Fine structure of cultured human gingival fibroblasts and demonstration of simultaneous synthesis of types I and III collagen

ArcnsoralBiol.Vol.25,pp.28310296 Pergamon PressLfd 1980. Printed in Great Britain

FINE STRUCTURE OF CULTURED HUMAN GINGIVAL FIBROBLASTS AND DEMONSTRATION OF SIMULTANEOUS SYNTHESIS OF TYPES I AND III COLLAGEN D. ENGEL*, H. E.

SCHROEDER~,

R.

GAYS

and J. CLAGETT*

Department of Periodontics* *and Center for Research in’ Oral Biology, University of Washington, Seattle, WA 98195, Department of Oral Structural Biology?, Dental Institute, University of Zurich, CH-8028, Zurich, Switzerland and the Institute for Dental Research& University Medical Center, Birmingham AL 35209, U.S.A.

Summary-A strain of human gingival fibroblasts, obtained from clinically normal gingiva, was examined by electron and immunofluorescent microscopy to determine: (i) their fine structure; (ii) whether the cultures also contained smooth muscle or endothelial cells; and (iii) the collagen synthesis activities of single cells. Cultured gingival fibroblasts were homogeneous in their fine structure with features similar to those reported for cultured human skin fibroblasts. They differed from fibroblasts in uiuo in that the cultured cells had less rough endoplasmic reticulum, no collagen-containing vacuoles and a more prominent microfilament network. Smooth muscle or endothelial cells were not seen in the cultures. By electron microscopy, none of the cells had cell-to-cell junctions typical of cultured smooth muscle cells, or Weibel-Palade bodies, which are unique to endothelial cells. By immunofluorescent microscopy, none of the cells contained type IV collagen, a phenotypic marker for endothelial cells. Furthermore, type III procollagen, a major product of smooth muscle cells, was present in only small amounts. Simultaneously staining for type I collagen and type III procollagen demonstrated that most cells contained both types of collagen, although some cells contained only type I. The latter finding suggests that functionally distinct subpopulations of human gingival fibroblasts may exist.

INTRODUCTION Collagen is a major component of the gingiva, and many aspects of gingival disease are due to qualitative

and quantitative changes in this connective tissue substance. The fibroblast is the principal collagenproducing cell in the gingiva. Fibroblasts derived from human gingiva may be propagated for long periods of time in culture (Tsutsui, Hirokawa and Maizumi, 1973), and cultured gingival fibroblasts are widely used to study various aspects of collagen synthesis. For instance, it has been reported that fibroblasts obtained from inflamed gingiva synthesize a different type of collagen in culture than do cells derived from clinically normal gingiva (Narayanan and Page, 1976; Narayanan, Page and Kuzan, 1978), and that fibroblasts obtained from patients with Dilantinassociated overgrowth of the gingiva synthesize a greater amount of collagen in vitro than do cells from the gingiva of normal subjects (Hassell et al., 1976). In spite of wide use in research, the fine structural features of human gingival fibroblasts have not been thoroughly characterized. It is not known how they compare morphologically with cultured fibroblasts from other tissues, and with gingival fibroblasts in vim Furthermore, there is no proof that fibroblast cultures obtained from gingival explants do not also contain smooth muscle and/or endothelial cells. It is known that both smooth muscle and endothelial cells grow well in culture, and that they synthesize significant amounts of collagen. Endothelial cells are typiCAB.25j5 -A

fied by synthesis of type IV collagen (Howard er al., 1976). Smooth muscle cells and fibroblasts synthesize both types I and III collagen, but in different ratios (Burke et al., 1977; Narayanan and Page, 1976). The fine structural features of cultured human smooth muscle cells and endothelial cells have recently been described (Gimbrone and Cotran, 1975; Haudenschild et al., 1975). Recent evidence indicates that at least two subpopulations of cells exist in cultures of gingival fibroblasts as approximately half of the cells are sensitive to inhibitory effects of prostaglandin E, and half are not (Ko, Page and Narayanan, 1977). Whether or not morphological heterogeneity exists, or if there are distinct subpopulations which synthesize different types of collagen (i.e., type I vs. type III) is not known. Therefore to characterize better cultured human gingival fibroblasts morphologically, to determine if the fibroblast cultures also contain other cell types, and to find out if subpopulations of gingival fibroblasts may exist with regard to collagen synthesis activity, we have done electron and immunofluorescent microscopic studies. MATERIALS AND

METHODS

Cell culture techniques

A gingival fibroblast cell strain was derived from an interdental biopsy of clinically noninflamed gingiva from a 24 yr old male using the explant method of 283

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Martin (1973). This cell strain, designated HGF.,, was confirmed to be diploid by karyotype analysis, and to be free of Mycoplasma and bacterial contamination. Monolayer cultures of these cells were maintained in Dulbecco-Vogt modified Eagle’s medium (Priest, 1969) supplemented with 10 per cent fetal calf serum (Flow Laboratories, Rockville, MD), 100 units/ml of penicillin and lOOpg/ml of streptomycin. Stock cultures were grown in plastic tissue culture flasks in an atmosphere of 5 per cent CO1 in air at 37°C. Upon reaching confluency, cultures were dispersed with 0.05 per cent trypsin in versene-buffered saline (pH 7.2) and transferred into 2 or 3 new flasks. Generally, this was done every 5 to 8 days. Culture medium was renewed twice a week. For immunofluorescent studies, 50 pg/ml of ascorbic acid was added to the medium, The culture ages of cells described in this paper were between 5 and 15 transfers after removal of the cells from the primary explant. Smooth muscle cells derived from human fetal artery were a gift from Dr. Gary Striker, University of Washington, and were cultured and passaged as described for the HGF* cells above. Electron microscopy Cells were seeded on 35 x 1Omm tissue culture dishes (no. 3001, Falcon Plastics, Oxnard, CA) and incubated as described above until reaching near confluency. At that time the cells were washed with medium lacking serum and fixed for 20 min in situ on the dishes with 2 per cent glutaraldehyde buffered with 0.05 M sodium cacodylate at pH 7.2. The cells were rinsed briefly with cacodylate buffer and postfixed for 1 h with a 1 per cent osmium tetroxide solution buffered with s-collidine (pH 7.3). The cells were then stained with aqueous 0.5 per cent uranyl-acetate in the dark for lOmin, rinsed with distilled water, dehydrated in a graded series of ethanol for 15 min, and infiltrated with Epon 812 directly in the tissue culture dish as described by Ross (1971). Briefly, this technique involves inverting BEEM capsules (Ted Pella, Altadena, CA) filled with Epon over partially polymerized Epon in the tissue culture dish. The Epon is then fully polymerized at 60°C for 48 h and the capsules, along with an underlying circular area of embedded cells in monolayer, are snapped free from the dish. The advantage of this technique is that the cells can be examined in the same morphologic and topographic relationships they had during culture, and no artifacts are introduced by trypsinizing or scraping the cells free of the plate for embedding. Ultrathin sections from these blocks were cut parallel to the plane of the monolayer using a diamond knife (DuPont) and an LKB-III ultratome. The sections were mounted on carbon-coated copper grids, stained with uranyl-magnesium acetate followed by lead citrate (Fraska and Parks, 1965; Reynolds, 1963), and the fibroblasts were examined and photographed with a Phillips EM-201 electron microscope. Measurements of cytoplasmic filament diameters were done on positive film copies of a series of 30 electron micrographs taken at a primary magnification of x 19,800 from cytoplasmic portions containing filaments in longitudinal and cross section, a magnification standard being included in the sample. Mul-

tiple measurements of filament thickness and diameter respectively were made at a final magnification of x 226,500. lmmunofluorescent microscopy Rabbit antibodies to bovine types I and III procollagens and type IV collagen were a gift from Dr. George Martin, National Institute of Dental Research and had been prepared and rendered specific by immuno-absorption as previously described (Nowack et al., 1976). The isolated antibodies show crossreactions with bovine and human tissue structures when studied by immunohistological techniques (Nowack et al.). In double-staining experiments, where we simultaneously stained with antibodies to type III procollagen and antibodies to type I collagen, the antibodies against human type I collagen were obtained from rats and purified by immunoabsorption. Monolayers of HGF, and fetal smooth muscle cells were cultured individually on culture chamber slides (Lab-Tek No. 4804, LabTek Products, Naperville, IL) for 36 h. The cells were then fixed for 20 min with 3 per cent formaldehyde in phosphate-buffered saline (PBS; pH 7.4), washed briefly in PBS, and immersed in ice-cold acetone for 10 min to render the cells permeable. The cells were then rinsed with PBS, air dried and treated for 30 min in a moist chamber with one of the specific anti-procollagen antibody preparations, or with the IgG fraction of normal rabbit serum (Cappel Labs, Downingtown, PA) as a negative control. After a thorough wash with PBS, the cells were stained with rhodamine-conjugated goat anti-rabbit IgG (Cappel Labs) for 30 min in a moist chamber, washed with PBS and mounted in tris-buffered glycerol (pH 7.0) for viewing and photography with a Zeiss model RA fluorescent microscope. In some experiments, we used a double-labelling technique described by Gay et al. (1976). Air-tied monolayers of HGF4 cells were labelled consecutively with rabbit antibodies to type III procollagen, rat antibodies to type I collagen, rhodamine-conjugated anti-rat globulin (Nordic Immunology, Tilbure, Netherlands, 1 :lO dilution) and finally, with a 1:30 dilution of fluorescein-isothiocyanate-conjugated antirabbit globulin (Behring-Werke, Marburg, Germany; or Cappel Labs). The sections were incubated with each antibody for 30min in a moist chamber and rinsed with PBS between each step. Finally, the monolayer cultures were extensively washed with PBS and sealed with a PBS-buffered glycerol. The fluorescent preparations were observed and photographed with a Leitz microscope with a KZ-filter system for fluorescein and a NZ-filter system for rhodamine fluorescence. RESULTS

Electron microscopy

Figure 1 shows the general morphology of cultured human gingival fibroblasts. In a plane parallel to that of cellular spreading, the cells exhibit fusiform or spindle-like profiles. The nuclei were usually elliptically shaped with a smooth nuclear membrane, although some indented forms were seen (Fig 2). The nuclei commonly contained 2 to 5 nucleoli, a feature

Cultured human gingival fibroblasts

characteristic for rapidly growing cells with a high rate of protein synthesis (Rhodin, 1974). Occasionally, mitotic division was observed (Fig. 1). In many instances, there appeared to be cell-to-cell contact (Figs. 1 and 2). However, we did not observe gap-type junctions or attachment sites such as Ross (1971) and Gimbrone and Cotran (1975) reported for cultured smooth muscle cells. The paranuclear cytoplasm contained most of the organelles; at the poles of the cell spindle, microfilaments and polyribosomes prevailed (Fig. 1). At higher magnification, the paranuclear polarization along the spindle axis of cytoplasmic organelles such as the rough endoplasmic reticulum, Golgi fields and mitochondria were more obvious (Fig. 2). Mitochondria were elongated, twisted, and occasionally branched. The rough endoplasmic reticulum (RER) comprised a network of narrow cisternae around the nucleus and parallel to the cell axis (Fig. 2). These RER-cisternae were often seen in tangential planes and then seemed slightly dilated. They regularly contained moderately electron dense, finely granular material (Fig. 3, left inset). The RER-membranes were studded with bound ribosomes which, in tangential sections, were aligned in circular, spiral patterns (Fig. 3, left inset). Each cell usually contained several paranuclear Golgi fields (Fig. 3B), composed of arrays of packed cisternae and numerous small vesicles (Figs. 2 and 3B). Single ribosomes and polyribosome aggregates consisting of up to 6 ribosomes were widely distributed between other cell organelles as well as in perinuclear regions (Fig. 2). A major cytoplasmic constituent was interdigitating bundles of filaments of similar size arranged parallel with the long axis of the cell (Figs. 2 and 4). Frequently, densely staining filament bundles were seen at the cell periphery (Figs. 4A and 5A) or, in more apical regions, radiating as a meshwork towards the cell surface (Fig. 4B). Individual filaments had a diameter of 7.6 + 1 nm. Microtubules were frequent either coursing through the cytoplasm or aligned along the cell periphery (Fig. 58). Membrane-bound lysosome-like bodies were a prominent component of most cells (Figs. l-4). They contained a highly electron dense, homogeneous or lamellated material (Figs. 3A and 4A), or were partly or completely empty. Large vesicles containing fine granular material of low electron density were observed near Golgi fields (Fig. 3A). We did not observe Weibel-Palade bodies, which are a unique marker of endothelial cells (Haudenschild et al., 1975). Vacuoles containing collagen or collagen-like fibrils were absent, although such vacuoles are common wrthin the cytoplasm of gingival fibroblasts in viva (Ten Cate, 1972). Signs of endocytotic activity were regularly ohserved over considerable distances along the cell membranes (Figs. 2, 5B and 5C). Small pinocytotic vesicles were aligned along the cell surface, often invaginating from it, and in several rows in tangential sections (Fig. 5, round inset). Whenever many vesicles were present, peripheral bundles of cytoplasmic filaments were absent. Immunojluorescent microscopy

We found no evidence of smooth muscle or endothelial cell contamination by electron microscopy, but

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this was not conclusive proof of their absence as only a small portion of cells in a culture could be examined. We therefore used immunofluorescent techniques which allowed the scrutiny of a higher proportion of the cells. When HGF.+ cells were exposed to antibodies to type I procollagen, the majority of the cells (97 per cent) showed bright perinuclear staining (Figs. 6A and B). Approximately 85 per cent of HGF4 cells stained weakly with antibody to type III procollagen (Fig. 6C and D), indicating that most, but not all, of the cells were synthesizing a small amount of type III procollagen. Exposing the fetal aortic smooth muscle cells to specific antibodies to types I and III procollagens produced staining patterns different from HGF* cells (Fig. 7). Most of the muscle cells were moderately to brightly stained with antibody to type III procollagen. The staining intensity with type I antibody was similar. None of the cells in the HGF, or smooth muscle cultures were stained by specific antibody to type IV collagen although thousands of cells were examined. Therefore it is unlikely that any endothelial cells were present. To determine the collagen synthesis activities of single fibroblasts, we used the double-labelling technique described in the Methods. By changing filter systems on the fluorescent microscope, we could sequentially observe whether a single cell had taken up both the rhodamine and the fluorescein stains and so determine whether the cell was producing both types I and III collagens, or only one (Fig. 8). Most of the cells were clearly labelled with antibody to both type I collagen (Fig. 8A) and type III procollagen (Fig. 8B), but more intensely to type I collagen. All cells positive for type III procollagen were also positive for type I collagen, but some cells producing type I were negative for type III (Fig. 8C and D). About 3 per cent of the cells were negative for both type I and type III. DISCUSSION

The fine structural appearance of cultured human gingival fibroblasts was remarkably homogeneous; they have many similarities with cultured human skin fibroblasts (Lucky et al., 1975) which include the overall shape of the cell, the polarized arrangement of cytoplasmic’organelles, the long, thin mitochondria, large number of lysosomes and autophagosomes, and ribosome distribution of the RER. These similarities, taken with their collagen-synthesis similarities (Narayanan and Page, 1976; Lichtenstein et al., 1975), suggest that basic biological observations made in experiments with gingival fibroblasts may hold true also for skin fibroblasts, and vice versa However, until more in vitro parameters can be examined such presumptions must be made cautiously. Tissue-specific differences, such as different replication rates and life span, have been reported for fibroblasts obtained from human lung and skin, although these cells are morphologically similar (Schneider et al., 1977). There are several structural differences between the cultured gingival fibroblasts and gingival fibroblasts in vivo (Ten Cate, 1972); the amount of granular en-

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D. Engel, H. E. Schroeder, R. Gay and J. Clagett

doplasmic reticulum is less in cultured fibroblasts suggesting that they are synthesizing products different from those of cells in oiuo, or are producing different quantities of protein. We did not observe the type of collagen-containing vacuoles observed in gingival fibroblasts in uiuo by Ten Cate (1972) and thought by him to arise in viuo from either the phagocytosis of matrix collagen fibrils, or from intracellular aggregation of excess collagen macromolecules. The absence of such structures indirectly supports the first alternative, because cultured fibroblasts synthesize and secrete large amounts of collagen, but lack an extracellular collagen matrix which could be engulfed by the cells. We did observe numerous lysosomes and autophagosomes. One of .the most striking features of the cultured fibroblasts was their dense microfilament component which was more prominent than in fibroblasts in uioo. Immunofluorescent studies using specific anti-actin reagents kindly provided by Dr. G. Gabbiani, University of Geneva shows that these microfilaments are composed of actin (unpublished). Their actin nature was also shown by Lazarides and Weber (1974). The biological role of microfilaments has been studied extensively and these structures are believed to be important in cell movement, maintenance of cell shape, epdocytosis and exocytosis (Allison, 1973). We suspect that the skeletal attributes of these filaments may be important in maintaining the structural integrity of the gingival fibroblast in vitro. We have recently found that these cultured cells are resistant to cytopathic changes when exposed to whole dental plaque and homogenized Actinomyces viscosus bacteria. Furthermore, the fibroblasts endocytose large amounts of bacterial substance and package the substance in phagocytic vacuoles (Engel, Schroeder and Page, 1978). We found that the microfilaments had a mean diameter of 7.6 nm. This is similar to the 7-9 nm measurements reported by Perdue (1973) for chick embryo fibroblast microfilaments and to the 6-8 nm measurements in pig myofibroblasts (Ross and Woodward, 1978), but slightly larger than the 6 nm filaments reported for cultured mouse fibroblast cell lines (Willingham et al., 1977), the 67nm myofilaments observed in cultured smooth muscle cells (Ross, 1971; Gimbrone and Cotran, 1975), and the 67 nm filaments of cultured human endothelial cells (Haudenschild et al., 1975). We did not find any evidence of contamination of the gingival fibroblast cultures with endothelial or smooth muscle cells. None of the cells contained endothelial-specific organelles, nor were they stained by specific antibody to type IV collagen, a phenotypic marker for endothelial cells. Furthermore, the intensity of staining with antibody to type III procollagen was much weaker in the gingival fibroblasts than in the control smooth muscle cells, which are known to produce large quantities of type III collagen. Although we recognize that intensity of staining is somewhat subjective, the difference between the fibroblast and smooth muscle cells stained for type III procollagen was very striking. Using electron microscopy, the gingival fibroblasts were found to lack the gap-type junctions which are a feature of cultured smooth muscle cells (Ross, 1971; Gimbrone and

Cotran, 1975). This finding, taken with the immunofluorescent observations make it very unlikely that smooth muscle cells were present in the gingival fibroblast cultures. A single gingival fibroblast is capable of synthesizing both types I and III collagen simultaneously. This agrees with an earlier report dealing with simultaneous synthesis of types I and III collagen by skinfibroblasts (Gay et al., 1976) and is yet another example of the similarities which exist between fibroblasts derived from human skin and gingiva. Many of the cells contained both types I and III collagen, but, it should be pointed out that some cells were producing only type I. Also, a few cells were found to be negative for type I collagen as well. These findings suggest that although gingival fibroblasts are morphologically homogeneous, there may be functionally different subpopulations as reflected by the type of collagen produced. Alternatively, the presence or absence of a given type of collagen within a cell may be a function of the maturational or differentiative state of that cell.

Acknowledgements-We

are grateful to Drs. R. C. Page and

A. S. Narayanan for their help in preparing this manuscript, to Drs. G. R..Martin and G. Gabbiani for providing us with specific antibodies, and to Dr. G. Striker for providing the smooth muscle cell cultures. This work was sup ported by Public Health Service Grants DE-02600, DE-02670, and DE-03301 from the National Institute of Dental Research.

REFERENCES

Allison A. C. 1973. The role of microfilaments and microtubules in cell movement, endocytosis and exocytosis. In: Locomotion of Tissue Cells. Ciba Foundation Symposium 14, pp. 109-148. Associated Scientific Publishers, Amsterdam. Burke J. M., Balian G., Ross R. and Bornstein P. 1977. Synthesis of types I and III procollagen and collagen by monkey aortic smooth muscle cells in vitro. Biochemistry 16, 3243-3249. Engel D., Schroeder H. E. and Page R. C. 1978. Morphological features and functional properties of human fibroblasts exposed to Actinomyces uiscosus substances. Infect. Immun. 19, 287-295. Fraska J. M. and Parks V. R. 1965. A routine technique for double staining ultrathin sections using uranyl and lead salts. J. Cell Biol. 25, 157-161. Gay S., Martin G. R., Muller P. K., Timpl R. and Kuhn K. 1976. Simultaneous synthesis of types I and III collagen by fibroblasts in culture. Proc. natl Ad. Sci. 73, 4037-4040.

Gimbrone M. A. and Cotran R. S. 1975. Human vascular smooth muscle in culture: growth and ultrastructure. Lab. Invest. 33, 16-27.

Hassell T. M., Page R. C., Narayanan A. S. and Cooper C. G. 1976. Diphenyl-hydantoin (Dilantin) gingival hyperplasia: drug-induced abnormality of connective tissue. Proc. natl Acad. Sci. 73,2909-2912. Haudenschild C. C., Cotran R. S., Gimbrone M. A. and Folkman J. 1975. Fine structure of vascular endothelium in culture. J. Ultrastruct. Res. 50, 22-32. Howard B. V., Macarak E. J., Gunson D. and Kefalides N. A. 1976. Characterization of the collagen synthesized by endothelial cells in culture. Pm. natl Ad. Sci. 73. 2361-2364.

Cultured human gingival fibroblasts Ko S. D., Page R. C. and Narayanan A. S. 1977. Fibroblast heterogeneity and prostaglandin regulation of subpopulations. Proc. natl Acad. Sci. 14. 3429-3432. Lazarides E. and Weber K. 1974. Actin antibody: the specific visualization of actin filaments in non-muscle cells. Proc. natl Acad. Sci. 71, 2268-2272. Lichtenstein J. R., Byers P. H., Smith B. D. and Martin G. R. 1975. Identification of the collagenous proteins synthesized by cultured cells from human skin. Biochemistry 14, 1589-1594. Lucky A. W., Mahoney M. J., Barrnett R. J. and Rosenberg L. E. 1975. Electron microscopy of human skin fibroblasts in situ during growth in culture. Exp. Cell Res. 92, 383-393. Martin G. M. 1973. Human skin fibroblasts. In: Tissue Culture: Methods and Applications. (Edited by Kruse P. F. and Patterson M. K.) pp. 394. Academic Press, New York. Narayanan A. S. and Page R. C. 1976. Biochemical characterization of collagens synthesized by fibroblasts derived from normal and diseased human gingiva. J. biol. Chem. 251,5464-5471. Narayanan A. S., Page R. C. and Kuzan F. 1978. Collagens synthesized in vitro by diploid fibroblasts obtained from chronically inflamed human connective tissue. Lab. Invest. 39, 61-65.

Nowack H., Gay S., Wick G., Becker U. and Timpl R. 1976. Preparation and use in immunohistology of antibodies specific for type I and type III collagen and procollagen. J. Immunol. Methods 12, 117-124.

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Perdue J. F. 1973. The distribution, ultrastructure, and chemistry of microfilaments in cultured chick embryo fibroblasts. J. Cell Biol. 58. 265-283. Priest J. H. 1969. Cytogenek, Medical Technology Series. pp. 172-174. Lea and Febiger, Philadelphia. Reynolds E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212.

Rhodin J. A. G. 1974. Histology, A Text and Atlas. p. 52, Oxford University Press, New York. Ross R. 1971. The smooth muscle cell II. Growth of smooth muscle in culture and formation of elastic fiberi. J. Cell Biol. 50, 172-186. Ross R. and Woodward M. 1978. Spatial orientation of microtubules in contractile fibroblasts in uiuo. Anat. Rec. 191, 169-181.

Schneider E. L., Mitsui Y., Au K. S. and Shorr S. S. 1977. Tissue-specific differences in cultured human diploid fibroblasts. Exp. Cell Res. 108, l-6. Ten Cate A. R. 1972. Morphological studies of fibrocytes in connective tissue undergoing rapid remodeling. J. Anat. 112,401-414.

Tsutsui T., Hirokawa Y. and Maizumi H. 1973. Long-term cultivation of fibroblasts derived from normal gingival tissues of human adults. Jap. J. med. Sci. Biol. 26, 169-177. Willingham M. C., Yamada K. M., Yamada S. S., Pouysseaur J. and Pastan I. 1977. Microfilament bundles and cell shape are related to adhesiveness to substratum and are dissociable from growth control in cultured fibroblasts. Cell 10, 375-380.

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Plate 1. Fig. 1. Composite micrograph displaying various features of cultured human fibroblasts. Note dividing cell in metaphase, the number of nucleoli, the distribution of cytoplasmic organelles and filament bundles along the cell periphery (arrows). x 4000 Plate 2. Fig. 2. Composite micrograph illustrating nuclear and cytoplasmic features at higher magnification. Note indented nucleus (left inset), the paranuclear distribution of rough endoplasmic reticulum, Golgi fields (G), occasional branching mitochondria (arrows), and the dense areas of polyribosomes (right inset). x 8900; left inset: x 4000; right inset: x 19,200 Plate 3. Fig. 3. Paranuclear cytoplasmic portions containing cross- and tangentially cut, in part dilated, rough endoplasmic reticulum cisternae and vesicles (arrows) with low electron dense contents (A), and several Golgi fields with narrow cisternae (B). Note circular, and spiral distribution of bound ribosomes at rough endoplasmic reticulum membranes (left inset) and dense body with lamellated inclusion (right inset). x 19,200; left inset: x 19,200; right inset: x 36,400 Plate 4. Fig. 4. Peripheral cytoplasmic portions with filament bundles arranged parallel to the cell surface (A, right inset), and a filament meshwork within the cytoplasm (A) and at cell periphery (B). Note numerous lysosomal bodies which are empty or contain varying amounts of electron dense material (left inset in A). x 19,200; left inset: x 19,200; right inset: x 36,400 Plate 5. Fig. 5. Cytoplasmic periphery. Cytoplasmic filaments oriented parallel to the cell surface are depicted at low magnification in the triangular inset and at higher magnification in the rest of micrograph 5A. Numerous pinocytotic vesicles are shown in a tangential section of the cell periphery (round inset) and in cytoplasmtc cross sections (5B and C). Note the parallel and longitudinally oriented microtubules (arrows), cytoplasmic filaments and rough endoplasmic reticulum (5B and C). x 36,400 Plate 6. Fig. 6. Immunofluorescent and darkfield photomicrographs of gingival fibroblasts. (A) Cells stained with specific antibody to type I procollagen. Staining is most intense in region around the nucleus. Note that one of the cells is negative for type I procollagen (arrow). (B) Darkfield of cells shown in 6A. (C) Cells stained with specific antibody to type III procollagen. The staining is of much weaker intensity than seen with type I antibody, indicating that these cells contain less type III procollagen than type I procollagen. (D) Darkfield for cells shown in 6C. x 400 Plate 1. Fig. 7. Immunofluorescent and darkfield photomicrographs of smooth muscle cells. (A) Cells stained with specific antibody to type III procollagen. Staining was much more intense than with gingival fibroblasts stained with the same antibody preparation, indicating the smooth muscle cells contain larger amounts of type III procollagen. (B) Darkfield of cells shown in 7A. (C) Cells stained with specific antibody to type I procollagen. All of the cells in this photograph show positive staining for type I procollagen, although the intensity of staining is much greater in two of the cells than in the third (arrow). (D) Darkfield of cells shown in 7C. A and B, x 160; C and D, x 400 Plate 8. Fig. 8. Immunofluorescent photomicrographs of gingival fibroblasts stained with rat antibody to type I collagen (A and C) and rabbit antibody to type III procollagen (B and D). Note that most of the cells contain both types I and III collagen, although the staining is weaker for type III. Also note that one of the cells which is positive for type I (C, arrow) is negative for type III (D, arrow). x 120

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