to a characteristic stacked platelet structure. Using TEM the macroperiod is faintly visible before mineral crystallites have formed and is more prominent after the apatite crystallization has started in the fibrils. In this step a needle-like structure of the newly formed apatitic crystals is visible.     

MT1-MMP-dependent, apoptotic remodeling of unmineralized cartilage: a critical process in skeletal growth

Skeletal tissues develop either by intramembranous ossification, where bone is formed within a soft connective tissue, or by endochondral ossification. The latter proceeds via cartilage anlagen, which through hypertrophy, mineralization, and partial resorption ultimately provides scaffolding for bone formation. Here, we describe a novel and essential mechanism governing remodeling of unmineralized cartilage anlagen into membranous bone, as well as tendons and ligaments. Membrane-type 1 matrix metalloproteinase (MT1-MMP)-dependent dissolution of unmineralized cartilages, coupled with apoptosis of nonhypertrophic chondrocytes, mediates remodeling of these cartilages into other tissues. The MT1-MMP deficiency disrupts this process and uncouples apoptotic demise of chondrocytes and cartilage degradation, resulting in the persistence of "ghost" cartilages with adverse effects on skeletal integrity. Some cells entrapped in these ghost cartilages escape apoptosis, maintain DNA synthesis, and assume phenotypes normally found in the tissues replacing unmineralized cartilages. The coordinated apoptosis and matrix metalloproteinase-directed cartilage dissolution is akin to metamorphosis and may thus represent its evolutionary legacy in mammals.     

Mechanics of cranial sutures using the finite element method

To investigate how cranial suture morphology and the arrangement of sutural collagen fibres respond to compressive and tensile loads, an idealised bone–suture–bone complex was analysed using a two-dimensional finite element model. Three suture morphologies were simulated with an increasing interdigitation index (I.I.): butt-ended, moderate interdigitated, and complex interdigitated. The collagen matrix within all sutures was modelled as an isotropic material, and as an orthotropic material in the interdigitated sutures with fibre alignment as reported in studies of miniature pigs. Static uniform compressive or tensile loading was applied to the complex. In interdigitated sutures with isotropic material properties, the orientation of the maximum (tensile) principal stresses within the suture matched the collagen fibre orientation observed in compressed and tensed sutures of miniature pigs. This suggests that randomly arranged sutural collagen fibres could optimise to an orientation most appropriate to withstand the predominant type of loading. A compression-resistant fibre arrangement imparted the highest suture strain energy relative to the isotropic and tension-resistant arrangements, indicating that this configuration maximises energy storage. A comparison across the different suture morphologies indicated that bone strain energy generally decreased with a decrease in I.I., irrespective of the sutural fibre arrangement. However, high bone stress at the interdigitation apices shifted to the limbs of the suture with an increase in I.I. These combined findings highlight the importance of suture morphology and anisotropy as properties having a significant influence on sutural mechanics.     

Matrix mineralization as a trigger for osteocyte maturation.

The morphology of the osteocyte changes during the cell's lifetime. Shortly after becoming buried in the matrix, an osteocyte is plump with a rich rough endoplasmic reticulum and a well-developed Golgi complex. This "immature" osteocyte reduces its number of organelles to become a "mature" osteocyte when it comes to reside deeper in the bone matrix. We hypothesized that mineralization of the surrounding matrix is the trigger for osteocyte maturation. To verify this, we prevented mineralization of newly formed matrix by administration of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) and then examined the morphological changes in the osteocytes in rats. In the HEBP group, matrix mineralization was disturbed, but matrix formation was not affected. The osteocytes found in the unmineralized matrix were immature. Mature osteocytes were seen in the corresponding mineralized matrix in the control group. The immature osteocytes in the unmineralized matrix failed to show immunoreactivity with anti-sclerostin antibody, whereas mature osteocytes in the mineralized matrix showed immunoreactivity in both control and HEBP groups. These findings suggest that mineralization of the matrix surrounding the osteocyte is the trigger for cytodifferentiation from a plump immature form to a mature osteocyte. The osteocyte appears to start secreting sclerostin only after it matures in the mineralized bone matrix.     

Sutural bone frequency in synostotic rabbit crania

This study tests the hypothesis that crania with synostosed sutures will have a significantly higher incidence of calvarial sutural bones than normal crania. Sutural bones were counted in seven calvarial sutures and compared among four groups of adult New Zealand white rabbit skulls: normal in-colony (NI) controls (N = 14), normal out-colony (NO) controls (N = 12), skulls with familial delayed onset (DO) coronal synostosis (N = 25), and skulls with experimentally immobilized coronal sutures (EI) (N = 20). Comparisons among groups were made with a Kruskal-Wallis one-way ANOVA and between groups with a Mann-Whitney U-test, using a Bonferroni correction for multiple comparisons. Significant differences (P > 0.05) were noted only in the coronal and sagittal sutures, with EI crania having the greatest number of coronal sutural bones; between group differences were undetectable for sagittal sutural bones. A post hoc two-sample binomial test for equal proportions showed that the distribution of coronal sutural bones among individuals across groups was even, while the distribution of sagittal sutural bones was significantly higher in EI crania. These results suggest that altered sutural forces of the calvaria contribute to an increased occurrence of sutural bones. However, the influence of inheritance on increased occurrence of sutural bones cannot be discounted, as reflected in the equivalent number of individuals across groups that possessed coronal sutural bones. Am J Phys Anthropol 102:555–563, 1997. © 1997 Wiley-Liss, Inc.     

A study of sutural bones in Anatolian-Ottoman skulls

In this study, a total of 302 adult Anatolian-Ottoman skulls were classified into three morphological forms (dolichocephalic, mesocephalic, brachycephalic), and were examined in order to find the incidence of sutural bones in each forms. In addition, the cranial capacity was measured in the skulls with and without sutural bones. Comparison of the incidence of sutural bones in each form of the skulls, and cranial capacity in skulls with and without sutural bones showed no significant difference. In most populations, the average measure of difference between Anatolian-Ottoman and the others was statistically significant. In conclusion, the incidence of sutural bones is well suited for comperative studies as an anthropological marker or an indicator of population distance. This study was presented in the 3rd National Anatomy Congress (6–9 Sept 1995) Izmir, Türkiye.     

A study of sutural bones in Gujarati (Indian) crania

370 adult crania were examined to find the incidence of sutural bones in Gujarati (Indian) crania and to compare it with other populations to establish the distance between them. The mean measure of difference between Indian and other populations was statistically significant. Comparison of cranial capacity in skulls with and without sutural bones showed no significant difference, and this is interpreted as indicating that sutural bones are not formed secondary to stress.     

Age-dependent properties and quasi-static strain in the rat sagittal suture

We measured the morphology of and performed tensile tests on sagittal sutures from rats of postnatal age 2 to 60 days. Using the properties measured ex vivo and a pressure vessel-based analysis, we estimated the quasi-static strain that had existed in the suture in vivo from 2 to 60 days. Sutural thickness, width, and stiffness per length were notable properties found to be age dependent. Sutural thickness increased 4.5-fold (0.11-0.50mm) between 2 and 60 days. Sutural width increased transiently between 2 and 20 days, peaking around 8 days; at 8 days, mean sutural width was 75% larger than mean sutural width at two days (0.35+/-0.07 (SD) vs. 0.20+/-0.06 mm). Sutural stiffness per length increased 4.4-fold (8.77-38.3N/mm/mm) between 2 and 60 days. The quasi-static sutural strain estimated to exist in vivo averaged 270+/-190 muepsilon between 2 and 60 days and was not age dependent. These findings provide data on the age-dependent sutural properties of infant to mature rats and provide the first estimate of quasi-static sutural strain in vivo in the rat. The findings show that during development the rat sagittal suture, as a structure, changes significantly and is exposed to quasi-static tensile strain in vivo due to intracranial pressure.     

Comparative morphology and functional interpretation of the sutures in the dermal skull roof of temnospondyl amphibians

Sutures in the dermal skull roof of several Palaeozoic temnospondyl amphibians were studied, including Archegosaurus decheni, Sclerocephalus haeuseri, Cheliderpeton latirostre (Archegosauridae), Acanthostomatops vorax (Zatrachydidae), Onchiodon labyrinthicus (Eryopidae), Micromelerpeton credneri and Branchierpeton amblystomum (Dissorophoidea). Lamellae, flat bevels, butt joints, steep walls, and grooves are the sutural types occurring. Morphological sutural differences cannot be used as taxonomically relevant characters, as they mainly differ in their functional manifestations. Similarities of sutural patterns in taxa not closely related are most probably based on convergence, therefore sutural morphology allows functional conclusions rather than revealing phylogenetic relationships. The basic sutural pattern is described in Micromelerpeton credneri. Different textures and obliquity patterns of the sutural surfaces are characteristic of each region of the skull roof. In all temnospondyls studied, the median plane and the margin of the skull roof show a nearly uniform sutural morphology; the circumorbital region is most complex. Although the skulls studied are akinetic, mesokinetic movements of dermal elements along their sutures must have been permitted to guarantee the dynamic stability of the skull construction.     

Midpalatal suture of osteopetrotic (op/op) mice exhibits immature fusion.

The midpalatal suture was observed histologically in both toothless osteopetrotic (op/op) and normal (control) mice. The normal mice had a mature sutural structure, which consists of a well-developed cartilage cell zone and palatal bone. In contrast, the thickness of the cartilage cell zone was substantially greater in the op/op mice than that in the controls. Moreover, the cartilage cells in the op/op mice were frequently found in the palatal bone as well as in the sutural space, exhibiting an imperfect fusion. It seems that immature fusion at the sutural interface in the op/op mice is related to a decrease in biting or masticatory force accompanied by the failure of tooth eruption in addition to an essential defect in osteoclast differentiation, which is a congenital symptom in op/op mice.     

Sutural complexity in artificially deformed human (Homo sapiens) crania.

The pattern of complexity of cranial sutures is highly variable both among and within species. Intentional cranial vault deformation in human populations provides a controlled natural experiment by which we were able to quantify aspects of sutural complexity and examine the relationship between sutural patterns and mechanical loading. Measures of sutural complexity (interdigitation, number, and size of sutural bones) were quantified from digitized tracings of 13 sutures and compared among three groups of crania (n = 70) from pre-European contact Peru. These groups represent sample populations deformed in 1) anteroposterior (AP) and 2) circumferential (C) directions and 3) an undeformed population. Intergroup comparisons show few differences in degree or asymmetry of sutural interdigitation. In the few comparisons which show differences, the C group is always more interdigitated than the other two while the AP group has more sutural bones. The sutures surrounding the temporal bone (sphenotemporal, occipitotemporal, and temporoparietal) most frequently show significant differences among groups. These differences are related to the more extreme binding of C type deformation and are consistent with hypothesized increases in tension at coronally oriented sutures in this group. The larger number of sutural bones in the AP group is consistent with the general broadening of the cranium in this group and with experimental evidence indicating the development of ossicles in areas of tension. We suggest that so few changes in sutural complexity occurred either because the magnitude of the growth vectors, unlike their direction, is not substantially altered or because mechanisms other than sutural growth modification are responsible for producing the altered vault shapes. In addition, the presence of fontanelles in the infant skulls during binding and the static nature of the binding may have contributed to the similarity in complexity among groups.     

Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones

Bone development requires the recruitment of osteoclast precursors from surrounding mesenchyme, thereby allowing the key events of bone growth such as marrow cavity formation, capillary invasion, and matrix remodeling. We demonstrate that mice deficient in gelatinase B/matrix metalloproteinase (MMP)-9 exhibit a delay in osteoclast recruitment. Histological analysis and specialized invasion and bone resorption models show that MMP-9 is specifically required for the invasion of osteoclasts and endothelial cells into the discontinuously mineralized hypertrophic cartilage that fills the core of the diaphysis. However, MMPs other than MMP-9 are required for the passage of the cells through unmineralized type I collagen of the nascent bone collar, and play a role in resorption of mineralized matrix. MMP-9 stimulates the solubilization of unmineralized cartilage by MMP-13, a collagenase highly expressed in hypertrophic cartilage before osteoclast invasion. Hypertrophic cartilage also expresses vascular endothelial growth factor (VEGF), which binds to extracellular matrix and is made bioavailable by MMP-9 (Bergers, G., R. Brekken, G. McMahon, T.H. Vu, T. Itoh, K. Tamaki, K. Tanzawa, P. Thorpe, S. Itohara, Z. Werb, and D. Hanahan. 2000. Nat. Cell Biol. 2:737-744). We show that VEGF is a chemoattractant for osteoclasts. Moreover, invasion of osteoclasts into the hypertrophic cartilage requires VEGF because it is inhibited by blocking VEGF function. These observations identify specific actions of MMP-9 and VEGF that are critical for early bone development.     

A Simple Histological Method for Identification of Osteoid Matrix in Decalcified Bone

The present experiment indicated that cyanuric chloride fixation was very useful in identifying osteoid matrix, which is difficult to distinguish from mineralized matrix in sections decalcified in the routine fashion. Small slices of bone from 3 mm to 5 mm thick were fixed with 0.5% cyanuric chloride in methanol containing 1% N-methyl morpholine for from 1 to 2 days at room temperature. EDTA decalcified sections were prepared and stained with hematoxylin and eosin. The regions presumed to be osteoid matrix were intensely eosinophilic. It was shown that the eosinophilic regions correspond precisely to the unmineralized osteoid matrix which was radiolucent by microradiography and devoid of silver by the von Kossa method in undecalcified serial sections.     

The effect of unilateral partial facial paralysis on nasofrontal sutural growth: an experimental study in the rabbit.

In a previous study in the rabbit, it was demonstrated that paralysis of the midfacial musculature results in decreased anteroposterior growth of the snout. At the end of growth, these animals showed macroscopically striking similarities to animals with unilateral fusion of the nasofrontal suture. In this study, whether nasofrontal sutural growth is unilaterally restricted in animals with unilateral partial facial paralysis was investigated. A left-sided partial facial paralysis was induced in sixteen 12-day-old New Zealand White rabbits. At the ages of 5, 9, 12, and 17 weeks, four animals were randomly assigned to be killed for analysis of nasofrontal sutural growth. In each animal, the left experimental side was compared with the right control side. By means of histomorphometric measurements, it was shown that diminished sutural growth activity was present on the left paralyzed side in periods of rapid growth. On the other hand, no significant alterations in sutural width were found. These findings seem to explain some of the macroscopic growth alterations (i.e., diminished anterior maxillary length) observed in rabbits with unilateral partial facial paralysis.     

Vertical distribution of elements in cells and matrix of epiphyseal growth plate cartilage determined by quantitative electron probe analysis

Quantitative electron probe analysis was performed on chick epiphyseal growth cartilage prepared by two anhydrous methods, ultrathin cryosections and freeze-dried epoxy-embedded tissue. Levels of Na, Mg, P, S, Cl, K, and Ca were determined in cytoplasm, mitochondria, extracellular matrix, matrix vesicles, and mineral nodules in four zones of the cartilage--proliferative, prehypertrophic, early hypertrophic, and early calcification. The exceptionally high levels of Na and K (up to 550 and 200 mmol/kg wet wt, respectively) found in the matrix are believed to be largely bound to fixed anions. Within cells, Na was higher than K (140 versus 20-34 mmol/kg wet wt), a condition that may reflect hypoxia. Ca and P were low in cells and unmineralized matrix. Ca and P were high in mitochondrial granules of the early hypertrophic zone and diminished in amount in the calcifying zone; the converse occurred in matrix vesicles. Mg was low to undetectable except in heavily mineralized structures (i.e., mitochondrial granules, matrix vesicles, and mineral nodules). S levels were high in matrix (approximately 400 mmol/kg wet wt) and increased slightly with maturation. The amount of S present greatly exceeds Ca levels and implies that sulfate, the predominant form of sulfur in proteoglycans, may serve as an ion-exchange mechanism for the passage of Ca through the matrix to sites where Ca and phosphate are precipitated.     

Morphostructural constraints and phylogenetic overprint on sutural frilling in Late Jurassic ammonites

The functional significance of frilled septa and complex sutures in ammonoids has generated ongoing debate. The 'classic' hypothesis envisages ammonoid shells and septa as designed for resisting ambient hydrostatic pressure, complex sutures being the evidence of strength in shells for colonization of deep habitats. Here we address the 'suture problem', focusing on the analysis and interpretation of variables in our database of Late Jurassic ammonites not included in previous studies, such as whorl height ( W h ), whorl shape ( S ), shell coiling ( WD ), taxonomic grouping and basic planispiral shell shape. The results indicate that sutural complexity, as measured by the fractal dimension ( D f ) value of the suture line, is positively correlated with W h , and that the sutures of oceanic shells tend to provide, for a given W h value, lower D f estimates than do those of neritic shells. No general trend of increase in sutural complexity was noted for specimens recovered from swell areas belonging to oceanic fringes with respect to those that inhabited epicontinental shelves. In fact, Perisphinctoidea, the clade best represented in the database analysed, shows a higher D f mean value in neritic species than in epioceanic ones. Significant differences in sutural complexity were detected for groups of ammonites classified according to shell shapes ( WD , S ). Oxycones and discocones, streamlined potential swimmers, show the highest D f mean values, while spherocones and cadicones, which were presumably vertical vagrants, present the lowest ones. This indicates that sutural complexity was more related to shell geometry than to bathymetry.     

Fusion Patterns in the Skulls of Modern Archosaurs Reveal That Sutures Are Ambiguous Maturity Indicators for the Dinosauria

The sutures of the skulls of vertebrates are generally open early in life and slowly close as maturity is attained. The assumption that all vertebrates follow this pattern of progressive sutural closure has been used to assess maturity in the fossil remains of non-avian dinosaurs. Here, we test this assumption in two members of the Extant Phylogenetic Bracket of the Dinosauria, the emu, Dromaius novaehollandiae and the American alligator, Alligator mississippiensis, by investigating the sequence and timing of sutural fusion in their skulls. As expected, almost all the sutures in the emu skull progressively close (i.e., they get narrower) and then obliterate during ontogeny. However, in the American alligator, only two sutures out of 36 obliterate completely and they do so during embryonic development. Surprisingly, as maturity progresses, many sutures of alligators become wider in large individuals compared to younger, smaller individuals. Histological and histomorphometric analyses on two sutures and one synchondrosis in an ontogenetic series of American alligator confirmed our morphological observations. This pattern of sutural widening might reflect feeding biomechanics and dietary changes through ontogeny. Our findings show that progressive sutural closure is not always observed in extant archosaurs, and therefore suggest that cranial sutural fusion is an ambiguous proxy for assessing maturity in non-avian dinosaurs.     

Biochemical and immunocytochemical characterization of mineral binding proteoglycans in rat bone

We examined biochemically and immunocytochemically the type and distribution of mineral binding proteoglycans (PGs) in rat mid-shaft subperiosteal bone using three monoclonal antibodies (MAb 1-B-5, 9-A-2, and 3-B-3) which specifically recognize unsulfated chondroitin, chondroitin 4-sulfate (C4-S) and dermatan sulfate (DS), and chondroitin 6-sulfate. Bone proteins were extracted from fresh specimens with a three-step technique: 4 M guanidine HCl (GdnCl), aqueous EDTA without GdnCl (E-extract), followed by GdnCl. Western blot analysis of SDS-polyacrylamide gel electrophoresis revealed that E-extract after chondroitinase ABC digestion reacted strongly with MAb 9-A-2 but not with MAb 1-B-5 or 3-B-3. After adehyde fixation, ethanolic trimethylammonium EDTA was used as a demineralizing agent for light and electron immunocytochemistry. This provided good retention of water-soluble PGs in the specimens. After chondroitinase ABC pre-treatment of tissue sections, MAb 9-A-2 specifically stained C4-S and/or DS in the walls of osteocyte lacunae and bone canaliculi in the mineralized matrix as well as in the unmineralized matrix such as pre-bone, vascular canals, and pericellular matrix surrounding osteocytes; the remainder of the mineralized matrix lacked staining. These results indicate that mineral binding PGs contain C4-S and/or DS and are exclusively localized in the walls of the bone lacuna and canaliculus.     

Evaluation of cranial bone suture autotransplants in the growing rabbit

A coronal bone suture segment was autotransplanted into an experimentally created defect in the nongrowing portion of the nasal bone in 12 5-week-old male New Zealand white rabbits. The animals were sacrificed 90 days postoperatively. In most specimens the transplants were well incorporated into the recipient site. The transplanted sutures appeared narrower radiographically, and the bony projections of the sutures were not as long as those seen in control sutures. In each case the transplanted sutural ligament had atrophied, and six of the transplants showed bony union across the sutural space. The results suggest that growth and biomechanical stresses are important for maintenance of the sutural ligament and for the stimulation of bone deposition.     

A study of sutural bones in different morphological forms of skulls

117 adult human skulls were classified for three morphological forms, i.e. dolichocephalic, mesocephalic and brachycephalic, and were examined for the presence of sutural bones in each form. Sutural bones occur in each forms of the skulls with no statistically significant differences. This finding is interpreted as indicating that sutural bones are not formed secondary to stress, otherwise the incidence of the sutural bones would have been differed in different morphological forms of skulls.