Ultrastructural cytochemistry of complex carbohydrates in osteoblasts,osteoid, and bone matrix

Summary Glycosaminoglycans (GAGs) and glycoproteins are essential components for osteogenesis. We have examined rat osteoblasts, osteoid, transitional zone, and fully calcified bone matrix, utilizing Spicer's high-iron diaminethiocarbohydrazide-silver protein (HID-TCH-SP) method for sulfated glycoconjugates and Thiéry's periodate-TCH-SP (PA-TCH-SP) method for vicinal glycol-containing glycoconjugates. HID-TCH-SP stained cytoplasmic granules of osteoblasts. Stain deposits in the extracellular matrix were observed in decreasing amounts in osteoid, the transitional zone, and fully calcified bone matrix. Enzyme digestion with testicular hyaluronidase removed most HID-TCH-SP stain deposits. PA-TCH-SP staining was observed with increasing intensity in rough endoplasmic reticulum, Golgi saccules, and cytoplasmic granules. Collagen fibrils in osteoid were weakly stained with PA-TCH-SP, and their staining appeared even weaker in fully calcified bone matrix. In contrast, collagen fibrils in calcified cartilage stained intensely with the PA-TCH-SP method. Focal circular profiles (0.1–0.5μm in diameter), which lacked collagen fibrils but reacted moderately with PA-TCH-SP, were frequently seen in the transitional zone and fully calcified bone matrix, but were only occasionally present in osteoid. The presence of testicular hyaluronidase-resistant GAG and acid phosphatase in these focal areas suggests that they represent sites of GAG degradation. The eventual loss of HID-TCH-SP staining in the bone matrix suggests that removal of sulfated glycoconjugates may be a requisite for expansion of initial calcification sites and/or complete calcification.     

Characteristic localization of carbohydrates in osteoclasts by lectin cytochemistry

Lectin cytochemistry was performed to clarify the process of glycosylation and the localization of glycocalyx in osteoclasts. Microslicer sections of decalcified rat tibiae were incubated in the presence of HRP-conjugated lectins (Con A, PNA, MPA, WGA, UEA-1). Lectin reactions in cell organelles revealed that glucose (Glc) and mannose (Man) are transferred to carbohydrate chains in nuclear envelopes, rough endoplasmic reticuli, and the cis and medial sides of the Golgi apparatus. N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), and/or N-acetylneuraminic acic (NANA) residues are transferred, in turn, in the Golgi apparatus. Lectin reactions detected in lysosomal structures suggest that some sugar residues are incorporated into carbohydrate chains of hydrolytic enzymes, such as acid phosphatase and arylsulfatase. Others would be transported to plasma membranes as glycocalyx. PNA and MPA reactions were most evident on ruffled borders of osteoclasts. On the other hand, cement-line-like structures on bone surfaces displayed Con A, MPA, and WGA positive reactions. The following factors suggest that osteoclasts actively metabolize sugar: characteristic localization of glycocalyx in osteoclasts reflect the polarity of osteoclasts, and carbohydrate complexes in cementline-like structures seem to play an important role in the coupling phenomenon in bone tissue.     

Immunoelectron microscopy of osteonectin and type I collagen in osteoblasts and bone matrix

Summary The pathway of production, secretion, and extracellular deposition of type I collagen and osteonectin was studied by immunoelectron microscopy using respective polyclonal antibodies. Protein A gold and immunogold methods yielded to similar results in human callus tissue used as a model of bone formation. The intracellular distribution of osteonectin in active osteoblasts is found as a faint immunolabeling of vesicular Golgi fields and some lamellae of rough endoplasmic reticulum. A more intensive labeling occurs in opaque cytoplasmic vesicles pointing to the process of secretion as some of the vesicles are connected with the basal cell membrane. Our type I collagen antibody did not label the respective intracellular compartments. Extracellulary, the type I collagen antibody showed a continuous labeling from the immature subcellular osteoid to the mineralized bone. Osteonectin antibodies were bound first to the deeper layer of osteoid maturation with intensity increasing below the mineralization front. Osteonectin is thought to be associated with mineralization of bone matrix.     

Mechanical stretch induced calcium efflux from bone matrix stimulates osteoblasts

The mechanisms by which bone cells sense critically loaded regions of bone are still a matter of ongoing debate. Animal models to investigate response to microdamage involve post mortem immunohistological analysis and do not allow real-time monitoring of cellular response during the emergence of the damage in bone. Most in vitro mechanical stimulation studies are conducted on non-bone substrates, neglecting the damage-related alterations in the pericellular niche and their potential effects on bone cells. The current study reports spontaneous efflux of calcium ions (Ca(2+)) (1.924±0.742 pmol cm(-2)s(-1)) from regions of devitalized bone matrix undergoing post-yield strains, induced by a stress concentrator. When these samples are seeded with MC3T3-E1 osteoblasts, the strain-induced Ca(2+) efflux from bone elicits cell response at the stress concentration site as manifested by activation of intracellular calcium signaling (increase in fluorescence by 52%±27%). This activity is associated with extracellular calcium because the intracellular calcium signaling in response to mechanical loading subsides when experiments are repeated using demineralized bone substrates (increase in fluorescence by 6%±10%). These results imply a novel perspective where bone matrix acts as an intermediary mechanochemical transducer by converting mechanical strain into a chemical signal (pericellular calcium) to which cells respond. Such a mechanism may be responsible for triggering repair at locations of bone matrix undergoing critical deformation levels.     

Ultrastructural cytochemistry of epithelial gland-like component in synovial sarcoma

In order to analyze the mucoid substance in the epithelial component of synovial sarcoma, electron microscopic and cytochemical studies were made on three of these neoplasms. The mucoid substances in the glandular lumen were intensely stained with ruthenium red (RR), appearing as granular, fibrillar and amorphous structures. RR staining of proteoglycans was diminished after treatment with chondroitinase AC or ABC, and was partially diminished by exposure to streptomyces hyaluronidase. Trypsin treatment did not affect RR staining of proteoglycans in the lumen. On thin sections stained with periodic acid-thiocarbo-hydrazide-silver proteinate (PA-TCH-SP), deposits of reaction product were observed on the mucoid substances within the lumen, and were localized in the Golgi complex, including the rough endoplasmic reticulum, small vesicle and lysosome-like dense body. Trypsin digestion decreased the stain intensity of PA-TCH-SP. These results indicate that the lumen of the gland-like component contains glycoproteins as well as proteoglycans mainly consisting of chondroitin sulfate and hyaluronic acid, and suggest that GERL (Novikoff) is closely related to production, storage and transport of glycoproteins in the cytoplasm of tumor cells.     

Effects of acute and chronic PTH stimulation on osteoblasts and the under-lying bone matrix

Calcified Tissue International -     

Development and transplantation of a mineralized matrix formed by osteoblasts in vitro for bone regeneration

The use of extracellular matrix materials as scaffolds for the repair and regeneration of tissues is receiving increased attention. The current study was undertaken to test whether extracellular matrix formed by osteoblasts in vitro could be used as a scaffold for osteoblast transplantation and induce new bone formation in critical size osseous defects in vivo. Human osteoblasts derived from alveolar bone were cultured in six-well plates until confluent and then in mineralization media for a further period of 3 weeks to form an osteoblast--mineralized matrix complex. Histologically, at this time point a tissue structure with a "connective tissue"-like morphology was formed. Type I collagen was the major extracellular component present and appeared to determine the matrix macrostructure. Other bone-related proteins such as alkaline phosphatase (ALP), bone morphogenetic protein (BMP)-2 and -4, bone sialoprotein (BSP), osteopontin (OPN), and osteocalcin (OCN) also accumulated in the matrix. The osteoblasts embedded in this matrix expressed mRNAs for these bone-related proteins very strongly. Nodules of calcification were detected in the matrix and there was a correlation between calcification and the distribution of BSP and OPN. When this matrix was transplanted into a critical size bone defect in skulls of immunodeficient mice (SCID), new bone formation occurred. Furthermore, the cells inside the matrix survived and proliferated in the recipient sites, and were traceable by the human-specific Alu gene sequence using in situ hybridization. It was found that bone-forming cells differentiated from both transplanted human osteoblasts and activated endogenous mesenchymal cells. This study indicates that a mineralized matrix, formed by human osteoblasts in vitro, can be used as a scaffold for osteoblast transplantation, which subsequently can induce new bone formation.     

Leptin increases extracellular matrix mineralization of human osteoblasts from heterotopic ossification and normal bone

Heterotopic ossification (HO) is the pathologic formation of bone in soft tissue. The exact pathomechanism is unknown but probably involves a disturbed osteoblast differentiation. Leptin, known as the obesity gene, may regulate normal osteoblast function in vitro. The aim of the present in vitro study was to further analyze the pathomechanisms of HO, including a possible role of leptin in ectopic bone formation. Human osteoblasts were cultivated either from normal bone or from resected HO. Both groups were incubated with increasing doses of leptin. Phenotype expression and mineralization of extracellular matrix were measured after 7, 14, and 21 days. In both groups, leptin increased both the formation of bone nodules and Ca-45 incorporation. This is the first study to analyze the effect of leptin on bone cells from ectopic ossification. Similar to the in vitro behavior of normal osteoblasts, cells from HO respond to leptin exposure with an increased mineralization of the extracellular matrix. This mechanism may be involved in the pathogenesis of ectopic bone formation in vivo.     

Nucleobindin is produced by bone cells and secreted into the osteoid, with a potential role as a modulator of matrix maturation

Nucleobindin (Nuc), also known as CALNUC, is a Ca²⁺-binding protein, located in the nucleus, the Golgi apparatus and the endoplasmic reticulum (ER). The presence of a signal sequence in Nuc suggests secretion from the cell and it has been found in bone extracellular matrix. Within the present study, molecular biological and morphological methods were combined to evaluate the synthesis and distribution of Nuc in and around cells of rat metaphyseal and calvarial bone. Northern blot analysis and in situ hybridization of bone tissues confirmed that the protein was a product of bone cells. By electron microscopy, immunolabeling for Nuc was seen in osteoid of newly formed bone, on all surfaces facing the various bone cells and also in compact bone. Intracellularly, the gold particles were found in the rough ER of osteoblasts, which suggested synthesis of the protein by these cells. Compared to bone sialoprotein and osteopontin, Nuc demonstrated different localization pattern in bone trabeculae, with the majority of labeling restricted to nonmineralized osteoid. Moreover, the role of Nuc during the mineralization process was investigated in rat calvaria-derived primary osteoblasts grown under osteogenic conditions. Semiquantitative RT-PCR and Northern blot analysis showed Nuc expression to be low during cell proliferation, upregulated during differentiation and matrix maturation, but subsequently downregulated during mineralization. In summary, our data show that Nuc was synthesized by osteoblasts and osteocytes, and secreted into the osteoid, suggesting a role as a modulator of matrix maturation in the mineralization process in bone.     

Ultrastructural features of the osteoid of patients with fibrogenesis imperfecta ossium

The osteoid of a patient with Fibrogenesis Imperfecta Ossium is described. Three iliac crest biopsies were taken; firstly before treatment, secondly after calcitriol therapy and finally after successful treatment with melphalan and prednisolone. In the pretreatment biopsy the osteoid was greatly enlarged, showed complete absence of the birefringence characteristic of oriented collagen fibers, and at ultrastructural level was shown to be composed of abnormal collagen fibrils. The fibrils were often curved and were extremely variable in thickness. Calcification within the osteoid took the form of calcospherites and spread of calcification from these to collagen fibrils was greatly delayed. In the second biopsy two aspects of osteoid ultrastructure were noted; some samples resembled the first biopsy, but others had a different organization. The osteoid of these samples had two regions: an inner region containing abnormal collagen fibrils and an outer region composed of moderately electron-dense amorphous material. The osteoblasts associated with this region were clearly highly biosynthetically active. The third biopsy, after treatment with Melphalan and prednisolone, showed a reversion to more normal bone ultrastructure with uniform, oriented collagen fibrils and prompt mineralization resulting in narrow osteoid seams. Remnants of the original abnormal osteoid were present in the marrow space as calcified debris. Reasons for the success of this therapeutic regime are unclear; however, some speculation is made as to the possible roles of the cytotoxic drug and the glucocorticoid in the regression of this condition.     

Radioautographic visualization of3H-fucose incorporation into glycoprotein by osteoblasts and its deposition into bone matrix

Summary The elaboration of bone matrix glycoprotein by osteoblasts of alveolar bone was investigated by radioautography after the intravenous injection of³H-fucose into young rats. At selected times after injection, animals were sacrificed by intracardiac perfusion and demineralized specimens were prepared for light and electron microscope radioautography. At 5 and 10 min after injection, when the blood fucose level was high, silver grains were restricted to the spheroidal and cylindrical saccules of the Golgi apparatus. At 20 min membrane-limited secretory granules were also labeled. By 35 min, the blood fucose level had dropped and silver grains were detected over the apical cortical cytoplasm, in association with secretory granules located therein. Some grains were present over osteoblast processes and the adjacent prebone matrix. By 4 h most of the silver grains had left the cell. At that time they were observed over prebone, adjacent to osteoblast processes, as well as over the prebone-bone junction where a distinct band of label was noted. In demineralized preparations an electron-dense granular material was present at the prebone-bone junction in association with collagen fibrils. These findings provide evidence that osteoblasts in alveolar bone synthesize fucose-containing glycoprotein and indicate that the addition of³H-fucose occurs in the Golgi apparatus. The glycoprotein passes to the apical cortical cytoplasm by way of membrane-limited secretory granules, is exteriorized, and accumulates at the site where prebone transforms into bone (the prebone-bone junction). Since this is also the site of the calcification front, the deposition of labeled glycoprotein may be related to the deposition of bone mineral.     

Adhesion,growth, and matrix production by osteoblasts on collagen substrata

Summary A number of studies have demonstrated the pivotal role of collagen molecules in modulating cell growth and differentiation. In order to analyze the direct effects of collagen type I on the osteoblastic phenotype, we have devised an in vitro culture system for studying the interactions between bovine collagen type I and Saos-2 cells, a human osteoblastic cell line. Saos-2 cells were cultured both on top of collagen-coated culture dishes as well as inside a three-dimensional collagen network. Plating on dishes treated with collagen induced maximal adhesion of Saos-2 cells after 24-hour incubation. Cells cultured on collagen gel matrix expressed about 2.5-fold more alkaline phosphatase when compared with untreated plastic dishes. On collagen-coated dishes the responsiveness of Saos-2 cells to parathyroid hormone was decreased, whereas no modifications were observed in the effect of vasoactive intestinal peptide on these cells. Using a microfluorimetric measurement of DNA, an increase of proliferation was observed in Saos-2 cells cultured on collagen gel Saos-2 cells were also able to colonize collagen sponges and in this three-dimensional network they were able to synthesize osteocalcin, as assessed both by immunocytochemistry and radioimmunoassay. In this study we have demonstrated that bovine collagen type I exhibits favorable effects on attachment and functional and growth activities of a human osteoblastic cell line, encouraging its use as a bone graft material.     

Secretory territories and rate of matrix production of osteoblasts

The secretory territories of rat osteoblasts on the parietal bone were measured directly using scanning electron microscopy. The mean territory of 4620 cells in 19 fields was 154 m² per osteoblast. The range for the fields was 136 to 177 m² per osteoblast. Four hundred cells were measured individually—for these the mean value per osteoblast was 143 m² with a standard deviation of 33. The daily rate of apposition over an 8 day period was 3.12 m (standard deviation 0.22) measured by tetracycline marking of the mineral front. This gave a daily matrix production rate of approximately 470 m³ per osteoblast.

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Zusammenfassung Die Ausscheidungsbereiche von Ratten-Osteoblasten des Scheitelbeines wurden mit dem Raster-Elektronenmikroskop direkt gemessen. Der durchschnittliche Bereich von 4620 Zellen in 19 Gesichtsfeldern war 154 m² per osteoblast. Der Streubereich lag in den verschiedenen Gesichtsfeldern zwischen 136 und 177 m² per Osteoblast. 400 Zellen wurden einzeln gemessen. Bei diesen war der Durchschnittswert per Osteoblast 143 m², mit einer Standard-Abweichung von 33. Die tägliche Anlagerungsrate während einer Periode von 8 Tagen war 3,12 m (Standard-Abweichung 0,22); sie wurde mittels Tetracyclinmarkierung der Mineralisierungsfront gemessen. Dies ergab eine tägliche Produktionsrate der Matrix von etwa 470 m³ per Osteoblast.

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Résumé Les territoires sécrétoires des ostéoblastes d'os pariétal de rats sont déterminées en utilisant la microscopie électronique à balayage. Le territoire moyen de 4.620 cellules, dans 19 territoires, est de 154 m² par ostéoblaste. Les valeurs extrêmes par champ varient de 136 à 177 m² par ostéoblaste. Quatre cent cellules sont mesurées individuellement; la valeur moyenne par ostéoblaste est de 143 m³ avec une déviation standard de 33. Le taux d'apposition journalier, mesuré par la tétracycline pendant 8 jours, est de 3.12 m (déviation standard 0.22). Ce qui correspond à une production matricielle journalière d'environ 470 m³ par ostéoblaste.

     

High bone turnover and accumulation of osteoid in patients with neurofibromatosis 1

Summary  

Although it is known that neurofibromatosis 1 (NF1) patients suffer from vitamin D deficiency and display decreased bone mineral density (BMD), a systematic clinical and histomorphometrical analysis is absent. Our data demonstrate that NF1 patients display high bone turnover and accumulation of osteoid and that supplementation of vitamin D has a beneficial effect on their BMD.     

Nucleoside diphosphate kinase in fetal rat bone tissue: increased expression in osteoblasts and localization in the matrix vesicle fraction

Received: March 18, 1998 / Accepted: April 6, 1998     

Formation of bone by isolated,cultured osteoblasts in millipore diffusion chambers

Summary Osteoblast-like and osteoclast-like cells freed from neonatal calvaria by sequential enzymatic digestion after 6–7 days in culture were placed in diffusion chambers and implanted in the peritoneal cavities of CD-1 mice. About half of the chambers also contained a dead calvarium to test for the need of an “inducer.” After 20 days, 11 of 18 chambers containing the osteoblast-like cells formed large foci of mineralized bone that corresponded to alkaline phosphatase activity throughout the chambers. Moreover, only type I (i.e., bone) collagen was formed. Occasional deposits of bone were found in only 3 of 22 chambers containing the osteoclast-like cells. The presence of dead bone did not affect any of the results. These data confirm the osteoblast-like nature of the isolated cell populations and demonstrate that these cells retain their differentiated function in culture.     

Ultrastructural observations of amorphous bone mineral in avian bone

Mineral from medullary bone of three avian species was examined with the electron microscope in order to clarify the ultrastructure of amorphous bone mineral (ABM) in a mineralized tissue. Powders from freeze-dried bone revealed bone mineral with morphology and behavior identical to synthetic amorphous calcium phosphate (ACP). These powders exhibited spherically shaped particles 180–400 Å in diameter with uniform electron density when viewed at low bem intensity. Thin sections of embedded freeze-dried bone also revealed spherically shaped particles 100–350 Å in diameter with electron beam sensitivity characteristic of ACP. Regions of bone mineral with irregular outline and morphology were observed which closely resemble the discoidal form of synthetic ACP. More electron-dense spherical particles (150 Å in diameter) could be seen budding from these regions. Some of these buds exhibited electronlucent centers characteristics of AMB. The inorganic nature of these features of bone mineral was confirmed using ultramicroincineration. Preliminary exploration of a freeze-substitution technique showed spherical bone mineral particles which were similar in morphology to those observed in freeze-dried samples. A limited degree of preservation of cellular material was observed using this freeze-substitution technique.