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

Bone, 9, 155-163 (1988) Printed in the USA . All rights reserved .

8756-3282/88 $3 .00 + .00 Copyright © 1988 Pergamon Press plc

Ultrastructural Analysis of Bone Nodules Formed In Vitro by Isolated Fetal Rat Calvaria Cells U . BHARGAVA,' M . BAR-LEV, 2 C .G . BELLOWS,' and 7 .E . AUBINI Medical Research Council Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Toronto, Ontario MSS IA8 Canada 2 Department of Oral Biology, School of Dental Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978 Israel I

Address Jor correspondence and reprints : Dr . J .E . Aubin, MRC Group in Periodontal Physiology, Room 4384 Medical Sciences Building, University of Toronto, Toronto, Ontario M5S 1A8 Canada . Abstract

cells that appear fibroblastic, as well as cells with diverse osteoblast properties, perhaps reflecting their stage of differentiation (Aubin et al ., 1982 ; Hcersche et al ., 1985) . Cells capable of fully expressing the osteoblast phenotype would be expected to deposit an organic mineralizing matrix with features typical of bone formed in vivo . Attempts to assay the bone forming capacity of such isolated cell populations often involved reimplantation of the cells back into animals, either in diffusion chambers (Simmons et al ., 1982 ; Bab et al ., 1984 ; Ashton et al ., 1984), onto chorioallantoic membranes (Nijweide et al ., 1982) or by inoculation intramuscularly (Groot et al ., 1983 ; Moskalewski et al ., 1983) . Earlier attempts to produce mineralizing bone matrices by isolated cells in vitro (Binderman et al ., 1974 . 1979 ; Williams et al ., 1980) have been generally either difficult to reproduce or the material deposited did not resemble bone . After Tenenbaum and Heersche (1981, 1982) demonstrated that folded chick periostea formed bone in vitro more reproducibly in the presence of ascorbic acid and Na-(3-glycerophosphate, Ecarot-Charrier et al . (1983) showed that non-enzymatically isolated mouse calvaria cells cultured with the same two additives produced a bone-like material . Using enzymatically isolated fetal rat calvaria cells, we (Bellows et al ., 1986) and others (Nefussi et al ., 1985) have shown that discrete, three dimensional nodular structures arc formed reproducibly when fetal rat calvaria cells are cultured with these same additives . The nodules formed resemble woven bone histologically and immunolabelling has documented the presence of type I and 111 collagen and osteonectin and the absence of type 11 collagen (Bellows et al ., 1986) . The fact that nodule numbers are related to plated cell densities has allowed us to initiate studies on the effects of a variety of regulatory agents that both stimulate (Bellows et al ., 1987) and inhibit (Antosz et al ., 1987) the number of nodules (amount of bone) formed . To use this system as a precise model for studies of regulation of osteogenesis, we felt it important to verify that the nodular structures exhibited features closely resembling bone formed in vivo . Thus, we have analyzed nodules by transmission electron microscopy (TEM) . X-ray diffraction analysis and electron microprobe analysis, to determine ultrastructural features of the cells, the nature of the organic matrix and the type of mineral crystal deposited .

When cells enzymatically digested from 21 d fetal rat calvaria are grown in ascorbic acid and Na (f-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 fibroblastlike 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. All the ultrastructural features observed indicated that nodules resemble true bone and therefore are an excellent model to investigate parameters of bone formation and bone cell differentiation and metabolism in vitro .

Introduction Populations of cells enzymatically isolated from fetal rat and mouse calvaria have been defined as osteoblast-like based on a number of biochemical features (Peck et al ., 1964 ; Wong and Cohn, 1974, 1975 : Rao et al ., 1977) . Such populations arc, however . heterogeneous and comprise 155

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Materials and Methods Cell culture

Cells were isolated by sequential enzyme digestion from 21 d fetal rat calvariae as described (Auhin et al ., 1982), plated in a-MEM + 15% fetal bovine serum (FBS) and antibiotics (100 pg/ml penicillin G, 50 pg/ml gentamicin and 300 ng/ml amphotericin B), and incubated at 37°C in 5% CO, in air in r-75 Falcon tissue culture flasks for 24 h to separate viable (attached) from nonviable cells and debris (unattached) . For nodule formation, cells were prepared and grown as described previously (Bellows et al ., 1986) . Briefly, after 24 h . populations 11-V were washed in PBS, trypsinized . and pooled . Cells were plated at 3 x 10 4 cells/35 mm Falcon tissue culture dish in a-MEM supplemented with 15% FBS, antibiotics as above, 50 µg/ml ascorbic acid and 10 mM Na (3-glyeerophosphate . Medium was changed every 2-3 days . Electron microscopy At d 17-23, cultures were scanned on a Leitz inverted phase contrast microscope . Individual nodules of approximately 0 .4 mm diameter were marked by encircling the bottom of the culture dish with a felt-tipped ink marker. Once marked, the nodules were fixed in situ in 1 .7% glutaraldehyde in O .IM sodium cacodylate buffer, pH 7 .2 at 4°C for 1 .5 h, post fixed in 1% osmium tetroxide in the same buffer on ice for I h and then dehydrated through aa graded series of ethanols up to 100% . Equal volumes of ethanol and Epon-Araldite were then added to the culture dishes for I h, followed by three 30 min infiltrations with 100% Epon-Araldite . Finally an overnight infiltration with Epon-Araldite was carried out in a dessicator over P 2 O, . Specimens were then embedded in fresh Epon-Araldite and polymerised at 60°C in a vacuum oven for 48 h . Before peeling the plastic base of the tissue culture dishes, the marked nodules were again marked on the top surface of epon . The plastic base was peeled oft and the marked nodules were cut out using a saw and re-embedded in Epon-Araldite . 60-70 nm sections were cut perpendicular to the cell layer using an LKB ultramicrotome, stained with 7 .7%u uranyl acetate and Reynolds lead citrate and examined using Philips 300 and Hitachi 7000 transmission electron microscopes . Micrographs were taken using Kodak electron microscope film 4489 .

U . Bhargava et al . : UItrasimcture of bone nodules formed in vitro

long dimension of the apatite crystal, was calculated from corrected B, (002) values using the Scherrer equation (Klug et al ., 1974) . Results When primary fetal rat calvaria cells are prepared and plated as described at approximately 3 x 10 4 cells/35 mm dish in medium supplemented with ascorbic acid and (3glycerophosphate, they reach confluency by about day 6 . By day 9, regions containing distinctly more polygonal cells are evident (Fig. la) . These areas increase in number and size until, by day 17-21, they are large (--0 .1-0 .5 mm'), very three-dimensional opaque nodular structures (Fig . lb), which we have shown previously to be mineralized, bone-like tissue (Bellows et al ., 1986) . Several nodules from 20-21 day cultures were marked and prepared for TEM as outlined in "Materials and Methods," The overall structural organization of a typical nodule cut in cross-section is shown in an EM montage (Fig . 2) . Areas of the cell multilayer not engaged in hone formation and the bottom portion of the nodule (side towards the tissue culture dish) comprised relatively fibreblast-like cells surrounded by collagenous matrix (Fig . 3) . The upper surface of the nodule (towards the culture medium) was lined by relatively cuboidal osteoblast-like cells (Figs . 4 and 5), on whose upper surface blebs, microvilli and other membrane protrusions, and coated pits were evident (Fig . 5) . Abundant Golgi and RER were also seen in

Electron microprobe and electron diffractionanalysis

Electron microprobe and electron diffraction analyses were performed on sections adjacent to those used for TEM . Electron microprobe analysis was done with a Link Analytical Model AN 10000 connected to a Philips EM 430 . X-raydiffraction analysis

Nodules from 21 d cultures were cut from the bottom of culture dishes with a scalpel and Freeze-dried . Lyophilized culture samples were analyzed with a Rigaku microdiffractometer with CuK radiation and a highly crystalline mineral fluoroapatite as a standard . The value of B, (002), the width at ' the maximum height of the hydroxyapatite (002) reflection, was measured using a step scanning procedure . D(002) . which is related to the crystal size and strain in the

Fig . 1 . (a) Phase contrast micrograph of two developing nodules (arrowheads) . A covering layer of plump osteoblast cells is evident and is at a different focal plane from the fibroblastic cell layer. (b) Phase contrast micrograph of a mineralized nodule . The nodule is opaque and three-dimensional . Note the osteoblasts covering the sides of the nodule (arrowheads) . Magnification : x55 .

U . Bhargava et al . : Ultrastructure of bone nodules formed in vitro

157

2 Fig. 2 . Electron micrograph montage illustrating the overall organization of a nodule cut in cross-section . Areas of the RC cell multilayer not engaged in bone formation and the bottom portion of the nodule (side towards the tissue culture dish) are composed of relatively fibroblast-like cells (arrows) surrounded by collagenous matrix . The upper surface of the nodule (towards the culture medium) is covered by a layer of more cuboidal osteoblast-like cells . Osteocyte-like cells are within the nodule and are completely surrounded by a dense collagenous matrix (arrowheads) . Mineralization has begun in this nodule (crossed arrow) ; an unmineralized osteoid seam is below the osteoblast layer of cells, while further into the nodule mineralization is further advanced . Magnification : x 480 .

these cells . Osteocyte-like cells completely surrounded by collagenous matrix were present further into the nodule (Figs . 4 and 6) . These cells displayed abundant RER and numerous cell processes extending through the extracellular matrix (Fig. 6) . A variety of junctional complexes were present between cells in the nodule . Electron dense adherens-type junctions with associated microfilament-rich cortical cytoplasm were frequently observed between adjacent osteoblasts (Figs . 7 and 8) and between osteocyte processes (Fig . 9) . In addition, junctions with the appearance of gap junctions were present between osteocyte processes (Figs . 10 and 11) . Banded collagen fibers, in orthogonal arrays, were densely packed throughout the nodule, right up to the lower surface of the ostcoblasts and completely surrounding osteocytes (Figs . 5 and 6) . An unmineralized osteoid seam underlay the osteoblast cell layer (Fig . 4), while further into the nodule mineral deposits were evident (Figs . 4 and 6) . In those regions showing mineralization . numerous vesicular structures with a hilayer membrane resembling matrix vesicles (Fig . 12a and b) and wispy needle-like mineral deposits (Fig . 13) were apparent . In more heavily mineralized areas, the mineral appeared very electron dense and amorphous (Fig . 14) . Mineralization was not observed in the cell multilayers of fibroblastic cells at the edge of the nodules or in other parts of the culture dish, despite the presence of banded collagen in those regions (e .g ., Fig . 3) . In certain culture dishes, mineralization occurred in fbroblastic regions of the cell layer, but examination with TEM and electron microprobe analysis revealed little matrix, and the mineral, chiefly consisting of

phosphorous, to be associated with areas of cell death (not shown) . To determine the nature of the mineral, we performed both electron diffraction analysis and X-ray diffraction analysis . Electron microprobe analysis of epon-araldite embedded sections of nodules revealed the mineral to consist of Ca and P (Fig . 15a) with small amounts of Os and Ph occurring from the fixation and staining procedures and Cu from the grid . Electron diffraction analysis (Fig . 15b) showed the mineral to be crystalline with the characteristic structure of hydroxyapatite . X-ray diffraction of freezedried powders prepared from several nodules confirmed that the mineral deposited in the nodules is hydroxyapatite and the D (002) of 120 ± 5 SEM (Table I) is characteristic of the early mineral deposited in embryonic chick bone (Sonar et at ., 1983) . Discussion Our observations clearly demonstrate that cells present in single cell suspensions prepared from fetal rat calvaria can grow and differentiate in vitro to produce nodular structures recognizable as hone. Previously, we described the histological appearance of the nodules ; i .e ., the overall characteristics of cells and matrix, the presence of high alkaline phosphatase activity associated with the covering osteoblastic layer, the intense Van Gieson staining of the matrix and Von Kossa staining, and by immunolabelling, verified the presence of type I and the absence of type II collagen (Bellows et al . . 1986) . We have now described ultrastructural details of the nodules and surrounding cellular



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U . Rhargnva et at Ulirastructure of bone nodules formed in vitro

Fig . 3 . The cell multilayer at the edge of a nodule . Note the fibroblastic cells and the surrounding collagenous matrix . Mineralization is not observed in these or other Fibroblastic areas in the culture dish . Magnification : x 2,11811 . Fig . 4 . The upper surface of the nodule is lined by relatively cuhoidal osteoblast-like cells (OR ; drown at higher magnification in Fig . 5) . Osteocyte-like cells (OC) completely surrounded by collagenous matrix are present further into the nodule . The orthogonal arrangement of collagen fibers can he seen larrowheads)- An smmineralized ostcoid seam underlies the osteoblast layer ; below that mineralization is heavier. Magnification : x 2,400



U . Bhargava et al . : Ultrastructure of bone nodules formed in vitro

159

Fig . 5 . The osteoblast-like cells at the upper surface of the nodule . The upper surface of these cells displays blebs, microvilli and other membrane protrusions . Abundant Golgi and RER are evident . Banded collagen fibers (arrows) . in an orthogonal packing, are present right up to the ventral cell surface . Early mineral deposits (arrowheads) are evident throughout the collagenous matrix . Magnification : x 8,500 . Fig . 6 . An osteocyte completely surrounded by dense collagenous matrix . Note the abundant RER and the cell processes (arrowheads) extending through the extracellular matrix . Early mineral deposits are present . Magnification : x 5,100 .



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U . Bhargava et al . : Ultrastnicture of bone nodules formed in vitro

Fig . 7 . Junctional complexes are abundant between cells in the nodule . Note the close proximity of the two osteoblastic cells at the upper surface of the nodule . Adherens-type junctions are evident between the cells (arrows) . Note also the microfilament-rich cortical (subplasma membrane) cytoplasm (arrowhead) . Magnification x 14,400 . Fig. 8 . Higher magnification of the junctions between the same two cells as shown in Figure 7 . Magnification : x41,600 . Fig. 9 . An adherens-type junction between a cell process of one osteocyte and the cell body of another (arrow) . Note the cortical microfilament bundles (arrowheads) . Magnification : x64,000 . Fig. 10. Gap junctions are present between two osteocyte processes deep within the collagenous matrix in the nodule (arrows) . Early mineralization is also evident in this micrograph . Note the wispy mineral deposits close to the cell processes (arrowheads) . Magnification : x 41,6(X), Fig. 1t . Another gap junction is shown between two cells (arrow) . Banded collagen fibers are present (arrowheads) . Magnification : x 84,000 .

U . Bhargava et al . : Ultrastructure of bone nodules formed in vitro

161

Fig. 12 . (a) and (b) Matrix vesicles (arrowheads) within the collagenous matrix . Magnification : x 50,000 . Fig . 13 . A feathery, early mineralization site within the collagenous matrix . x 70,000 . Fig . 14 . Heavier mineralization is associated with banded collagen fibers further into the nodule . x 50,000 . areas which strengthen our conclusion that the tissue formed in vitro is bone . The overall morphology of the nodule, the relationship of cells to matrix, and matrix-mineral characteristics, are reminiscent of a true osseous structure . The presence of a well-developed Golgi, and abundant rough ER and mitochondria, are indicative of biosynthetically active cells in general and osteoblastic cells in situ (Weinger et al ., 1973 ; Holtrop, 1975) . The nodule surface comprised a continuous layer of cuboidal cells, observed to be attached to each other via numerous adherens junctions . Both adherens junctions and junctions resembling gap junctions were seen between cell processes from adjacent osteocytes . Junctional complexes were also observed between cells of the osteoblast layer and osteocytes located further into the nodule . Thus, it appears that tight associations have been made between the cells in vitro and cells within the nodule may receive nutrients and information via the gap junctions present, consistent with the concept that cells within bone form an integrated network (Doty and Schofield, 1972 ; Holtrop, 1972 ; Doty, 1981 ; Matthews and Davis, 1985) . In this regard, the junctional morphology of the cells in the nodule resembles that described for bone

cells in vivo with respect to gap junctions (Holtrop and Weinger, 1972 ; Whitson, 1972 ; Holtrop, 1975 ; Miller et al ., 1980 ; Doty, 1981) . Whether the gap junctions formed in these cultured cells are truly functional remains to be investigated and is of interest based on the observations of communicational (dye transporting) gap junctions in calvaria (Jeansonne et al ., 1979) . Junctions of the adherenstype with associated microtilaments and electron dense plaques (Volk and Geiger, 1986) were also observed between osteoblasts, and osteoblasts and osteocytes in the nodules . Although gap junctions between cells in bone in vivo have been well-described, no comparable studies describing adherens-type junctions have been done . It seems possible from the abundance of microfilaments observed in some of the published micrographs that at least some of the junctional complexes may be adherens-type . The latter junctional type appears now to be widespread in many cell types, including those forming tight cell-cell associations where they help to define cell polarity and maintain cell shape (Volk and Geiger, 1986) . It is also interesting that the upper surfaces of the osteoblasts (towards the medium) were noticeably convoluted and contained blebs, microvilli-like structures and coated pits, which may he present



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U . 13hargava et al . : Ultrastructure of bone nodules formed in vitro

Ca

Cu

P

I

CU

k

O .Y

15a Fig . 15 . (a) Electron microprobe profile of a mineralized area of a nodule . The mineral consists of phosphorous (P) and calcium (Ca) .

Traces of lead (Pb) and osmium (Os) from the fixation and staining procedures and copper (Cu) from the grid are present . (h) Electron diffraction pattern of the mineral deposit shown in A . The mineral has a crystalline structure that is characteristic of hydroxyapatite . to increase cell surface area and increase nutrient uptake as the nodules increase in size and volume . Highly organized, densely packed, orthogonally arranged, cross-handed collagen was seen throughout the nodule . In nodules undergoing mineralization, initial mineralization areas were marked by feathery or needle-like crystals . Most of the mineral was found in association with collagen fibers cut either lengthwise or in cross-section, but mineral-containing vesicular structures morphologically identical to matrix vesicles (Anderson . 1984 ; Boskey, 1981) were also observed . An unmineralized osteoid scam was seen directly under the osteoblast layer . SEM studies have confirmed the dense collagenous nature of this material (Nefussi et al ., 1985) . Mineral deposits were not normally seen in fibroblastic areas adjacent to nodules or in other non-nodular areas of the culture dish, despite the abundant collagenous matrix in those regions . Only when obvious necrotic cells were present was mineralization seen in non-nodular areas and

Table 1 . X-Ray diffraction analysis of bone nodule samples . Sampling spot 1

Mean

D-002

R,,,

D-002( A)

0 .85' 0 .73° 0-78° 0 .75°

0 .76° 0.63 1 0 .69° 0 .65°

107 130 118 126 120A t 5

The mean size of 120A ± 5 .0 SEM is indicative of hydroxyapatite crystals .

this mineral was amorphous in nature and found not to he hydroxyapatite by electron microprobe analysis (not shown) . In contrast, the mineral deposited in nodules was verified to he hydroxyapatite by electron microprobe analysis and electron diffraction (Mrose, 1975) . In addition . X-ray diffraction analysis indicated that the crystal dimensions were the same as those found in early mineral deposits in embryonic chick bone (Bonar et al ., 1983) . Thus, the specificity of location of the mineral and the nature of the deposited crystal are characteristic of intramemhranous osteogenesis in vivo (Arsenault and Ottensmeyer, 1984) and point to a highly regulated, rather than non-specific, mineralization process in our in vitro model system . Previously we have documented the reproducibility and quantitative aspects of nodule formation by primary and subcultured rat calvaria cells (Bellows et al . . 1986, 1987 ; Antosz et al ., 1987) . The observations we report here indicate that the nodular structures formed in vitro by isolated rat calvaria cells comprise cells and matrix identifiable as bone at the ultrastructural level . Moreover, the fact that mineral is deposited onto banded collagen within the nodules, but not in adjacent areas of fibrohlastic cells implies that efficient regulatory mechanisms are in play in this system in vitro . Therefore, this is an excellent model in which to investigate in vitro parameters of bone formation and its regulation by hormones and growth factor.

A(knowledgments : We thank Mr. I) . Holmyard and Dr . H .C . Tenenbaunr for performing the electron microprobe and electron (fit -

Traction analysis and Dr. M . Grynpas for performing the X-ray diffraction analysis .

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U . Bhargava et at . : Ultrastructure of bone nodules formed in vitro

References Anderson H .C . : Mineralization by matrix vesicles . Scanning Electron Microscopy 2 :953-964, 1984 . Antosz M .E ., Bellows C .G . and Aubin i .F, . : Biphasic effects of epidermal growth factor on bone formation by isolated rat calvaria cell populations in vitro, J . Bone Mineral Res . 2:385-393, 1987 . Arsenault A .L . and Ortensmever FP. : Visualization of early intramembranous ossification by electron microscopic and spectroscopic imaging . J . Cell Biol. 98 :911-921, 1984. Ashton B .A ., Eaglesom C .C., Bab I . and Owen M,L . : Distribution of tibroblastic colony-forming cell in rabbit hone marrow and assay of their osteogenic potential by an in vivo diffusion chamber method . Caln) : 7iss . Lit. 36 :83-86, 1984 . Aubin 1.E . . Hecrsche J .N .M ., Merrilees M .J . and Sodas J . : Isolation of hone cell clones with differences in growth, hormone responses and cxtracellular matrix production . .1 . Cell Biol . 192 :452-461, 1982 . Bab I ., Ashton B .A ., Syftestad G .D . and Owen M .E . : Assessment of an in vivo diffusion chamber method as a quantitative assay for osteogenesis . Calrif. Tiss . Ira . 36 :77-82, 1984 . Bellows C .G . . Aubin 1 .E . . Hecrsche J .N .M . and Antosz M .E . : Mineralized bone nodules formed in vitro from enzymatically released rat calvaria cell populations . Calrif. Tiss. Inn . 38 :143-154, 1986 . Bellows C .G., Aubin 1 .E ., Heersche J .N .M . : Physiological concentrations of glucocorticoids stimulate formation of bone nodules from isolated rat calvaria cell in vitro . Endocrinology 121 :1985-1992 . 1987 . Binderman 1 . . Duksin D ., Harell A ., Katzir E . and Sachs L . : Formation of bone tissue in culture from isolated hone calls . J . Cell Biol . 61 :427-439, 1974 . Binderman I ., Greene R.M . and Pcnnypacker J.P. : Calcification of differentiating skeletal mesenchyme in vitro . Science 206 :222-225, 1979 . Bonar L .C ., Roufosse A .H . . Sabine W.K., Grynpas M .D. and Glimcher M .J . : X-Ray diffraction studies of the crystallinity of bone mineral in newly synthesized and density fractional bone . Cabal : Ti.rs . Inr . 35 :202= 09, 1983 . Boskey A .L . : Current concepts of the physiology and biochemistry of calcitication . 111 . Basic science and pathology . Clin . Orthop . 157 :225-257, 1981 . Doty S .B . : Morphological evidence of gap junctions between bone cells . Catch' . Tiss . Izn . 33 :509-512, 1981 . Doty S .R . and Schofield B .H . : Metabolic and structural changes within osteocytes of rat bone, In : Calcium, parathyroid hormone and the calcitonin .r . R .V. Talmage and P.L . Munson, eds . Amsterdam : Exccrpta Mee dica pp . 353 364 . 1972 . Ecarot-Charrier B ., Glorieux F.H ., van der Rest M . and Pereira G . : Ostcohlasts isolated from mouse calvaria initiate matrix mineralization to culture. J . Cell Blot. 96 :639-643 Grant C .G . . Moskalewski S ., Schetft J.P. and Boonekamp P.M . : Electron mic'oscopy of bone formed by syngeneic transplanted calvarial osteoblasts . Cell Biol . Int . Rep . 7 :577. 1983 . Hancox N .M . : In : Biology of Bone . New York : Cambridge University Press . t972 . Heersche J .N .M ., Aubin I .E ., Grigoriadis A .E . and Mmiva Y. : Hormone responsiveness of hone cell populations . Searching for answers in vivo and in vitro . In : The Chemisrn' and Biology of Mineralized Tissues. W.T. Butler . ed . Birmingham : Ebsco Media, Inc . pp. 286-29x, 1985 . Holtrop M .E . : The Ultrastructure of Bone- Annals CYin . and Lab . Sri. 5 :264-271 . 1975 . Holtrop M . F . and Weinger J . M . : Ultrastructural evidence for a transport system in bone . In : Calcium, parathyroid hormone and the calcironins . R.V. lalmage and P.L . Munson .. eds . Amsterdam : Exce pta Medico pp . 365-374, 1972 . Jeansonne B .G., Feagin LA . . McMinn R.W . Shoemaker R.L . and Rehn

W.S . : Cell-to-cell communication of osteoblasts . J. Dent . Res . 58:14151423 . 1979 . King A .P. and Alexander I .E . : X-Ray diffraction procedures for polycrystalline and amorphous materials . 2nd edition . New York : John Wilev and Sons, pp . 687-692, 1974 . Matthews J .L_ and Davis W.L . : Ultrastruchu'al Aspects of Osteogenesis . In : the Chemistry and Biology of Mineralized Tissues . W.T . Butler- ed . Birmingham : Ebsco Media, Inc . pp . 15-21, 1985. Miller S .C ., Bowman B .M ., Smith 3 .M . and Jee W.S .S . : Characterization of endasteal bone-lining cells from fatty marrow bone sites in adult beagles. Annt . Rec . 198 :163-173, 1980 . Moskalewski S ., Boonekamp PM . and Scharf: J .P. : Bone formation by isolated calvarial osteoblasts in syngeneic and allogenic transplants : light microscopic observations . Am . J. Anat, 167 :249-263 .. 1983 . Morse M .E . : Minerals . In : Mineral Powder Diffraction File: Data B ook. J .C .P.D .S . International Center for Diffraction Data, Pennsylvania . Ref. No . 9-432 . p . 446, 1975 . Nefussi J .-R ., Bay-Lcfcvrc M .L., Boulekhachc H . and Forest N . : Mineralizalion in vitro of matrix formed by osteoblasts isolated by collagenase digestion . Differentiation 29 :160-168, 1985 . Nijweide P.J ., van Iperen-van Gent A .S ., Kawilarang-de Haas R .W.M ., van der Plas A . and Wassenaar A .M . : Bone formation and calcification by isolated osteoblastic-like cells . .1 . Cell Binl. 93 :318-323, 1982 . Peck W.A ., Birge S .J, and Fedak S .A . : Bone cells : biochemical and biological studies after enzymatic isolation . Science 146:1476-1477 . 1964 . Ran L .G_. Ng B ., Brunette D .M . and Hecrsche J .N .M . : Parathyroid hormone and prostaglandin E,-response in a selected population of bone cells after repeated storage and subculture at -80°C . Endocrinology 100 :1223-1241, 1977, Simmons D .J . . Kent G .N ., Jilka R .L ., Scott D .h1 . . Fallon M . and Cohn D .V. : Formation of bone by isolated, cultured osteohlasts in millipore diffusion chambers . Calcif. Tiss- Int . 34:291-294, 1982 . ienenbaum H .C . Role of organic phosphate in mineralization of hone in vitro . J . Dent Res . 60 :1568-1571 . 1981 . Tenenbaum H .C . and Heersche J .N .M . : Differentiation of osteoblasts and formation of mineralized hone in vitro . Calrif. Turn Inr . 34 :76-79, 1982 . Volk T- and Geiger B- : A-CAM : A 135-KD receptor of intercellular adherensjunctions . I . Immunoelectron microscopic localization and biochemical studies . J . Cell Riot 103 :1441-1450 . 1986 . Weinger J .M . and Holtrop M .E . : An ultrastructural study of bone cells : The occurrence of microtubules, microfilaments and tight junctions . Calrif. Tiss Res . 14 :15 29, 1973 . Whitson SW : Tight junction formation in the cistern . Clin . Orr/toped . Rel, Res . 86 :206-213, 1972 . Williams D .C ., Boder G .B . . Tooney RE_. Paul D .C ., Hillman C .C . . King K .1 . ., Van Frank R .M . and Johnson C .C . : Mineralizatinn and metabolic response in serially-passaged adult rat bone cells . Calcif Tiss . Lu . 30223-246, 1980 . Wong G . and Cohn D V. : Separation of parathyroid hormone and calcitonin-sensitive cells from non-responsive bone cells . Nature 252^13715, 1974 . Wong G . and Cohn D .V. : target cells in bone for parathormone and catcitonin are different : enrichment for each cell type by sequential digestion of mouse calvaria and selective adhesion to polymeric surfaces . pros. Nar. Acad . Set. 72 :3167-3171 . 1975 .

Received: September 14, 1987 Rerised: November20, 1987 Acrepied: December I . 1987