Jane E. Aubin

Ultrastructural analysis of bone nodules formed in vitro by isolated fetal rat calvaria cells

When cells enzymatically digested from 21 d fetal rat calvaria are grown in ascorbic acid and Na beta-glycerophosphate, they form discrete three-dimensional nodular structures with the histological and immunohistochemical appearance of woven bone. The present investigation was undertaken to verify that bone-like features were identifiable at the ultrastructural level. The nodules formed on top of a fibroblast-like multilayer of cells. The upper surface of the nodules was lined by a continuous layer of cuboidal osteoblastic cells often seen to be joined by adherens junctions. Numerous microvilli, membrane protrusions, and coated pits could be seen on the upper surface of these cells, their cytoplasm contained prominent RER and Golgi membranes, and processes extended from their lower surfaces into a dense, highly organized collagenous matrix. Some osteocyte-like cells were completely embedded within this matrix; they also displayed RER and prominent processes which extended through the matrix and often made both adherens and gap junctional contacts with the processes of other cells. The fibroblastic cells not participating in nodule formation were surrounded by a less dense collagenous matrix and, in contrast to the matrix of the nodules, it did not mineralize. An unmineralized osteoid-like layer was seen directly below the cuboidal top layer of cells. A mineralization front was detectable below this in which small, discrete structures resembling matrix vesicles and feathery mineral crystals were evident and frequently associated with the collagen fibrils. More heavily mineralized areas were seen further into the nodule. Electron microprobe and electron and X-ray diffraction analysis confirmed the mineral to be hydroxyapatite.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of numbers of osteoprogenitors present in isolated fetal rat calvaria cells in vitro

When maintained in long-term cell culture in the presence of ascorbic acid and organic phosphate, single cell suspensions isolated from fetal rat calvaria form discrete, three-dimensional bone nodules. We have used limiting dilution analysis in microtiter wells to determine the number of osteoprogenitor cells expressing the capacity to form bone in the isolated mixed population, to examine the possibility of cooperativity among cell types in bone nodule formation, and to determine the effects of dexamethasone on osteoprogenitor cells. Cells plated at very low densities and screened for the presence or absence of bone nodules revealed a linear relationship (r = -00.997) between the number of cells plated and the number of bone nodules formed. The complete limiting dilution analyses showed that 1 of every 335 plated cells (0.30% of the cell population) has the capacity to form a bone nodule under standard culture conditions and when the actual numbers of nodules were quantitated from the same plated cell populations the ratio of nodules formed to plated cells was similar. Comparison of data from 13 different isolates of cells in which cells were plated into 35-mm dishes and number of nodules were determined indicated a mean +/- 95% confidence interval of one nodule for every 301 +/- 61 plated cells, consistent with the data obtained from the limiting dilution experiments. Dexamethasone increased the number of bone-forming cells to 1 in 225 cells, in contrast to 1 in 340 cells in the same population grown without added dexamethasone. The results suggest that approximately 0.30% of the cells in isolated rat calvaria populations are osteoprogenitor cells, that one osteoprogenitor cell gives rise to one bone nodule, that cooperativity between different cells in vitro is not necessary for bone formation, and that dexamethasone stimulates the expression of osteoprogenitor cells.

Determination of the capacity for proliferation and differentiation of osteoprogenitor cells in the presence and absence of dexamethasone

Osteoprogenitor cells present in single-cell suspensions prepared from fetal rat calvaria (RC) form discrete mineralized three-dimensional bone nodules when cultured long-term in the presence of ascorbic acid and beta-glycerophosphate. These cells (CFU-O) constitute less than 1% of the total cell population under standard culture conditions and their number is increased in the presence of dexamethasone. Using the formation of the bone nodule as a marker for CFU-O, we have now analyzed the proliferation and differentiation capacity of these CFU-O by redistribution and continuous subculture experiments in the presence and absence of dexamethasone. Cell redistribution experiments showed no increase in nodule number after one population doubling with either treatment. After 5.4 population doublings of the entire RC population, nodule number increased up to 2.0-fold in control cultures and 4.5-fold in cultures containing 10 nM dexamethasone. Continuous subculture experiments in which cultures were split 1:3 every 3 day for up to seven subcultures showed that nodule number decreased in parallel with the split ratio in the absence of dexamethasone, while with dexamethasone nodule number was elevated above the number present in primary cultures for 1 or 2 subcultures after which nodule number decreased with the split ratio. Bone nodules were present for up to 18 population doublings. Measurements of nodule area by automated image analysis showed that dexamethasone increased nodule size and that nodule size decreased from primary to 1st to 2nd subculture with or without dexamethasone. The data suggest that dexamethasone selectively stimulates the proliferation of osteoprogenitor cells and that these progenitor cells have a limited capacity for generating daughter cells capable of expressing the bone phenotype.

A band of F-actin containing podosomes is involved in bone resorption by osteoclasts

Isolated rabbit osteoclasts cultured on devitalized thin bone slices excavate resorption lacunae that can be visualized with brightfield or phase-contrast microscopy. Superimposition of the brightfield images of such resorption lacunae and the fluorescence images of the corresponding osteoclasts after fixation and staining with rhodamine-conjugated phalloidin revealed that a bright fluorescent band of F-actin-containing podosomes precisely outlined the resorption lacunae in stationary osteoclasts. When the resorption lacunae were being extended laterally, the clearly delineated band of podosomes corresponded to the advancing edge of the resorbing osteoclast and the most recently excavated part of the lacunae. Reshaping and reorganization of the bright bands preceded development of the lateral boundary of the lacunae. Podosomes forming these bands were highly dynamic, changed in size and location, and appeared and disappeared continuously. Their lifespan varied between 2 and 12 min. Similar bands were also seen in vivo in bone-resorbing osteoclasts on the endocranial surface of growing calvariae. Podosomes disappeared in osteoclasts treated with calcitonin, resulting in the disruption of the fluorescent bands. Our results suggest that podosomes are an essential part of the resorption apparatus of osteoclasts.

Continuously growing bipotential and monopotential myogenic, adipogenic, and chondrogenic subclones isolated from the multipotential RCJ 3.1 clonal cell line

The clonal multipotential RCJ 3.1 cell line, which gives rise to myotubes, adipocytes, chondrocytes, and osteoblasts, contains different progenitor subpopulations. By limiting dilution analysis, of 296 single colonies identified, approximately 20% contained a single recognizable cell type, approximately 10% contained two cell types, and approximately 1% contained three cell types. We recloned RCJ 3.1 and isolated continuously growing subclones, including four novel bipotential (adipocytes/chondrocytes; adipocytes/myotubes and chondrocytes/myotubes) cell populations, whose phenotypes bred true. In the bipotential subclones, single colony analyses confirmed the presence of single cells which could both self-renew the bipotential progenitors and give rise to their respective committed monopotential lineages. Eight subclones were restricted to a single cell lineage and were considered monopotential; one of these is a novel cell line differentiating into cartilage. Thus, we have isolated unique monopotential and bipotential progenitor cell lines which provide a valuable model for studying the mechanisms leading to lineage restriction in mesenchymal populations.

Multiple forms of SppI (secreted phosphoprotein, osteopontin) synthesized by normal and transformed rat bone cell populations: Regulation by TGF-β

Metabolic labeling has revealed that rat bone cell populations in culture synthesize several forms of the secreted phosphoprotein, SppI. Most cell populations produced two major [32PO4]-labeled forms that behaved anomolously on SDS-PAGE migrating at 60 kDa and 56 kDa on 10% gels and 55 kDa and 44 kDa on 15% gels. Minor forms of intermediate sizes were also resolved. In normal bone cells the 60 kDa form was predominant and was the only form produced by the clonal bone cell line, RCA 11, whereas the 56 kDa a form predominated in the transformed bone cell line, ROS 17/2.8. In all populations [35S]-methionine-labeling revealed SppIs at approximately 60 kDa but no 56 kDa form. Each form of SppI was specifically cleaved by thrombin which generated fragments of approximately 28 kDa. Transforming growth factor beta 1 increased SppI mRNA levels 3 to 6-fold within 24 h in the normal bone cells, but no increase occurred in the ROS 17/2.8 cells. The elevated expression of SppI was reflected in a selective increase in the synthesis of the [32PO4]-and [35S]-methionine-labeled 60 kDa SppIs.

Cytoskeleton rearrangements during calcitonin-induced changes in osteoclast motility in vitro

Although relatively little is known about osteoclastic degradation of bone, observations both in vivo and in tissue culture in vitro suggest that osteoclast motility is required. Osteoclasts with abundant motile activity and responsiveness to a bone-regulating hormone, calcitonin, have recently been isolated from rat femur and maintained in culture for short periods of time. Using indirect immunofluorescence with antibodies to tubulin and myosin and NBD-phallacidin staining for F-actin, we have studied cytoskeleton distributions in such osteoclasts, either untreated or treated with calcitonin. Untreated and unresponsive cells were well spread on the substratum and displayed striking motility in time-lapse cinematography. In these cells a fine network of microfilaments but no stress fibers could be seen. Ruffles, both at the cell periphery and on the dorsal cell surface, stained intensely for F-actin. In contrast, myosin staining appeared in a relatively diffuse dotty pattern that diminished toward the cell periphery and was absent from the ruffled borders. Microtubules labeled in a pattern similar to that typically seen in a variety of mononuclear cells with microtubules radiating from the perinuclear region toward the cell periphery. In cells that responded to calcitonin by ceasing motile activity and retracting cytoplasm, microtubules were not detectably altered. Both myosin and actin labeling, however, changed dramatically, with retraction fibers labeled brightly for actin but not for myosin. Early in cytoplasmic retraction, myosin stained most intensely as a ring at the cell periphery at the base of retraction fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Subclone heterogeneity in a clonally-derived osteoblast-like cell line

To analyze the phenotypic diversity of a clonal rat osteosarcoma cell line (ROS 17/2) we have subcloned the cell line and characterized four subclones, ROS 17/2-A.II, A.III, A.V, and A.XIV. The subclones retained many of the characteristics of the parent clone that are considered typical of normal osteoblast-like cells; they responded to parathyroid hormone and isoproterenol, and had a negligible response to prostaglandin E2 as measured by their respective changes in cyclic AMP concentration. In addition up to a 75% decrease in 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) binding was observed over a four-fold increase in cell density. The morphologies of the subclones varied from spindle-shaped, fibroblast-like to cuboidal. Doubling times varied from 24 to 48 hours, and basal alkaline phosphatase (AP) levels differed by as much as 10 times over the initial 3 months in culture. After 6 months (approximately 100 PDL), the population doubling time of subclone A.XIV decreased from approximately 48 to approximately 20 hrs and there was a 2.5 to 3-fold increase in saturation density. This cell line was designated A.XIV.1 and was compared to a thawed sample from frozen stock of the original A.XIV isolate, designated A.XIV.2. These two populations, the parent cell line (ROS 17/2) and subclone A.V had similar growth properties, but differed with respect to changes in their alkaline phosphatase activity (AP) with time in culture: that is, all clones increased AP with time but there was a three to five-fold difference in their respective AP levels at various times in culture. All clones except A.V exhibited decreased AP activity upon reaching their saturation densities.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of dexamethasone on expression and maintenance of cartilage in serum-containing cultures of calvaria cells.

SummaryThe effects of dexamethasone on the ability of cells enzymatically isolated from 21-day fetal rat calvaria to produce cartilage in vitro has been investigated. Primary cultures of single-cell suspensions of rat calvaria were grown for up to 28 days in vitro in α-minimal essential medium containing 15% fetal bovine serum, 50 μ/ml ascorbic acid, 10 mM Na β-glycerophosphate and dexamethasone at concentrations of 1 μM to 1 nM. Two types of nodules were present in dexamethasone-containing cultures. One has been characterized previously as bone (Bellows et al. 1986). The second morphologically resembled hyaline cartilage, possessed a strong Alcian blue-positive matrix and contained type-II, but not type-I, collagen. Both bone and cartilaginous nodules were spatially distinct and developed in isolation from each other. Cartilaginous nodules were found in the highest number at a dexamethasone concentration of 100 nM. Time-course experiments revealed that while the number of bone nodules increased continuously at least to day 28, the number of cartilaginous nodules remained constant after cultures had reached confluency. When cells were isolated separately from frontal and parietal bones and suturai regions, the greatest number of cartilaginous nodules developed from parietal bones. Since 21-day fetal rat calvaria contains 2 distinct patches of cartilage at the periphery of the parietal bones, it seems likely that this cartilaginous tissue is the origin of the cartilage cells. The results demonstrate that cultures of rat calvaria cells contain chondrocytes and possibly chondroprogenitor cells that are distinct from osteoprogenitors. Results support previous data that 100 nM dexamethasone permits the expression of and maintains the phenotype of chondrocytes in serum-containing cultures in vitro.

Effect of cells of epithelial rests of Malassez and endothelial cells on synthesis of glycosaminoglycans by periodontal ligament fibroblasts in vitro

Cultures of fibroblast-like cells (PLF) and epithelial rest cells (PLE) prepared from explants of porcine periodontal ligament synthesized and secreted four glycosaminoglycans (GAG) in differing proportions. The PLF produced predominantly chondroitin sulfate (greater than 60%) with smaller amounts of hyaluronic acid (HA) (17%), dermatan sulfate (13%), and heparan sulfate (7%), whereas PLE produced predominantly HA (greater than 80%). In coculture and under conditions of reciprocal transfer of conditioned media neither cell type affected the others GAG synthesis. Endothelial cells (EC), however, or their conditioned growth media, were able to stimulate increased GAG synthesis, especially HA, in PLF. A similar result was obtained with smooth muscles cells (SMC) cultured in EC growth media but here again PLE were unable to stimulate GAG synthesis by SMC. These findings suggest that the spectrum of GAG found in whole ligament results both from independent production by, and from interaction between, the different cell types within the ligament. The results also provide support for a general hypothesis that loose connective tissues, which are rich in HA, are formed and maintained under the influence of epithelial, including endothelial, cells.