Regulation of parathyroid hormonelike protein production in cultured normal and malignant keratinocytes.

We have recently demonstrated that parathyroid hormone-like protein (PLP) production by cultured human squamous carcinoma cells (SCC) can be modulated by co-culture with fibroblasts. The interaction of SCC with fibroblasts, possibly occurring during the invasive phase of SCC, may be the stimulus for enhanced PLP production, thus contributing to the genesis of humoral hypercalcemia of malignancy in this type of cancer (Cancer Res 50:3589-3594, 1990). In the present study we show that the fibroblast-induced increase in PLP level in the medium of SCC-4 cells is paralleled by an increase in PLP messenger ribonucleic acid (mRNA) expression in these cells. We also found that the inhibition of secretion of PLP by monensin for 2 h resulted in a marked increase in immunodetectable PLP intracellularly, suggesting that secretion of PLP was a fast process. The modulation of the production of PLP by calcium and hydrocortisone was further examined in SCC-4 cells and was compared to that in normal keratinocytes and in SCC-9 cells. PLP levels in conditioned media were highest in poorly differentiating SCC-4 cells, intermediate in moderately differentiating SCC-9 cells, and lowest in normal keratinocytes showing high differentiating capacity. Furthermore, in each of the cell types used, PLP production was highest in cultures grown under low calcium conditions; at both calcium concentrations used, the presence of hydrocortisone reduced the PLP release into the medium. This reduction was probably due to a direct effect of hydrocortisone on PLP synthesis because the expression of PLP mRNA was also reduced in the presence of hydrocortisone when tested in SCC-4 cells. In conclusion, our findings indicate that the induction of differentiation in both normal and malignant keratinocytes is associated with the inhibition of PLP production.

Light and electron microscopic demonstration of osteocalcin antigenicity in embryonic and adult rat bone.

Of the noncollagenous proteins in bone, about 20% consists of osteocalcin. This vitamin K-dependent protein can be found in adult bone, but its presence in embryonic bone could not be demonstrated unequivocally by biochemical methods. Therefore, we used light and electron microscopic immunohistochemical methods to investigate whether osteocalcin antigenicity could be demonstrated in radii of 20-day-old rat embryos. The results show that osteocalcin antigenicity can be demonstrated in the bone matrix of adult bone and in the shaft and endochondral bone matrix of embryonic bone. It could not be demonstrated in calcified cartilage matrix. In bone the antigenicity was observed in the early foci of calcification, i.e., the mineralization nodules.

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.

Morphologic features of bone in human osteopetrosis.

Trabecular bone biopsies obtained from six patients with malignant osteopetrosis, one patient with benign osteopetrosis, and two controls were examined by light and electron microscopy. Osteopetrotic osteoclasts showed little to no signs of active involvement in bone resorption. Ruffled borders and clear zones were absent in most cells. In all patients there were large numbers of osteoclasts. Numbers of osteoblasts, bone lining cells, and bone marrow stromal cells were extremely low in all patients with malignant osteopetrosis. In six of the patients a mineralized layer of amorphous organic material lacking collagen fibrils was seen covering large areas of the bone or cartilage matrix. We suggest that this layer represents a pathological calcification on which subsequently organic material has accumulated. The abnormalities in osteopetrotic bone are discussed in the light of the pathogenesis of this disease.

Polyethyleneimine as a contrast agent for ultrastructural localization and characterization of proteoglycans in the matrix of cartilage and bone.

We examined the presence of proteoglycans in the extracellular matrix of cartilage and bone in fetal mouse radii at the ultrastructural level, using the cationic dye polyethyleneimine (PEI). After staining with this dye, the proteoglycans appeared as granules in the uncalcified bone matrix and as extended winding structures in the cartilage matrix. PEI-positive material was removed after treatment of the tissue with chondroitinase ABC. Inhibition of the proteoglycan synthesis by beta-D-xyloside resulted in smaller PEI-positive windings in the cartilage matrix. These observations suggest that the winding, PEI-positive structures represent proteoglycan aggregates. No loss of PEI-positive material in the calcified cartilage matrix was seen, suggesting that proteoglycans do not need to be removed to make the matrix calcifiable.

Better histology and biochemistry for osteoblasts cultured on titanium-doped bioactive glass: bioglass 45S5 compared with iron-, titanium-, fluorine- and boron-containing bioactive glasses.

In the present study we used an established cell culture model to compare Bioglass 45S5 with four other bioactive glasses. Small substitutions or additions of certain ions like iron, titanium, fluorine or boron modified the basic 45S5 glass network. We used several histological and biochemical parameters to interpret the results found in terms of the used model. Regarding 45S5 as a reference, we found that osteoblasts cultured on iron-doped bioactive glass showed a more flattened morphology, and both lower proliferation rate and osteoblast expression. Osteoblasts cultured on titanium-doped glasses also showed a flattened morphology, but higher proliferation and remarkably higher osteoblast expression. On fluorine- and boron-containing glasses the osteoblasts showed a rather compact morphology, a normal proliferation but only moderate osteoblast expression. With microprobe analysis it was shown that the formation of calcium and phosphorus on titanium-doped glass was relatively lower and the release of sodium slower when compared with 45S5. Osteoblasts cultured on titanium-doped bioactive glasses demonstrated superior histological and biochemical parameters when compared with the other glass types. Further research into the physico-chemical properties and the in vivo behaviour of doped bioactive glasses is recommended.

Behaviour of fetal rat osteoblasts cultured in vitro on bioactive glass and nonreactive glasses.

We examined the behaviour of fetal rat osteoblasts cultured upon bioactive glass and nonreactive glasses, and the supposed stimulatory effects of bioactive glass on osteoblasts. Nonreactive glass cultures showed flattened cells with almost no dorsal ruffles. Bioactive glass cultures showed compact cells with dorsal ruffles and filapodia resulting in the formation of a denser cell layer. For confluent nonreactive glass cultures the osteoblast expression was mainly concentrated in the clustered cells which were formed upon the monolayer, whereas for confluent bioactive glass cultures the osteoblast expression was more generally distributed. The production of type I collagen, osteocalcin and an osteoblast-specific antigen was shown by immunocytochemistry for all cultures, although differences in distribution were observed. The bioactive layer of bioactive glass is responsible for a better osteoblast-like morphology, a higher proliferation rate and generally a better osteoblast expression.

Osteoblasts develop from isolated fetal mouse chondrocytes when co-cultured in high density with brain tissue.

Chondrocytes from the hypertrophic and proliferative zones of 16-day-old fetal murine metatarsal bones were enzymatically dissociated and cultured in a high-density type of culture, exposed to the gas phase. We ascertained that no cells of the perichondrium were included in the cell suspension. Control cultures formed a solid cartilaginous mass, of which all the chondrocytes were alkaline phosphatase positive and the matrix started to calcify after 4 days. After 6 days, nearly the entire matrix was calcified. When co-cultured with pieces of cerebral tissue, some chondrocytes had transdifferentiated into osteoblasts after 4 days. They had started to form osteoid. After 6 and 11 days part of the cartilage had been replaced by bone, especially in the periphery of the cultures, but also in areas in the center. The bone matrix was partly calcified. Osteoblasts and bone matrix were identified as such electron microscopically. The nature of the bone matrix was also confirmed by immunohistochemical demonstration of collagen type I and osteocalcin. These results show that enzymatically isolated chondrocytes are able to become osteoblasts when properly stimulated. This supports the concept of chondrocytes being responsible for (part of) the endochondral bone formation in the marrow cavity of long bones.

Osteoblasts develop from isolated fetal mouse chondrocytes when co-cultured in high density with brain tissue.

Chondrocytes from the hypertrophic and proliferative zones of 16-day-old fetal murine metatarsal bones were enzymatically dissociated and cultured in a high-density type of culture, exposed to the gas phase. We ascertained that no cells of the perichondrium were included in the cell suspension. Control cultures formed a solid cartilaginous mass, of which all the chondrocytes were alkaline phosphatase positive and the matrix started to calcify after 4 days. After 6 days, nearly the entire matrix was calcified. When co-cultured with pieces of cerebral tissue, some chondrocytes had transdifferentiated into osteoblasts after 4 days. They had started to form osteoid. After 6 and 11 days part of the cartilage had been replaced by bone, especially in the periphery of the cultures, but also in areas in the center. The bone matrix was partly calcified. Osteoblasts and bone matrix were identified as such electron microscopically. The nature of the bone matrix was also confirmed by immunohistochemical demonstration of collagen type I and osteocalcin. These results show that enzymatically isolated chondrocytes are able to become osteoblasts when properly stimulated. This supports the concept of chondrocytes being responsible for (part of) the endochondral bone formation in the marrow cavity of long bones.

Electron microscopical x-ray microanalysis of mineralization nodules and several other tissue compartments in fetal bone.

Several chemical components are involved in bone and cartilage mineralization. Acid glycosaminoglycans (GAG) form one class of these components. In this study, the possible presence of sulfated GAG in areas of beginning bone mineralization was investigated. We analyzed areas containing acid groups, which were made visible with positive colloidal ThO2, by means of x-ray microanalysis. Four tissue compartments were internodular matrix, and noncalcified cartilage matrix. Sulfur proved to be present in a higher concentration in the mineralization nodules than in the osteoid, whereas no significant difference could be demonstrated between the sulfur concentrations in mineralized internodular matrix and osteoid. The ratio of the sulfur content between the mineralization nodules and the cartilage matrix is significantly lower than the corresponding thorium ratio. This difference suggests that acid groups different from sulfate groups occur in a relatively larger amount in mineralization nodules than in cartilage matrix.

An electron microscopic examination for the presence of acid groups in the organic matrix of mineralization nodules in foetal bone.

Several biochemical and histochemical investigations of the role of glycosaminoglycans (GAG) in bone and cartilage mineralization have been performed, but the exact relationship between GAG and mineralization is not clear. We performed an electron microscopic histochemical study of the composition of organic bone matrix at sites of beginning mineralization in mouse foetal radii, directing special attention to the presence and location of GAG. To demonstrate these substances, use was made of positive colloidal thorium dioxide and ruthenium red. The results demonstrate the presence of material containing acid groups in the young mineralization nodules. It is concluded that part of the demonstrated acid groups might be sulphate groups of glycosaminoglycans.

Positive colloidal thorium dioxide as an electron microscopical contrasting agent for glycosaminoglycans, compared with ruthenium red and positive colloidal iron.

In electron microscopy Thorotrast has been used as a specific contrasting agent for acid glycosaminoglycans. Because of its high atomic number, thorium (Z=90) gives good contrast in the electron microscope, but at present it is less frequently used for this purpose. We prepared a positive colloidal solution of ThO2 without stabilizers to compare its properties with those of ruthenium red and positive colloidal iron for contrasting fetal mouse epiphyseal cartilage. The results indicate that colloidal ThO2, which is easy to prepare in any laboratory, gives better results than ruthenium red and colloidal iron do in this kind of cartilage. Furthermore, as judged from data in the literature and obtained in our laboratory, it penetrates this tissue better than Thorotrast does, probably because of the absence of stabilizers.

Transdifferentiation of hypertrophic chondrocytes into osteoblasts in murine fetal metatarsal bones, induced by co-cultured cerebrum.

The fate of hypertrophic chondrocytes in 17-day-old metatarsal bones of fetal mice was studied in a culture system in which these cells were kept confined to their lacunae. Because the periosteum had been stripped off, osteoclasts could not invade the long bone and resorb the lacunar walls. The majority of the hypertrophic chondrocytes stayed alive and dedifferentiated gradually into cells with the appearance of stromal cells. When the long bones were co-cultured with pieces of cerebrum, the chondrocytes transdifferentiated into osteoblasts. We followed this process from day to day. The cells produced bone matrix that immunostained for collagen type I and osteocalcin. To exclude with certainty the possibility that the intralacunar osteoblasts had derived from remaining periosteal osteoprogenitor cells that invaded the lacunae, the long bones were pre-cultured with cytochalasin D, which inhibits cell proliferation and migration. After removal of the drug this effect persisted until after transdifferentiation had occurred. This proved that the bone matrix producing osteoblasts inside the cartilage lacunae were transdifferentiated chondrocytes. The transdifferentiation stimulating factor from brain tissue is still unknown.

Histological and biochemical evaluation of osteoblasts cultured on bioactive glass, hydroxylapatite, titanium alloy, and stainless steel.

We investigated the behavior of fetal rat osteoblasts cultured on four bone replacing materials: bioactive glass, hydroxylapatite, a titanium alloy, and stainless steel. The cultures were histologically examined for individual cell morphology and osteoblast expression after several periods of time using scanning electron, fluorescence, and normal light microscopy. Other cultures were used for biochemical determinations of alkaline phosphatase activity (APA) and DNA content. Osteoblasts cultured on bioactive glass showed a better osteoblast-like morphology and a higher proliferation rate, leading to confluent cultures with higher cell density and a generally better expression of the osteoblast phenotype in comparison with the other substrates. The confluent bioactive glass cultures also showed significantly higher DNA content and APA as well as the calculated APA/DNA ratio. Based on the evaluation of histological and biochemical parameters we conclude that osteoblasts cultured on bioactive glass show a generally better osteoblast character than on the other materials.

An electron microscopic study on the presence of proteoglycans in the mineralized matrix of rat and human compact lamellar bone.

The presence of proteoglycans (PGs) was studied in compact lamellar rat and human bone at the electron microscopic level. With the cationic dye cuprolinic blue (CB1), PGs could be demonstrated in the mineralized bone matrix. The amounts of PGs appeared to be equal in the different lamellae and osteons. More CBl-positive material was found in the outermost lamella of the cortex, in the perilacunar matrix around the osteocyte lacunae, and around the canaliculi. Enzyme digestion with chondroitinase ABC demonstrated that the CBl-positive rods consisted of PGs. These observations amplify biochemical studies in which PGs have been isolated from the mineralized bone matrix. The presence of CBl-positive rods in the mineralized matrix suggest that PGs do not have to be removed completely to make the matrix calcifiable.

Influence of beta-D-xyloside on growth and histological aspect of long bones in chicken embryos.

It is known that beta-D-xylosides interfere with the proteoglycan synthesis in several tissues. A possible influence of this disturbed synthesis on the matrix formation of bone and cartilage has not been described light microscopically. In the present study we used 10-day-old chicken embryos which were exposed in ovo to a final concentration of 0.5 mM beta-D-xyloside. After 3, 6, 9, 20, 25, 31, 35 and 40 days, lengths of several skeletal elements were determined and the middle metatarsal bones were processed for light microscopical demonstration of acidic groups. The results demonstrate that beta-D-xyloside inhibits growth of long bones and induces synthesis of a cartilage matrix with a very low concentration of chondroitin sulphate. It has no noticeable influence on the amount of acidic groups in the organic bone matrix. Despite the absence of chondroitin sulphate, the cartilage matrix becomes mineralized normally.

Isolation and culture of smooth muscle cells from human umbilical cord arteries.

A short method is described for obtaining a large number of pure vascular smooth muscle cells in culture. The smooth muscle cells were isolated from human umbilical cord arteries digested twice by an enzyme mixture of collagenase, trypsin, elastase, and DNAase with addition of alpha-tosyl-lysyl chloromethane. Primary cell culture and first subculture were not contaminated by endothelial cells, no Factor VIII being produced. The cultures consisted of smooth muscle cells as appeared from phase contrast and electron microscopy.

Changes in proteoglycans of ageing and osteoarthritic human articular cartilage: an electron microscopic study with polyethyleneimine.

BACKGROUND: Ageing and osteoarthritic (OA) cartilage show characteristic alterations in chondrocyte morphology and in the composition and content of matrix proteoglycans (PGs). Data concerning matrix components are mostly of biochemical nature. Ultrastructural histochemistry is needed to gain more information about distribution of these altered matrix components. METHODS: We used the cationic dye polyethyleneimine (PEI) to visualize at the EM level alterations in the distribution and dimensions of PGs of human healthy young, healthy aged, and OA articular cartilage. RESULTS: Young cartilage contained PEI-positive granules in the superficial layer and big winding PEI-positive structures in the deeper layers. In the healthy aged tissue, PEI-positive granules were observed throughout the matrix and smaller winding structures were present in the deeper layer. In OA cartilage both types of PEI-positive structures were absent in the superficial layer. Deeper in the matrix PEI-positive granules could be demonstrated. Moreover, PEI-positive angular structures were observed in the deeper zones. CONCLUSIONS: The differences in PEI-positive structures are a good reflection of the differences in PGs between young, ageing, and OA cartilage as demonstrated in biochemical studies. PEI, used at the EM level, gave more precise information concerning the localized changes in quality, quantity, and location of PGs in articular cartilage during ageing and disease.