Structural analysis of the frontal and parietal bones of the human skull.

Bone specimens were collected from the frontal and parietal bones of 4 adult, human skulls. The microstructure was characterized using microcomputed tomography (micro-CT) at about 6-µm resolution to map the change of porosity as a function of the depth, P(d), from the inner surface nearest to the brain to the outer surface nearest to the skin. A quantifiable method was developed using the measured P(d) to objectively distinguish between the three layers of the skull: the outer table, diploë , and inner table. The thickness and average porosity of each of the layers were then calculated from the measured porosity distributions, and a Gaussian function was fit to the P(d) curves. Morphological parameters were compared between the two bone types (frontal and parietal), while accounting for skull-to-skull variability. Parietal bones generally had a larger diploë accompanied by a thinner inner table. The arrangement of the porous vesicular structure within the outer table was also obtained with micro-CT scans with longer scan times, using enhanced parameters for higher resolution and lower noise in the images. From these scans, the porous structure of the bone appeared to be randomly arranged in the transverse plane, compared to the porous structure of the human femur, which is aligned in the loading direction.

Prickle1 regulates differentiation of frontal bone osteoblasts.

Enlarged fontanelles and smaller frontal bones result in a mechanically compromised skull. Both phenotypes could develop from defective migration and differentiation of osteoblasts in the skull bone primordia. The Wnt/Planar cell polarity (Wnt/PCP) signaling pathway regulates cell migration and movement in other tissues and led us to test the role of Prickle1, a core component of the Wnt/PCP pathway, in the skull. For these studies, we used the missense allele of Prickle1 named Prickle1Beetlejuice (Prickle1Bj). The Prickle1Bj/Bj mutants are microcephalic and develop enlarged fontanelles between insufficient frontal bones, while the parietal bones are normal. Prickle1Bj/Bj mutants have several other craniofacial defects including a midline cleft lip, incompletely penetrant cleft palate, and decreased proximal-distal growth of the head. We observed decreased Wnt/ß-catenin and Hedgehog signaling in the frontal bone condensations of the Prickle1Bj/Bj mutants. Surprisingly, the smaller frontal bones do not result from defects in cell proliferation or death, but rather significantly delayed differentiation and decreased expression of migratory markers in the frontal bone osteoblast precursors. Our data suggests that Prickle1 protein function contributes to both the migration and differentiation of osteoblast precursors in the frontal bone.

The Morphogenesis of Cranial Sutures in Zebrafish.

Using morphological, histological, and TEM analyses of the cranium, we provide a detailed description of bone and suture growth in zebrafish. Based on expression patterns and localization, we identified osteoblasts at different degrees of maturation. Our data confirm that, unlike in humans, zebrafish cranial sutures maintain lifelong patency to sustain skull growth. The cranial vault develops in a coordinated manner resulting in a structure that protects the brain. The zebrafish cranial roof parallels that of higher vertebrates and contains five major bones: one pair of frontal bones, one pair of parietal bones, and the supraoccipital bone. Parietal and frontal bones are formed by intramembranous ossification within a layer of mesenchyme positioned between the dermal mesenchyme and meninges surrounding the brain. The supraoccipital bone has an endochondral origin. Cranial bones are separated by connective tissue with a distinctive architecture of osteogenic cells and collagen fibrils. Here we show RNA in situ hybridization for col1a1a, col2a1a, col10a1, bglap/osteocalcin, fgfr1a, fgfr1b, fgfr2, fgfr3, foxq1, twist2, twist3, runx2a, runx2b, sp7/osterix, and spp1/ osteopontin, indicating that the expression of genes involved in suture development in mammals is preserved in zebrafish. We also present methods for examining the cranium and its sutures, which permit the study of the mechanisms involved in suture patency as well as their pathological obliteration. The model we develop has implications for the study of human disorders, including craniosynostosis, which affects 1 in 2,500 live births.

The BMP ligand Gdf6 prevents differentiation of coronal suture mesenchyme in early cranial development.

Growth Differentiation Factor-6 (Gdf6) is a member of the Bone Morphogenetic Protein (BMP) family of secreted signaling molecules. Previous studies have shown that Gdf6 plays a role in formation of a diverse subset of skeletal joints. In mice, loss of Gdf6 results in fusion of the coronal suture, the intramembranous joint that separates the frontal and parietal bones. Although the role of GDFs in the development of cartilaginous limb joints has been studied, limb joints are developmentally quite distinct from cranial sutures and how Gdf6 controls suture formation has remained unclear. In this study we show that coronal suture fusion in the Gdf6-/- mouse is due to accelerated differentiation of suture mesenchyme, prior to the onset of calvarial ossification. Gdf6 is expressed in the mouse frontal bone primordia from embryonic day (E) 10.5 through 12.5. In the Gdf6-/- embryo, the coronal suture fuses prematurely and concurrently with the initiation of osteogenesis in the cranial bones. Alkaline phosphatase (ALP) activity and Runx2 expression assays both showed that the suture width is reduced in Gdf6+/- embryos and is completely absent in Gdf6-/- embryos by E12.5. ALP activity is also increased in the suture mesenchyme of Gdf6+/- embryos compared to wild-type. This suggests Gdf6 delays differentiation of the mesenchyme occupying the suture, prior to the onset of ossification. Therefore, although BMPs are known to promote bone formation, Gdf6 plays an inhibitory role to prevent the osteogenic differentiation of the coronal suture mesenchyme.

Activation of FGF signaling mediates proliferative and osteogenic differences between neural crest derived frontal and mesoderm parietal derived bone.

BACKGROUND: As a culmination of efforts over the last years, our knowledge of the embryonic origins of the mammalian frontal and parietal cranial bones is unambiguous. Progenitor cells that subsequently give rise to frontal bone are of neural crest origin, while parietal bone progenitors arise from paraxial mesoderm. Given the unique qualities of neural crest cells and the clear delineation of the embryonic origins of the calvarial bones, we sought to determine whether mouse neural crest derived frontal bone differs in biology from mesoderm derived parietal bone. METHODS: BrdU incorporation, immunoblotting and osteogenic differentiation assays were performed to investigate the proliferative rate and osteogenic potential of embryonic and postnatal osteoblasts derived from mouse frontal and parietal bones. Co-culture experiments and treatment with conditioned medium harvested from both types of osteoblasts were performed to investigate potential interactions between the two different tissue origin osteoblasts. Immunoblotting techniques were used to investigate the endogenous level of FGF-2 and the activation of three major FGF signaling pathways. Knockdown of FGF Receptor 1 (FgfR1) was employed to inactivate the FGF signaling. RESULTS: Our results demonstrated that striking differences in cell proliferation and osteogenic differentiation between the frontal and parietal bone can be detected already at embryonic stages. The greater proliferation rate, as well as osteogenic capacity of frontal bone derived osteoblasts, were paralleled by an elevated level of FGF-2 protein synthesis. Moreover, an enhanced activation of FGF-signaling pathways was observed in frontal bone derived osteoblasts. Finally, the greater osteogenic potential of frontal derived osteoblasts was dramatically impaired by knocking down FgfR1. CONCLUSIONS: Osteoblasts from mouse neural crest derived frontal bone displayed a greater proliferative and osteogenic potential and endogenous enhanced activation of FGF signaling compared to osteoblasts from mesoderm derived parietal bone. FGF signaling plays a key role in determining biological differences between the two types of osteoblasts.

Transforming growth factor-beta1 stimulates chondrogenic differentiation of posterofrontal suture-derived mesenchymal cells in vitro.

BACKGROUND: Evidence from animal studies has associated transforming growth factor (TGF)-beta signaling with both normal and premature cranial suture fusion. However, the mechanisms whereby this pleiotropic cytokine mediates suture fusion remain uncertain. The authors established cultures of suture-derived mesenchymal cells from normally fusing (posterofrontal) and patent (sagittal) sutures and examined the in vitro effects of TGF-beta1 on these distinct cell populations. METHODS: Skulls were harvested from 80 5-day-old mice. Posterofrontal and sagittal sutures were dissected, and cultures of suture-derived mesenchymal cells were established. The mitogenic, osteogenic, and chondrogenic effects of recombinant TGF-beta1 were then assessed on posterofrontal and sagittal suture-derived mesenchymal cells (1 to 10 ng/ml). Quantitative real-time polymerase chain reaction was used to examine the effects of TGF-beta1 on gene expression. RESULTS: TGF-beta1 significantly decreased proliferation of both posterofrontal and sagittal suture-derived mesenchymal cells, by bromodeoxyuridine incorporation assays (n = 6). TGF-beta1 also inhibited osteogenesis in both suture-derived mesenchymal cells determined by alkaline phosphatase activity and mineralization (n = 3 for all assays). During chondrogenic differentiation, TGF-beta1 markedly increased expression of chondrocyte-specific gene markers in posterofrontal suture-derived mesenchymal cells (Sox9, Col II, Aggrecan, and Col X) (p

Molecular and cellular characterization of mouse calvarial osteoblasts derived from neural crest and paraxial mesoderm.

BACKGROUND: Cranial skeletogenic mesenchyme is derived from two distinct embryonic sources: mesoderm and cranial neural crest. Previous studies have focused on molecular and cellular differences of juvenile and adult osteoblasts. METHODS: To further understand the features of mouse-derived juvenile osteoblasts, the authors separated calvarial osteoblasts by their developmental origins: frontal bone-derived osteoblasts from cranial neural crest, and parietal bone-derived osteoblasts from paraxial mesoderm. Cells were harvested from a total of 120 mice. RESULTS: Interestingly, the authors observed distinct morphologies and proliferation potential of the two populations of osteoblasts. Osteogenic genes such as alkaline phosphatase, osteopontin, collagen I, and Wnt5a, which was recently identified as playing a role in skeletogenesis, were abundantly expressed in parietal bone-derived osteoblasts versus frontal bone-derived osteoblasts. In addition, fibroblast growth factor (FGF) receptor 2, and FGF-18 were more highly expressed in the parietal bone-derived osteoblasts, suggesting a more differentiated phenotype. In contrast, FGF-2, and adhesion molecules osteoblast cadherins and bone morphogenetic protein receptor IB, the bone tissue-specific type receptor were overexpressed in frontal bone-derived osteoblasts compared with parietal bone-derived osteoblasts. CONCLUSIONS: The authors observed that although neural crest-derived osteoblasts represented a population of less differentiated, faster growing cells, they formed bone nodules more rapidly than parietal bone-derived osteoblasts. This in vitro study suggests that embryonic tissue derivations influence postnatal in vitro calvarial osteoblast cell biology.

Concerted action of Msx1 and Msx2 in regulating cranial neural crest cell differentiation during frontal bone development.

The homeobox genes Msx1 and Msx2 function as transcriptional regulators that control cellular proliferation and differentiation during embryonic development. Mutations in the Msx1 and Msx2 genes in mice disrupt tissue-tissue interactions and cause multiple craniofacial malformations. Although Msx1 and Msx2 are both expressed throughout the entire development of the frontal bone, the frontal bone defect in Msx1 or Msx2 null mutants is rather mild, suggesting the possibility of functional compensation between Msx1 and Msx2 during early frontal bone development. To investigate this hypothesis, we generated Msx1(-/-);Msx2(-/-) mice. These double mutant embryos died at E17 to E18 with no formation of the frontal bone. There was no apparent defect in CNC migration into the presumptive frontal bone primordium, but differentiation of the frontal mesenchyme and establishment of the frontal primordium was defective, indicating that Msx1 and Msx2 genes are specifically required for osteogenesis in the cranial neural crest lineage within the frontal bone primordium. Mechanistically, our data suggest that Msx genes are critical for the expression of Runx2 in the frontonasal subpopulation of cranial neural crest cells and for differentiation of the osteogenic lineage. This early function of the Msx genes is likely independent of the Bmp signaling pathway.

TGFbeta-mediated FGF signaling is crucial for regulating cranial neural crest cell proliferation during frontal bone development.

The murine frontal bone derives entirely from the cranial neural crest (CNC) and consists of the calvarial (lateral) aspect that covers the frontal lobe of brain and the orbital aspect that forms the roof of bony orbit. TGFbeta and FGF signaling have important regulatory roles in postnatal calvarial development. Our previous study has demonstrated that conditional inactivation of Tgfbr2 in the neural crest results in severe defects in calvarial development, although the cellular and molecular mechanisms by which TGFbeta signaling regulates the fate of CNC cells during frontal bone development remain unknown. Here, we show that TGFbeta IIR is required for proliferation of osteoprogenitor cells in the CNC-derived frontal bone anlagen. FGF acts downstream of TGFbeta signaling in regulating CNC cell proliferation, and exogenous FGF2 rescues the cell proliferation defect in the frontal primordium of Tgfbr2 mutant. Furthermore, the CNC-derived frontal primordium requires TGFbeta IIR to undergo terminal differentiation. However, this requirement is restricted to the developing calvarial aspect of the frontal bone, whereas the orbital aspect forms despite the ablation of Tgfbr2 gene, implying a differential requirement for TGFbeta signaling during the development of various regions of the frontal bone. This study demonstrates the biological significance of TGFbeta-mediated FGF signaling cascade in regulating frontal bone development, suggests that TGFbeta functions as a morphogen in regulating the fate of the CNC-derived osteoblast and provides a model for investigating abnormal craniofacial development.

Increased presynaptic dopamine function in Asperger syndrome.

The etiology of Asperger syndrome is essentially unknown, but abnormality of the dopamine system has been shown in clinically overlapping disorders. The present study was designed to investigate the presynaptic dopamine function in Asperger syndrome. Eight healthy, drug-free males with Asperger syndrome and five healthy male controls were examined with positron emission tomography using 6-[18F]fluoro-L-DOPA ([18F]FDOPA) as a tracer. In the Asperger syndrome group, the [18F]FDOPA influx (Ki) values were increased in the striatum, i.e. in the putamen and caudate nucleus and in the frontal cortex. The results indicate that the dopamine system is affected in subjects with Asperger syndrome. Partially similar results have also been obtained in schizophrenia, suggesting an overlap not only of the clinical features but also of pathogenesis.

Experimental study of the frontal sinus development on Goettingen miniature pigs.

According to the literature, the development of the frontal sinus cavity is a result of the active immigration of cells from the ethmoidal complex into the os frontale. This migration theory is in contrast to the operative outcome of Apert's syndrome patients, after fronto-orbital advancement. When a fronto-orbital advancement at the age of a few months is performed in these patients while the frontal suture is yet closed, a sinus developed even the distance between nasal root and frontal bone bing up to 2 cm. In order to study the development of the frontal sinus, an animal study on 12 five-week-old infant Goettingen minipigs (GMP) was conducted, which did not have any clinical or histological signs of a frontal sinus development to investigate the development of the frontal sinus in "orthotopically" transplanted frontal bone with an open frontal suture. A comparison was made to a control group. The macro- and microscopical comparison with a control group revealed that the orthotopical transplants in the occipital bone developed epithelium-lined sinus, beginning from the thirty-fifth week. Based on these histomorphological results, a development scheme for the genesis of the sinus frontalis as a model were drawn.

Transient chondrogenic phase in the intramembranous pathway during normal skeletal development.

Calvarial and facial bones form by intramembranous ossification, in which bone cells arise directly from mesenchyme without an intermediate cartilage anlage. However, a number of studies have reported the emergence of chondrocytes from in vitro calvarial cell or organ cultures and the expression of type II collagen, a cartilage-characteristic marker, in developing calvarial bones. Based on these findings we hypothesized that a covert chondrogenic phase may be an integral part of the normal intramembranous pathway. To test this hypothesis, we analyzed the temporal and spatial expression patterns of cartilage characteristic genes in normal membranous bones from chick embryos at various developmental stages (days 12, 15 and 19). Northern and RNAse protection analyses revealed that embryonic frontal bones expressed not only the type I collagen gene but also a subset of cartilage characteristic genes, types IIA and XI collagen and aggrecan, thus resembling a phenotype of prechondrogenic-condensing mesenchyme. The expression of cartilage-characteristic genes decreased with the progression of bone maturation. Immunohistochemical analyses of developing embryonic chick heads indicated that type II collagen and aggrecan were produced by alkaline phosphatase activity positive cells engaged in early stages of osteogenic differentiation, such as cells in preosteogenic-condensing mesenchyme, the cambium layer of periosteum, the advancing osteogenic front, and osteoid bone. Type IIB and X collagen messenger RNAs (mRNA), markers for mature chondrocytes, were also detected at low levels in calvarial bone but not until late embryonic stages (day 19), indicating that some calvarial cells may undergo overt chondrogenesis. On the basis of our findings, we propose that the normal intramembranous pathway in chicks includes a previously unrecognized transient chondrogenic phase similar to prechondrogenic mesenchyme, and that the cells in this phase retain chondrogenic potential that can be expressed in specific in vitro and in vivo microenvironments.

Osteoclastic resorption of equine cranial and postcranial bone in vitro.

To address possible differences in the resorbability of cranial and postcranial bone, slices of equine frontal bone and leg (first phalanx or third metacarpus) were seeded with embryonic chick bone cells and cultured for 20-24h. After removing the cells and drying the specimens, the areas and volumes of more than 800 resorption pits in each set were measured using a video-rate reflection confocal microscope system. Relative mineralization densities were determined by quantitative electron backscattering analysis. The mean mineralization density was greater in the leg bone, but the mean depths for resorption pits in frontal bone were smaller (median volume/area ratios, experiment 1 and experiment 2: 1.98 microm frontal and 3.79 microm leg versus 2.70 microm and 4.20 microm, respectively; P < 0.0001, Mann-Whitney), even though the areas were greater in the frontal (medians, 286 microm2 and 324 microm2, versus 242 microm2 and 201 microm2; P < 0.0001). This study has shown a difference between cranial and postcranial equine bone in the shape and size of resorption pits formed in vitro. Overall, it has shown that cranial bone may be resorbed at least as readily as postcranial bone. This result is counter to the clinical impression that cranial bone has a greater staying power than postcranial bone when used as a grafting material.

Matrix mineralization and the differentiation of osteocyte-like cells in culture.

Osteocyte-like cells were prepared by sequentially treating calvaria from newborn rats with collagenase and chelating agents. On a reconstituted gel of basement membrane components, cells from the third collagenase digest displayed a round shape and expressed the highest level of alkaline phosphatase with minimal osteocalcin deposition into the matrix. On the other hand, cells derived from the interior after EDTA treatment exhibited well-developed dendritic cell processes and expressed essentially no alkaline phosphatase. The latter population also showed quite distinct characteristics such as higher extracellular activities of casein kinase II and ecto-5'-nucleotidase and the extracellular accumulation of a large amount of osteocalcin associated with mineral. These diverse phenotypic and protein expressions as well as the sites from which each population of cells were recovered strongly suggest that we have isolated osteoblastic and osteocytic cells. Bone sialoprotein II was extracellularly phosphorylated by casein kinase II in osteocytic cells but not in osteoblastic cells. We discuss the possibility that differentiation of young osteocytes from osteoblasts may facilitate the biochemical sequence of mineral deposition in the bone matrix.

Expression of collagen, osteocalcin, and bone alkaline phosphatase in a mineralizing rat osteoblastic cell culture.

Rat calvaria bone cells isolated by collagenase digestion form a bone-like matrix which mineralizes in vitro in the presence of beta-glycerophosphate, in less than 2 weeks. The purpose of this work was to investigate, in this mineralizing rat osteoblastic cell culture, the synthesis of collagen, osteocalcin, and bone alkaline phosphatase (ALP). The results obtained indicate (1) After 15 days in culture, the extracellular-matrix contains collagen type I, V, and to some extent type III. Metabolic labeling at day 14, during the phase of nodules mineralization as well as new nodules formation, shows that collagen types I and type V are synthesized; (2) During the phase of cell growth, no osteocalcin could be detected in the medium, however, at the point of nodule formation, the osteocalcin level reached values of 3.55 +/- 1.39 ng/ml, followed by a 30-fold increase after nodules became mineralized. At day 14, after metabolic labeling, de novo synthesized osteocalcin was chromatographed on an immunoadsorbing column. With urea-SDS PAGE the apparent molecular weight was determined to be 9,000 daltons. (3) Specific activity of ALP was found to be 10 nmol/min/mg of proteins at cell confluence. At day 15, when nodules are mineralized, this activity was increased by 40-fold. The Michaelis constant was 1.58 10(-3) M/L. ALP was inhibited by L-homoarginine and levamisole but not by L-phenylalanine. ALP was shown to be heat sensitive at 56 degrees C with two slopes of inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

[Histochemical study on effect of radix Salviae miltiorrhizae on growth of isolated cells from embryonic chicken frontal bone cultured in vitro].

12-day embryonic chicken frontal bone digested with trypsin to prepare the suspension of isolated bone cells. 3 x 10(6) cells were harvested altogether. The cells were divided equally into five parts. Then the Eagle medium and 0.1%, 0.2%, 0.4% and 0.8% Radix Salviae Miltiorrhizae in Eagle medium were added respectively and cultured in 5% CO2 incubator. It was observed under the inverted microscope every day. At the 26th day of culture, the cells were studied. The specimens were stained with H. E., Alcian Blue-Sirius Red, Alizarin Red S staining and alkaline phosphatase-acid phosphatase reaction for comparison. It was found that the maturation of the osteoblast-like cells could be accelerated by Radix Salviae Miltiorrhizae. Secretion of the collagenous substance, positive alkaline phosphatase reaction and deposition of mineral on the collagenous substance, forming bone nodules were found to be enhanced. But unduly high concentration of Radix Salviae Miltiorrhizae could lead to inhibition of osteoblast-like cell growth. The optimal concentration of Radix Salviae Miltiorrhizae was 0.2% in culture medium.

Differential serum dependence of cultured osteoclastic and osteoblastic bone cells.

Sequential collagenase digestion of mice calvariae provides populations of bone cells that express either osteoclasts (OC) or osteoblastic (OB) activities after growth for 6 days in similar culture conditions consisting of minimal essential medium supplemented with 10% fetal calf serum (FCS). The OC characteristics (acid phosphatase activity and hyaluronate synthesis, and their stimulation by PTH) were recovered in the cell populations released early from calvariae, but these also contained OB cells and numerous spindle-shaped alkaline phosphatase positive cells that resembled fibroblasts. We have attempted to select for growth of OC cells in these early populations by exploiting differences in growth requirements of OC, OB, and fibroblastic cells. We find that after growth for 6 days in low serum (2% FCS), OC cell populations demonstrated a threefold increase in OC activity/cell, and cell yield was reduced to one-third of that obtained in 10% FCS. Spindle-shaped cells were absent in 2% FCS and OB marker activities (alkaline phosphatase and citrate decarboxylation) were reduced threefold. In contrast to OC cells, high serum (10% FCS) favored the growth and phenotypic expression of OB cells (late populations). Cell yield and OB marker activities/cell were twofold higher in OB cells grown in 10% FCS vs 2% FCS, whereas growth but not phenotypic expression was retained at 5% FCS. These data suggest that differential serum dependence of OC and OB cells may provide a basis for further enrichment for each cell type following sequential digestion.

Ossification of the skull of the growing hamster. Autoradiographic observations.

Desmal ossification of the roof of the skull and of the alveolus of the incisor teeth of the mandible of growing hamsters and secondary ossification of the mandible were followed by autoradiography. Matrix production patterns as known from chondral ossification can be confirmed for desmal ossification as well. The secondary cartilage of the mandible shows an extremely low glycine turnover. These cells and the surrounding intercellular substance show different incorporation patterns as known for the epiphyseal cartilage of long bones. Secondary cartilage does not have growth functions for the mandible.

Fine structure of fetal rat calvarium; provisional identification of preosteoclasts.

This is a study of the fine structure of cells of the 20-day fetal rat calvarium. Special attention is given to identifying and characterizing preosteoclasts. These cells are relatively common and located largely, but not exclusively, at the endocranial bone surface. The preosteoclasts are characterized by abundant mitochondria, an incomplete perinuclear Golgi apparatus, and variable-shaped dense granules. The dense granules are unique in appearance in that they contain an internal dense matrix surrounded by a clear halo. Most granules are circular in shape but some are elongate or tubular in form. Granules with identical appearance are observed in osteoclasts. The preosteoclasts are mononucleate, or occasionally binucleate. It is suggested that because preosteoclasts are morphologically distinctive dna relatively abundant, it should be feasible to separate these cells from a heterogeneous cell isolate.

Effects of antimicrotubular agents on the fine structure of the Golgi complex in embryonic chick osteoblasts.

Embryonic chick frontal bones were cultured in the presence of colchicine or vinblastine and subsequently examined by tranmission electron microscopy. In control cultures the osteoblasts showed a large Golgi complex consisting of dictyosomes arranged in a well-defined juxtanuclear area. Microtubules were particularly numerous within this Golgi area although they could be observed throughout the cytoplasm. Colchicine and vinblastine caused the disappearance of cytoplasmic microtubules, while bundles of 10 nm diameter filaments appeared more frequently. In addition, cell polarity was lost and the Golgi complex became disorganized, with the dictyosomes randomly dispersed in the cytoplasm and showing a decreased number of cisternae and an increased number of vacuoles, the latter generally lacking stainable material. Increased number of autophagosomes were also noted. These findings indicate that microtubules function in the organization of the Golgi complex in osteoblasts. In view of the well documented role of this organelle system in collagen secretion it is suggested that previously observed secretory disturbances produced by antimicrotubular drugs may be due to a defective transfer of material to the dictyosomes and/or a defect in the packaging and transport of such material away from them.