Immunocytochemical demonstration of extracellular matrix proteins in isolated osteocytes.

Cultures of isolated osteocytes may offer an appropriate system to study osteocyte function, since isolated osteocytes in culture behave very much like osteocytes in vivo. In this paper we studied the capacity of osteocytes to change their surrounding extracellular matrix by production of matrix proteins. With an immunocytochemical method we determined the presence of collagen type I, fibronectin, osteocalcin, osteopontin and osteonectin in cultures of isolated chicken osteocytes, osteoblasts and periosteal fibroblasts. In osteoblast and periosteal fibroblast cultures, large extracellular networks of collagen type I and fibronectin were formed, but in osteocyte populations, extracellular threads of collagen or fibronectin were only rarely found. The percentage of cells positive for osteocalcin, osteonectin and osteopontin in the Golgi apparatus, on the other hand, was highest in the osteocyte population. These results show that osteocytes have the ability to alter the composition of their surrounding extracellular matrix by producing matrix proteins. We suggest this property is of importance for the regulation of the calcification of the bone matrix immediately surrounding the cells. More importantly, as osteocytes depend for their role as mechanosensor cells on their interaction with matrix proteins, the adaptation of the surrounding matrix offers a way to regulate their response to mechanical loading.

Adhesive properties of isolated chick osteocytes in vitro.

Different functions have been proposed for osteocytes over time, but it is now generally accepted that their most important task lies in the sensing of strain caused by mechanical loading on bone. The fact that mechanical strain can be sensed as deformation of the extracellular matrix or as fluid shear stress along the cell, in the space between cell membrane and extracellular matrix, requires that osteocytes have close (specialized) contact with the bone matrix. We studied to which extracellular matrix proteins isolated chicken osteocytes adhere and whether this adhesion is mediated by specific cell adhesion receptors called integrins. The adhesive properties of the osteocytes were compared with that of osteoblasts. Osteocytes (and osteoblasts) adhere to the same substrates (i.e., collagen types I and II, collagen fibers, osteopontin, osteonectin, fibronectin, fibrinogen, thrombospondin, and laminin). Cell spreading varied between substrates, from all cells rounded on thrombospondin to all cells fully spread out on osteopontin, osteonectin, vitronectin, fibronectin, fibrinogen, and laminin. The percentage of osteocytes adhered was equivalent to that of osteoblasts adhered on all substrates except osteopontin and vitronectin, where osteocytes adhered less. The adhesion of osteocytes and osteoblasts to osteopontin, osteonectin, vitronectin, and fibrinogen was strongly inhibited, and to fibronectin and laminin moderately, by an RGD peptide. No RGD inhibition was found on collagen. An antibody against chicken integrin alpha v beta 3, the monoclonal antibody (MAb) 23C6, did not interfere with the adhesion of osteocytes and osteoblasts to matrix proteins, whereas an MAb against chicken integrin subunit beta 1 (CSAT) strongly inhibited adhesion to all substrates. Labeling with osteocyte-specific MAbs (OB7.3, OB37.4, and OB37.11) also did not hinder the adhesion of osteocytes to collagen type I, vitronectin, and osteopontin. Adhesion sites on osteocytes were small compared with the large adhesion plaques of osteoblasts, as demonstrated by interference reflection microscopy and immunocytochemically by staining for vinculin. Osteocyte adhesion is analogous to osteoblast adhesion with regard to the range of extracellular matrix proteins to which they adhere. The adhesion is mediated by the integrin subunit beta 1, but other integrins or nonintegrin adhesion receptors are also involved. Osteocytes make contact with the extracellular matrix via small attachment points which colocalize with vinculin. This connection between the bone matrix and the cytoskeleton may be important for osteocytic sensing of mechanical strain, as it supplies a transduction route of extracellular (mechanical) signals into intracellular messages.

Characteristics and properties of osteocytes in culture.

Although the osteocyte is the most abundant among the highly differentiated cells of mature bone (osteocytes, lining cells, osteoblasts, and osteoclasts), its properties and functions are the least known and understood. Here we isolated osteocytes from mixed populations of bone cells liberated from fetal chick calvariae by alternate treatments with collagenase and EDTA. The osteocytes were removed from the bone cell populations by binding them via an osteocyte-specific antibody (MAb OB 7.3) to magnetic beads and removing the beads together with the coupled osteocytes from the population using a magnet. Isolated osteocytes were found to be highly differentiated, postmitotic cells that required their typical stellate morphology in culture. Osteocyte populations had alkaline phosphatase (ALP) activity somewhat lower than that of the osteoblast-like cell populations from which they were separated by the immunodissection procedure. On the single-cell level, the ALP activity was highly variable. Parathyroid hormone (PTH) receptors were found to be present on osteocytes as well as on osteoblast-like cells, but not on fibroblast-like cells of the outer periosteum. In response to PTH, osteocytes increased their intracellular levels of cAMP, as did the osteoblast-like cells. Osteocytes appeared to be somewhat more sensitive to PTH than osteoblasts. When seeded onto dentin slices, osteocytes did not corrode the dentin surface to any appraisable degree. We therefore found no evidence to support the notion that osteocytes play a role in the calcium homeostasis through osteocytic osteolysis. Whether osteocytes play an important role in perceiving and transducing hormonal and/or mechanical stimuli remains open for future research.

Function of osteocytes in bone.

Although the structural design of cellular bone (i.e., bone containing osteocytes that are regularly spaced throughout the bone matrix) dates back to the first occurrence of bone as a tissue in evolution, and although osteocytes represent the most abundant cell type of bone, we know as yet little about the role of the osteocyte in bone metabolism. Osteocytes descend from osteoblasts. They are formed by the incorporation of osteoblasts into the bone matrix. Osteocytes remain in contact with each other and with cells on the bone surface via gap junction-coupled cell processes passing through the matrix via small channels, the canaliculi, that connect the cell body-containing lacunae with each other and with the outside world. During differentiation from osteoblasts to mature osteocyte the cells lose a large part of their cell organelles. Their cell processes are packed with microfilaments. In this review we discuss the various theories on osteocyte function that have taken in consideration these special features of osteocytes. These are 1) osteocytes are actively involved in bone turnover; 2) the osteocyte network is through its large cell-matrix contact surface involved in ion exchange; and 3) osteocytes are the mechanosensory cells of bone and play a pivotal role in functional adaptation of bone. In our opinion, especially the last theory offers an exciting concept for which some biomechanical, biochemical, and cell biological evidence is already available and which fully warrants further investigations.

Inhibition of Ca2+ uptake in freshwater carp, Cyprinus carpio, during short-term exposure to aluminum.

In carp exposed to pH 5.2 in fresh water, the Ca2+ influx from the water is reduced by 31% when compared to fish in water of neutral pH. At pH 5.2, the Ca2+ influx but not Na+ uptake is decreased by aluminum (Al). Al reduces Ca2+ influx dose-dependently: a maximum 55% reduction was observed after 1-2 h exposure to 200 micrograms.1(-1) (7.4 microM) Al. Branchial Ca2+ efflux is less sensitive to Al and affected only by exposure for more than 1 h to high Al concentrations. Na+ influx is not affected by concentrations Al up to 400 micrograms.1(-1). Na+ efflux, similarly to Ca2+ efflux, increased when fish were exposed for more than 1 h to 400 micrograms.1(-1) Al.

Regulation of secretion of the teleost fish hormone stanniocalcin: effects of extracellular calcium.

The release in vivo and in vitro of stanniocalcin (STC) from the corpuscles of Stannius (CS) of the rainbow trout and the European eel was studied. Intraperitoneal injection of CaCl2 (2.45 mmol.kg-1 fish) leads to an elevation of both ionic and total calcium in the plasma and results in the release of STC from the CS into the blood. Release of STC in vitro is not affected at "physiological" (1.0-1.5 mM) or lower Ca2+ levels in the incubation medium. High levels of Ca2+ (2.5 mM and higher), however, stimulate the release of STC, in particular that of stored STC. We hypothesize that variations in extracellular Ca2+ in the normocalcaemic range do not directly regulate STC release.

Inverse relationships between steroid concentration and volume in preovulatory follicles of the golden hamster.

In order to investigate variations in the microenvironment of oocytes within a cohort of maturing follicles the follicular volumes as well as the intrafollicular concentrations of oestradiol (E2) and progesterone (P) were measured in the golden hamster. At 10 h before ovulation the follicular volumes varied from 0.009 to 0.037 mm3 (mean +/- SD: 0.0187 +/- 0.0071 mm3; n = 36). Large follicles (greater than 0.025 mm3; n = 8) contained statistically significantly lower E2 and P levels (30.1 +/- 10.4 and 517 +/- 113 mumol/l, respectively) than the medium sized group (less than 0.025 and greater than 0.015 mm3; n =20): 46.9 +/- 16.0 (P less than 0.02) and 919 +/- 264 (P less than 0.0001) mumol/l, respectively. Small follicles (less than 0.015 mm3) showed the highest steroid levels: 97.0 +/- 33.3 and 1590 +/- 517 mumol/l for E2 and P (P less than 0.001 versus the medium sized group values). Correlation coefficients for the steroid concentrations and the follicular volumes appeared to be -0.674 for E2 and -0.612 for P (P less than 0.001). At the time studied a positive correlation between E2 and P concentrations in the follicles was found: r = 0.655 (P less than 0.001). The mean ratios of intrafollicular over serum steroid concentrations appeared to be approx 36 x 10(3) in the case of E2 and about 17 x 10(3) in the case of P. These results clearly show that there is an inverse relationship between follicular volume and intrafollicular steroid concentrations. The presence of a fine regulatory mechanism for a collective maturation of follicles is hypothesized.