Deposition of collagen type I onto skeletal endothelium reveals a new role for blood vessels in regulating bone morphology.

Recently, blood vessels have been implicated in the morphogenesis of various organs. The vasculature is also known to be essential for endochondral bone development, yet the underlying mechanism has remained elusive. We show that a unique composition of blood vessels facilitates the role of the endothelium in bone mineralization and morphogenesis. Immunostaining and electron microscopy showed that the endothelium in developing bones lacks basement membrane, which normally isolates the blood vessel from its surroundings. Further analysis revealed the presence of collagen type I on the endothelial wall of these vessels. Because collagen type I is the main component of the osteoid, we hypothesized that the bone vasculature guides the formation of the collagenous template and consequently of the mature bone. Indeed, some of the bone vessels were found to undergo mineralization. Moreover, the vascular pattern at each embryonic stage prefigured the mineral distribution pattern observed one day later. Finally, perturbation of vascular patterning by overexpressing Vegf in osteoblasts resulted in abnormal bone morphology, supporting a role for blood vessels in bone morphogenesis. These data reveal the unique composition of the endothelium in developing bones and indicate that vascular patterning plays a role in determining bone shape by forming a template for deposition of bone matrix.

Bone matrix calcification during embryonic and postembryonic rat calvarial development assessed by SEM-EDX spectroscopy, XRD, and FTIR spectroscopy.

Bone mineral is constituted of biological hydroxyapatite crystals. In developing bone, the mineral crystal matures and the Ca/P ratio increases. However, how an increase in the Ca/P ratio is involved in maturation of the crystal is not known. The relationships among organic components and mineral changes are also unclear. The study was designed to investigate the process of calcification during rat calvarial bone development. Calcification was evaluated by analyzing the atomic distribution and concentration of Ca, P, and C with scanning electron microscopy (SEM)-energy-dispersive X-ray (EDX) spectroscopy and changes in the crystal structure with X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy. Histological analysis showed that rat calvarial bone formation started around embryonic day 16. The areas of Ca and P expanded, matching the region of the developing bone matrix, whereas the area of C became localized around bone. X-ray diffraction and FTIR analysis showed that the amorphous-like structure of the minerals at embryonic day 16 gradually transformed into poorly crystalline hydroxyapatite, whereas the proportion of mineral to protein increased until postnatal week 6. FTIR analysis also showed that crystallization of hydroxyapatite started around embryonic day 20, by which time SEM-EDX spectroscopy showed that the Ca/P ratio had increased and the C/Ca and C/P ratios had decreased significantly. The study suggests that the Ca/P molar ratio increases and the proportion of organic components such as proteins of the bone matrix decreases during the early stage of calcification, whereas crystal maturation continues throughout embryonic and postembryonic bone development.

Embryonic ablation of osteoblast Smad4 interrupts matrix synthesis in response to canonical Wnt signaling and causes an osteogenesis-imperfecta-like phenotype.

To examine interactions between bone morphogenic protein (BMP) and canonical Wnt signaling during skeletal growth, we ablated Smad4, a key component of the TGF-ß-BMP pathway, in Osx1(+) cells in mice. We show that loss of Smad4 causes stunted growth, spontaneous fractures and a combination of features seen in osteogenesis imperfecta, cleidocranial dysplasia and Wnt-deficiency syndromes. Bones of Smad4 mutant mice exhibited markers of fully differentiated osteoblasts but lacked multiple collagen-processing enzymes, including lysyl oxidase (Lox), a BMP2-responsive gene regulated by Smad4 and Runx2. Accordingly, the collagen matrix in Smad4 mutants was disorganized, but also hypomineralized. Primary osteoblasts from these mutants did not mineralize in vitro in the presence of BMP2 or Wnt3a, and Smad4 mutant mice failed to accrue new bone following systemic inhibition of the Dickkopf homolog Dkk1. Consistent with impaired biological responses to canonical Wnt, ablation of Smad4 causes cleavage of ß-catenin and depletion of the low density lipoprotein receptor Lrp5, subsequent to increased caspase-3 activity and apoptosis. In summary, Smad4 regulates maturation of skeletal collagen and osteoblast survival, and is required for matrix-forming responses to both BMP2 and canonical Wnt.

Apoptotic and anti-apoptotic factors in early human mandible development.

The regulators of apoptosis Bcl-2, Bax, caspase-3, p53, and Hsp70 were analyzed immunohistochemically in the developing human mandible of eight human conceptuses from weeks 5 to 10 of gestation. During this period, all proteins displayed an increased pattern of expression in the mandible ectomesenchyme and in newly formed bone, except for caspase-3, which showed decreased expression in the ectomesenchyme, but appeared first in the ossification zone at the 7th wk of development. Simultaneously, the oral epithelium showed weak (p53) to strong (hsp70) expression of all proteins investigated, while in Meckel's cartilage cells, bcl-2 was expressed weakly and hsp70 was expressed moderately. Cells on the surface of the forming bone were predominantly bax positive, and only occasionally bcl-2 positive. Only a few cells on the surface and inside the bony spicules co-expressed bax and bcl-2. Terminal deoxynucleotidyl transferase (TdT)-mediated biotin-dUTP nick-end labelling (TUNEL)-positive cells were found to be apoptotic osteoblasts. The expression of all proteins investigated changed dynamically during early mandible development and the subsequent differentiation of Meckel's cartilage and bone. While interactions between those factors might be associated with the survival of Meckel's cartilage, in the ossification zone they might participate in the control of cell numbers, mineralization, and bone remodelling. Among many other factors, precise orchestration of pro- and anti-apoptotic factors contributes to normal mandible development.

Fetal bovine bone cells synthesize bone-specific matrix proteins.

We isolated cells from both calvaria and the outer cortices of long bones from 3- to 5-mo bovine fetuses. The cells were identified as functional osteoblasts by indirect immunofluorescence using antibodies against three bone-specific, noncollagenous matrix proteins (osteonectin, the bone proteoglycan, and the bone sialoprotein) and against type 1 collagen. In separate experiments, confluent cultures of the cells were radiolabeled and shown to synthesize and secrete osteonectin, the bone proteoglycan and the bone sialoprotein by immunoprecipitation and fluorography of SDS polyacrylamide gels. Analysis of the radiolabeled collagens synthesized by the cultures showed that they produced predominantly (approximately 94%) type I collagen, with small amounts of types III and V collagens. In agreement with previous investigators who have employed the rodent bone cell system, we confirmed in bovine bone cells that (a) there was a typical cyclic AMP response to parathyroid hormone, (b) freshly isolated cells possessed high levels of alkaline phosphatase, which diminished during culture but returned to normal levels in mineralizing cultures, and (c) cells grown in the presence of ascorbic acid and beta-glycerophosphate rapidly produced and mineralized an extracellular matrix containing largely type I collagen. These results show that antibodies directed against bone-specific, noncollagenous proteins can be used to clearly identify bone cells in vitro.

Maspin is involved in bone matrix maturation by enhancing the accumulation of latent TGF-beta.

UNLABELLED: Maspin, a serine protease inhibitor, is expressed by formative osteoblasts. The repression of maspin expression in osteoblastic cells decreased the level of latent TGF-beta in the extracellular matrix, whereas the overexpression of maspin increased latent TGF-beta. These findings suggest that maspin plays an important role in bone matrix formation, particularly in the accumulation of latent TGF-beta. INTRODUCTION: Maspin is a serine protease inhibitor that exhibits tumor suppressive and anti-angiogenic activities. This study was performed to elucidate a possible role for maspin in bone formation. MATERIALS AND METHODS: We performed immunohistochemical analysis of the expression of maspin during endochondral ossification. We evaluated the expression of maspin mRNA and protein in ROS 17/2.8 cells and primary rat osteoblastic cells by RT-PCR, immunocytochemistry, and Western blot analysis. We also examined the accumulation of TGF-beta in the extracellular matrix of cultured ROS 17/2.8 cells after transfection with vectors expressing either maspin or maspin antisense. RESULTS: We observed expression of maspin by active osteoblasts in vivo. Rat osteoblastic cells also expressed maspin mRNA and protein in vitro. Moreover, the accumulation of latent TGF-beta in the extracellular matrix significantly decreased in cultures exposed to an anti-maspin antibody and when cells were transfected with a maspin antisense-expressing vector. In contrast, accumulation of latent TGF-beta in the extracellular matrix increased after transfection of cells with a vector expressing maspin. CONCLUSIONS: These findings suggest that maspin expressed in active osteoblasts plays an important physiological role during maturation of the bone matrix, and in particular, during the process of accumulation of latent TGF-beta in the extracellular matrix.

The presence of PHOSPHO1 in matrix vesicles and its developmental expression prior to skeletal mineralization.

PHOSPHO1 is a phosphoethanolamine/phosphocholine phosphatase that has previously been implicated in generating inorganic phosphate (P(i)) for matrix mineralization. In this study, we have investigated PHOSPHO1 mRNA expression during embryonic development in the chick. Whole-mount in situ hybridization indicated that PHOSPHO1 expression occurred prior to E6.5 and was initially restricted to the bone collar within the mid-shaft of the diaphysis of long bones but by E11.5 expression was observed over the entire length of the diaphysis. Alcian blue/alizarin red staining revealed that PHOSPHO1 expression seen in the primary regions of ossification preceded the deposition of mineral, suggesting that it is involved in the initial events of mineral formation. We isolated MVs from growth plate chondrocytes and confirmed the presence of high levels of PHOSPHO1 by immunoblotting. Expression of PHOSPHO1, like TNAP activity, was found to be up-regulated in MVs isolated from chondrocytes induced to differentiate by the addition of ascorbic acid. This suggests that both enzymes may be regulated by similar mechanisms. These studies provide for the first time direct evidence that PHOSPHO1 is present in MVs, and its developmental expression pattern is consistent with a role in the early stages of matrix mineralization.

Embryonic bone matrix formation is increased after exposure to a low-amplitude capacitively coupled electric field, in vitro.

In order to investigate the mechanism(s) of electric field-stimulated osteogenesis, we have developed an in vitro model in which embryonic chick tibiae have consistently demonstrated increased bone matrix formation in response to a low amplitude (estimated 10(-5) V/m in the serum-free culture medium), capacitively coupled, 10 Hz sinusoidal electric field. Initial applications of this model revealed that 72 h of continuous exposure to the electric field increased tibial collagen production by 29% compared to untreated controls, P less than 0.01. Additional studies further revealed: (a) that when electric field exposure was limited to 30 min/day during the 72 h in vitro incubation, embryonic bone matrix formation was increased by 83%, compared to non-treated controls (P less than 0.001), suggesting an inductive mechanism; (b) that the osteogenic response to electric field exposure in vitro was not unique to embryonic chick tibiae, since a similar response was also seen with newborn mouse calvaria (+133%, P less than 0.02); (c) that electric field-exposure-stimulated chick bone matrix formation was associated with increased bone cell proliferation; and (d) that this mitogenic response to in vitro electric field exposure could also be observed with embryonic chick calvarial cells in monolayer, serum-free cultures.

A molecular analysis of matrix remodeling and angiogenesis during long bone development.

The replacement of cartilage by bone is the net result of genetic programs that control chondrocyte differentiation, matrix degradation, and bone formation. Disruptions in the rate, timing, or duration of chondrocyte proliferation and differentiation result in shortened, misshapen skeletal elements. In the majority of these skeletal disruptions, vascular invasion of the elements is also perturbed. Our hypothesis is that the processes involved in endochondral ossification are synchronized via the vasculature. The purpose of this study was to examine carefully the events of vascular invasion and matrix degradation in the context of chondrocyte differentiation and bone formation. Here, we have produced a 'molecular map' of the initial vascularization of the developing skeleton that provides a framework in which to interpret a wide range of fetal skeletal malformations, disruptions, and dysplasias.

Relationships between cellular condensation, preosteoblast formation and epithelial-mesenchymal interactions in initiation of osteogenesis.

Initiation of osteogenesis or bone formation is dependent on cell and tissue interactions. We investigated the events between 4 and 7 days of incubation that translate epithelial-mesenchymal signalling into overt differentiation of osteoblasts and deposition of bone in the mandibles of chick embryos. Condensation of mandibular mesenchyme (the membranous skeleton), visualized with PNA-lectin, occurred at H.H. mid-26 (5.75 days), lasted 12 h and preceded osteoblast differentiation by 1.5 days. As determined from 3D-reconstruction all mandibular membrane bones arose from a single condensation closely associated with the stomodeal epithelium. The finding that the osteogenic condensation in the mandibular arch is a major branch of a common condensation that provides osteogenic mesenchyme to both maxillary and mandibular arches establishes a closer link between mechanisms controlling development of the skeleton in these two arches than previously suspected. Preosteoblasts (alkaline phosphatase-positive cells) form in the mandible at H.H. early 25, which is before condensation but after the epithelial-mesenchymal interaction upon which preosteoblast formation and condensation depend--neither form in isolated mesenchyme, whereas both form after recombination of mesenchyme and epithelium. Tenascin was present in the mandibular epithelium only at H.H. stage 19 but not in the mesenchyme at any age. Therefore, the epithelial-mesenchymal interaction controls initiation of osteogenesis at the preosteoblast stage. Preosteoblasts then condense, transform into osteoblasts and deposit bone matrix. Differentiation of preosteoblasts precedes condensation which amplifies their number. This is in contrast with chondrogenesis where condensation triggers prechondroblast differentiation.

The primary calcification in bones follows removal of decorin and fusion of collagen fibrils.

To elucidate the mechanisms of primary calcification in bone, ultrastructural changes in collagen fibrils, as well as cytochemical alteration of proteoglycan, especially decorin, were investigated morphologically in 19-day postcoitum embryonic rat calvariae. Below the osteoblast layer, calcification of the osteoid area increased in direct proportion to its distance from the osteoblasts. In the uncalcified osteoid area, collagen fibrils near matrix vesicles possessed sharp contours and were a uniform 50 nm in diameter. Immunoelectron microscopy revealed decorin to be abundantly localized in the vicinity of the collagen fibrils. In the osteoid area undergoing the process of calcification, collagen fibrils tended to fuse side by side. Where calcification was progressed, this fusion was even more so. Some very large fibrils exhibited complicated contours, 400 nm or more in diameter. Although the calcification at this stage affected areas both inside and outside of the collagen fibrils, the interior areas manifested a lower density of calcification. The immunolocalization of decorin was also much decreased around these fibrils. Thus, primary calcification in bone matrix follows the removal of decorin and fusion of collagen fibrils. This phenomenon may aid in the process of calcification and bone formation, because (1) inhibitors of calcification, such as decorin, are removed, (2) the fusion of collagen fibrils provides the room necessary for rapid growth of mineral crystals, and (3) the soft elastic bone matrix containing abundant fused collagen fibrils less subjective to calcification is safe for both maternal and embryonic bodies and is convenient for subsequent bone remodeling.

Stimulation of bone matrix apposition in vitro by local growth factors: a comparison between insulin-like growth factor I, platelet-derived growth factor, and transforming growth factor beta.

Many recent in vitro studies have shown effects of insulin-like growth factor I (IGF I), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF beta) on the proliferation and differential functions of bone-forming osteoblasts; however, the question whether these factors might ultimately lead to a net increase or decrease in bone formation has been difficult to assess. In this study, we have used an autoradiographic method based on the incorporation of [3H]proline into freshly synthesized bone matrix to determine the overall effects of these factors on bone matrix apposition in 21-day-old fetal rat calvariae. IGF I, PDGF, and TGF beta increased bone matrix apposition in a dose-dependent manner up to 2-fold within 48 h. In addition, they partially or completely reversed the inhibition of bone matrix apposition observed with PTH. Exogenously added TGF beta was significantly more potent than equimolar concentrations of PDGF or IGF I in stimulating bone formation. Matrix apposition was greatest when IGF I, PDGF, and TGF beta were added simultaneously to the culture medium, indicating that these factors can enhance each other in stimulating bone formation. In conclusion, our results provide direct evidence that IGF I, PDGF, and TGF beta are capable of stimulating bone formation in vitro.

Lumican is a major proteoglycan component of the bone matrix.

MC3T3-E1 mouse calvaria cells are a clonal population of committed osteoprogenitors that in the presence of appropriate supplements form a mineralized bone matrix. The development of the MC3T3-E1 cells can be divided into three major stages, namely, proliferation, differentiation, and mineralization. Recently, using the cDNA microarray technology we found lumican to be abundantly expressed during the mineralization and differentiation stages of the MC3T3-E1 development and not during the proliferation stage. Lumican has been shown to play essential roles in regulating collagen fibril formation in different extracellular matrices but its expression in the developing bone matrix remains elusive. By examining the expression profile of this gene during the different stages of MC3T3-E1 development, utilizing the 'real-time' PCR technology, we observed that the expression of lumican increases as the osteoblast culture differentiates and matures, suggesting that lumican may be involved in regulating collagen fibrillogenesis in bone matrices. Using immunostaining, we observed that during the early embryonic development of mouse (E11 to E13), lumican is mainly expressed in the cartilaginous matrices. However, in the older embryos (E14 to E16), the expression of lumican is more prominent in the developing bone matrices. Our data suggest that lumican is a significant proteoglycan component of bone matrix, which is secreted by differentiating and mature osteoblasts only and therefore it can be used as a marker to distinguish proliferating pre-osteoblasts from the differentiating osteoblasts.

An electron microscopical study on the presence of proteoglycans in the calcified bone matrix by use of cuprolinic blue.

With Cuprolinic Blue (CBl) as contrasting agent, PGs could be demonstrated in mouse fetal bone matrix. Large CBl-positive rod-like structures proved to be present in and outside the calcification nodules in regions of beginning mineralization. In further developed bone also smaller rods were present in the mineralized matrix. The CBl-positive rods were sensitive to chondroitinase ABC and hyaluronidase. Under the circumstances we chose, this indicates that these structures are PGs containing chondroitin and/or dermatan sulphate. The fine filamentous and granular material in the nodules was still present after digestion with these enzymes, but disappeared after treatment with pronase. This is an indication that this material mainly contains proteins.

Microcinematographic and autoradiographic kinetic studies of bone cell differentiation in vitro: matrix formation and mineralization.

Matrix formation and mineralization have been reported in vitro with cells isolated from rat calvaria bones by collagenase digestion (Nefussi et al., 1985). In the current study, kinetics of bone nodule formation and osteoblastic cell differentiation were studied in this in vitro system using an improved microcinematographic device and flash and follow-up labeling autoradiographic techniques. Microcinematographic analysis showed the formation of bone nodules within 24 h. The initial event observed was the change in the top cells layer which became alkaline phosphatase positive. Matrix synthesis occurred a few hours after this. The autoradiographic results demonstrated the formation of an integrated system where osteoblasts and osteocytes were active and synthesized a collagen matrix and mineralized it in a similar time sequence than in vivo.

The timing of the onset of osteogenesis in the tibia of the embryonic chick.

Haematoxylin, Alcian Blue-Chlorantine Fast Red (ABCR) and the Ralis osteoid-specific stain were employed to closely follow the histogenesis of the tibia of the embryonic chick so as to provide an accurate description of the onset of ossification. An overview of the major cytological events preceding osteogenesis in the tibia was obtained from hindlimbs of embryos of H. H. (Hamburger and Hambilton, '51) stages 16-26 (2.5-5 days of incubation) stained with ABCR. A description of the cytological changes in the periosteum as it develops from the perichondrium and an analysis of the timing of the onset of osteoid deposition was obtained from the tibiae of accurately aged and staged embryos of H. H. stages 28-32 (5.5-8 days). These tibiae were stained specifically for the detection of osteoid: the freshly-secreted, unmineralized product of fully-differentiated osteoblasts. The perichondrium transformed into a bi-layered periosteum at H. H. late stage 29 (6.5 days) while osteoid was first detected adjacent to the hypertrophic cartilage of H. H. stage 30 (6.5-7 days) tibial diaphyses. These results, correlated with the immunoflourescent studies of Von der Mark et al. ('76a,b), which revealed the presence of Type I (bone-type) collagen-synthesizing cells in the perichondria of tibiae from embryos of H. H. stage 28 (5.5-6 days), demonstrated that the onset of determination of cells for osteogenesis and the cytodifferentiation of the periosteum are not temporally coupled.

Initiation of secondary cartilage in the mandible of the Syrian hamster in the absence of muscle function.

The functioning lower jaw is a prerequisite of the ongoing secondary chondrogenesis in the mammalian mandibular condyle. Does the articular function also initiate secondary chondrogenesis in the mandible? The angular process of the fetal mammalian mandible possesses a large secondary cartilage without any apparent articular function. Past studies have shown that the anlage of the lower jaw of the mouse embryo grown in organ culture produces condylar and angular cartilages as in vivo. In order to clarify further the capacity of the mandibular anlage to initiate secondary chondrogenesis in a non-functional environment, mandibular arch explants taken from the prenatal hamster before any cartilage or bone formation was apparent were grown in organ culture. Both primary and secondary cartilage could be found in them within 9-10 days. The results thus indicate that initiation of the condylar and/or angular secondary cartilaginous development in the rodent mandible occurs in the absence of jaw-opening function, although the pertinent literature indicates that function maintains cartilaginous differentiation in the condyle.

Immunohistochemical localization of parathyroid hormone-related protein in developing mouse Meckel's cartilage and mandible.

In order to clarify the role of parathyroid hormone-related protein (PTHrP) during Meckel's cartilage and mandibular development, an immunohistochemical study of PTHrP and its receptor, PTH/PTHrP receptor, was designed to examine their localization in the anterior region of Meckel's cartilage including the rostrum, which is known to contribute to the development of the mandible. Meckel's cartilage was first observed on day 13 of gestation and PTHrP was faintly localized in the chondrocytes. On day 16 of gestation, at the stage of elongation and initiation of endochondral ossification in Meckel's cartilage, PTHrP was localized in the chondrocytes located in the area showing interstitial growth and in and around the nuclei of hypertrophic chondrocytes undergoing endochondral ossification. At day 18 of gestation, endochondral ossification was spread over the entire area proximal to the molar region in Meckel's cartilage, except in the mesial fusion site formed by immature chondrocytes. PTHrP was localized in the osteoblasts adjacent to the calcified matrix, but had disappeared from the chondrocytes forming Meckel's cartilage. The localization of PTH/PTHrP receptor was similar to that of PTHrP. These results show that localization of PTHrP is spatially and temporally related to the growth of Meckel's cartilage.

Influence of demineralized bone matrix's embryonic origin on bone formation: an experimental study in rats.

BACKGROUND: There are results suggesting that differences regarding bone-inducing potential, in terms of amount and/or rate of bone formation, exist between demineralized bone matrices (DBMs) of different embryonic origins. PURPOSE: The aim of the present study was to examine whether the embryonic origin of DBM affects bone formation when used as an adjunct to guided tissue regeneration (GTR). MATERIALS AND METHODS: Endomembranous (EM) and endochondral (ECH) DBMs were produced from calvarial and long bones of rats, respectively. Prior to the study the osteoinductive properties of the DBMs were confirmed in six rats following intramuscular implantation. Following surgical exposure of the mandibular ramus, a rigid hemispheric Teflon capsule loosely packed with a standardized quantity of DBM was placed with its open part facing the lateral surface of the ramus in both sides of the jaw in 30 rats. In one side of the jaw, chosen at random, the capsule was filled with EM-DBM, whereas in the other side ECH-DBM was used. Groups of 10 animals were sacrificed after healing periods of 1, 2, and 4 months, and undecalcified sections of the capsules were produced and subjected to histologic analysis and computer-assisted planimetric measurements. RESULTS: During the experiment increasing amounts of newly formed bone were observed inside the capsules in both sides of the animals' jaws. Limited bone formation was observed in the 1- and 2-month specimens, but after 4 months of healing, the newly formed bone in the ECH-DBM grafted sides occupied 59.1% (range 45.6-74.7%) of the area created by the capsule versus 46.9% (range 23.0-64.0%) in the EM-DBM grafted sides (p =.01). CONCLUSION: It is concluded that the embryonic origin of DBM influences bone formation by GTR and that ECH-DBM is superior to EM-DBM.

Immunocytochemical localization and biochemical characterization of large proteoglycans in developing rat bone.

This study used biochemical and light and electron microscopic immunohistochemical methods to localize and characterize large hyaluronate-binding proteoglycans in the developing mandible of fetal rats at embryonic day 15 (Day 15) to Day 18 using a monoclonal antibody (MAb) 5D5. This antibody is derived from bovine sclera and specifically recognizes the core protein of large proteoglycan such as versican, neurocan and brevican, but not that of aggrecan. At the light microscopic level, MAb 5D5 moderately stained the extracellular matrices among osteoblasts at the centers of ossification in Day 15 mandible specimens. Weaker staining was observed in osteoblasts, whereas Meckel's cartilage lacked staining. Ultrastructural immunocytochemistry showed the presence of immunogold particles over unmineralized matrices among osteoblasts and their intracellular organelles. In Day 16 to 18 specimens, bone nodules were recognized in LR gold sections before immunostaining, but, after immunostaining, consistently appeared devoid of mineral crystals and were seen as a demineralized structure that had an electron dense periphery within which fine filamentous and granular material were present. The appearance of these structures was created by the demineralization of thin sections on grids during immunostaining. Specific immunogold staining was clearly seen over the demineralized structures corresponding to bone nodules. The majority of immunogold particles tended to localize inside of the structures. Bone proteins were extracted from fresh, Day 18 specimens with a three-step technique: 4 M guanidine HCl (GdnCl,-extract), aqueous EDTA without GdnCl (E-extract), followed by GdnCl. Western blot analysis of SDS-polyacrylamide gel electrophoresis after chondroitinase ABC digestion, showed that G1-extract gave a 5D5 reactive band greater than 400 kDa, whereas E-extract produced two major reactive populations of small molecular size with core proteins approximately 63 and 74 kDa. These results indicate that the large proteoglycan having smaller molecular weight is preferentially localized to bone nodules and may correlate with bone matrix mineralization.