The effect of beta-aminoproprionitrile on the periodontal ligamentII. Radioautographic study of collagen secretion from fibroblastsкод для вставкиСкачать
THE ANATOMICAL RECORD 209:41-52 (1984) The Effect of Beta-Aminoproprionitrile on the Periodontal Ligament: II. Radioautographic Study of Collagen Secretion From Fibroblasts MOON-IL CHO AND PHILIAS R. GARANT Department of Oral Biology and Pathology, School of Dental Medicine, State University ofNew York at Stony Brook, Stony Brook, NY 11794 ABSTRACT Fibroblasts are distributed evenly throughout the periodontal ligament IPDL) of normal mice. In mice fed beta-aminoproprionitrile (0-APN) the fibroblasts undergo aggregation to form palisades of closely juxtaposed cells abutting pools of acellular collagenous matrix. Individual fibroblasts within these aggregates retain their polarized cytoplasmic organization and continue to synthesize and secrete collagen. However, unlike normal PDL fibroblasts, the P-APN-treated cells appear immobilized by well-developed cellto-cell adherens-type junctions along their lateral surfaces. We studied collagen secretion from P-APN-treated fibroblasts by light and electron microscopic radioautography after injection of 3H-proline. Newly synthesized collagen was secreted from the distal ends of the P-APN-aggregated fibroblasts as a distinct band of labeled material, resembling the pattern of matrix deposition seen in osteogenesis and dentinogenesis. The radioactive band of collagenous matrix was displaced further away from the fibroblasts at 2 and 4 days after 3Hproline injection a s more collagen was secreted. This pattern of radiolabeled collagen secretion confirmed previous observations that PDL fibroblasts are highly polarized and that collagen secretory granules are extruded from the distal or secretory pole of the cell. In normal PDL the even distribution of fibroblasts and the complex interrelationship of their distal cell processes leads to a diffuse pattern of silver grain deposition, masking the oriented flow of new collagen from the distal ends of individual fibroblasts. Analysis of electron microscopic radioautographs revealed that newly synthesized collagen was packaged and secreted from p-APN-treated fibroblasts via the normal cytoplasmic pathways but a t a slower rate. Periodontal ligament (PDL) fibroblasts are highly polarized cells with a n extensive network of microtubules which function in collagen secretion-granule translocation (Cho and Garant, 1981b,c,d). The appearance of the PDL fibroblast Golgi apparatus and the formation and maturation of collagen secretion granules is closely related to the macromolecular assembly and cross-linking of the procollagen chains (Cho and Garant, 1981a). On the basis of the above findings, one could predict that agents that interfere with collagen cross-linking inside the cell would produce changes in secretory granule maturation and perhaps alter the rate of col- 0 1984 ALAN R. LISS, INC. lagen secretion. Furthermore, interference with collagen cross-linking in the extracellular environment could cause changes in cell shape or cell distribution, since cell-to-matrix interactions are important factors in determining cellular behavior (Bellairs et al., 1982). Beta-aminoproprionitrile (p-APN) interferes with lysyl oxidase, preventing the formation of lysyl-derived aldehyde groups on the collagen alpha chains (Deshmukh and Nimmi, 1968; Bornstein, 1970). Because of Received September 22,1983; accepted November 16, 1983. 42 M.-I. CHO AND P.R. GARANT the reduced number of aldehyde groups, the collagen alpha chains fail to become fully cross-linked, and, therefore, the tensile strength of the collagen fibers is greatly reduced (Fry et al., 1962). The pathologic consequences of this molecular defect are most pronounced in connective tissues exposed to increased stress such as in articulations, muscle insertions, and in the periodontal ligament of functional teeth (Gardner et al., 1958; Ponseti and Shepard, 1954; Krikos, 1959; Barrington and Meyer, 1966; Sciaky and Ungar, 1961; Levene, 1973). The classic histologic lesion that develops in the periodontal ligament within 1-2 weeks after the onset of 6-APN administration is called the “lathyritic body.” It is characterized by an aggregation or palisading of the PDL fibroblasts in rows bordering on acellular amorphous matrix (Sciaky and Ungar, 1961; Barrington and Meyer, 1966; Yoshikawa, 1971; Sims, 1977; Baden and Bouissou, 1983; Krikos et al., 1958; Gardiner et al., 1958). In a recent publication (Cho and Garant, 1983) we described the ultrastructural appearance of 0-APN-treated fibroblasts of the periodontal ligament. We noted that although the fibroblasts became aggregated to form typical “lathyritic bodies,” individual fibroblasts maintained their polarity and appeared to contain a normal complement of cytoplasmic organelles. In fact, the polarity of the cells was easier to detect in the pAPN-induced fibroblast aggregates, a feature which we thought could be used to advantage to study collagen secretion from PDL fibroblasts. Furthermore, we noted changes in the appearance of the Golgi apparatus, which suggested a decreased rate of collagen secretion. In this paper we report our findings on collagen secretion from 0-APN-treated PDL fibroblasts and compare these results to those published for normal PDL fibroblasts and colchicine-treated PDL fibroblasts (Cho and Garant, 1981c,d). MATERIALS AND METHODS Ultrastructural Study Five male Balb-C mice weighing 15 to 17 gm were fed a powdered Purina rat chow diet containing 0.4% P-APN for 2 weeks. The mice were then anesthetized by i.p. injection of Nembutal (1mg/lO g m body weight) prior to intracardiac perfusion of 70-80 ml of 2% glutaraldehyde in a modified Tyrode solution, pH 7.4 (Sjostrand, 1967). The maxillae were then dissected free of the surrounding tissues in a dish containing 4% glutaraldehyde and 5% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4 (Karnovsky, 1965).The cleaned maxillae were fixed in Karnovsky’s fixative for a n additional 3 4 h, rinsed in 0.1 M cacodylate buffer, pH 7.4, for 15 min, and decalcified in 0.1 M EDTA containing 3% glutaraldehyde, pH 7.4, for about 2-3 weeks a t 4°C. The first interdental areas were then dissected out and cut into two identical pieces in a dish containing 0.1 M cacodylate buffer, pH 7.4. After washing in the same buffer with several changes for 45-60 min, the pieces of tissue were postfixed in 1%Osmium tetroxide in S-collidine buffer, pH 7.4, for 1.5 h. En-block staining was carried out for 1 h a t room temperature in 1% uranyl acetate in 0.1 M maleate buffer, pH 6.2, after rinsing in the same buffer, pH 5.2, for 15 min. Tissues were then dehydrated in a graduated series of cold ethanols and propylene oxide prior to infiltration with Epon mixture at room temperature for 4 h and placed in flat embedding molds containing fresh Epon mixture. Polymerization was accomplished a t 60°C for 48 h. For ultrastructural study, ultrathin sections (60-80 nm in thickness) were cut on a Porter-Blum MT2 ultramicrotome equipped with a DuPont diamond knife. The sections were doubly stained for 25 min with freshly prepared saturated uranyl acetate in 50% ethanol and for 7 min with lead citrate (Reynolds, 1963). The stained sections were then examined and photographed with a JEMlOOB electron microscope. For light and electron microscopic radioautography 11 male Balb-C mice weighing 1517 gm were fed powdered Purina rat chow containing 0.4% 0-APN for 2 weeks. They were then injected with L-(2, 3-3H)proline(SA = 40 Ciimmol; New England Nuclear, Inc., Boston, MA) a t a concentration of 1mCi per 0.1 ml of sterile saline solution per mouse. All injections were made via the jugular vein, in animals anesthetized with ether, a t a standard time of between 8:30 and 9:30 A.M. The mice were killed, one per time period, a t 3, 10,20, 30 min, 1 , 2 , 4 , 8 , 2 4 h, 2 and 4 days after injection of the tritiated proline, by proline, by intracardiac perfusion of 2% glutaraldehyde in Tyrode’s solution. The tissues were subsequently processed as described above, except for the omission of en-block staining with uranyl acetate. For light microscopic radioautographs, COLLAGEN SECRETION FROM 0-APN-TREATED FIBROBLASTS three to four 1-pm sections from each block (four blocks per animal) were mounted on glass slides, coated with Kodak NTM-3 liquid emulsion, exposed for 3 to 8 days at 4"C, and developed as previously described (Garant and Cho, 1979b). The radioautographs were subsequently stained with 1% toluidine blue in sodium veronal acetate buffer. For electron microscopic radioautography the loop method of Car0 and Van Tubergen (1962) was employed. Thin sections, about 100 nm in thickness from four blocks for each time period, were coated with a crystalline monolayer of Ilford L4 emulsion, exposed a t 4"C, and developed with either Microdal-X or a physical developer as described by Kopriwa (1973). The simple quantitative analysis of EM radioautographs described by Nadler (1971)was used. Silver grains were counted or. micrographs with a final magnification of x 30,000. The following compartments were analyzed: RER, Golgi complex (cisternae, saccules, intermediate vesicles, and secretory granules), intracellular collagen fibrils, extracellular collagen fibrils, nuclei, mitochondria, and cell periphery. One point was recorded when the grain was clearly encompassed by the organelle in question. When a grain was shared by two organelles, each compartment received a half point. RESULTS Light Microscopic Radioautography In P-APN-treated PDL, radioautographs obtained 10 min after 3H-proline injection showed silver grains a t the periphery of the cell bodies where the RER is located (Fig. 1). The juxtanuclear Golgi complex, observed as a pale staining zone, and the extracellular matrix were relatively free of silver grains (Fig. 1).At 30 min, most of the silver grains were concentrated over the supranuclear cytoplasm, i.e., the region of the Golgi complex (Fig. 2). A small number of grains were 10cated over distal cell processes or the adjacent extracellular matrix at 30 min (Fig. 2). By 8 h, the majority of silver grains were concentrated over the extracellular collagen matrix close to the distal cell processes (Fig. 3). The grain distribution in Figure 3 is reminiscent of that observed adjacent to a n osteoblast or odontoblast layer, and thus it demonstrates a similar oriented flow of secretory material from the secretory pole (distal end) of the polarized fibroblasts. At 2 days (Fig. 4)and 4 days (Fig. 5) the concentrated 43 band of radioactive collagen was located progressively farther away from the fibroblasts, presumably pushed away by newly secreted collagenous matrix. As not all fibroblasts were oriented in the same direction, one band formed on each side of the palisaded fibroblasts. There were few grains over the lateral intercellular spaces or the cell bodies a t these later time periods. The difference in grain distribution over the extracellular matrix can be appreciated by examination of Figure 6, which is a radioautograph of normal PDL 2 h after injection of 3H-proline. The difference in grain distribution is presumably due to the difference in the fibroblast distribution pattern and not to differences in mobility of the newly synthesized collagen precursors. Electron Microscopic Radioautography The percentage distribution of silver grains over various organelles with time is summarized in Table 1 and presented in graphic form in Figure 13. At 3 min after injection of 3H-proline the great majority of radioactivity was concentrated over the RER (Fig. 7). At 10 min most silver grains (72.5%) were still located over the RER, but a small number of grains (15.5%) were present over the immature side of the Golgi apparatus, near the RER (Fig. 8). The RER was still heavily labeled a t 20 min (58.1% of silver grains), while the Golgi complex reached its maximum labeling of 34.8% (Fig. 9). Presecretory granules (Cho and Garant, 1981a) were still unlabeled a t 20 min. The disappearance of silver grains over the RER was slower than that previously observed in normal PDL fibroblasts At 30 min, (Fig. 13)(Cho and Garant, 1981~). 55.2% of all silver grains were still located over the RER. Despite the slower decline of labeling of the RER and Golgi complex, a small number of presecretory granules and collagen secretion granules appeared to be labeled a t 30 min (Fig. 10). The later observation was in agreement with a decrease in the number of silver grains over the Golgi complex from 34.8% a t 20 min to 28.7% at 30 min. Thus, at about 30 min after 3H-proline injection, labeled collagen precursors were secreted from PDL fibroblasts. At 1 h the number of silver grains over the extracellular matrix increased to 29.5% up from 8.4% a t 30 min. At 1 and 4 h, silver grains positioned over the extracellular matrix were associated with striated collagen fibrils near the distal cell processes (Figs. 11, 12). The 44 M.4. CHO AND P.R. GARANT 45 COLLAGEN SECRETION FROM p-APN-TREATED FIBROBLASTS TABLE 1. Distribution of silver grains at various times over PAPN-treated PDL fibroblasts in electron microscopic radioautographs of 3H-proline uptake 10 min 20 min 30 min l h 2h 8h 24 h RER Golgi ICC ECC Nuclei Mito. Autoph. Cell periph. 8781 72.i!i2 54.83 577 58.1 44.9 588 55.2 29.2 378 34.2 16.6 188 19.2 3.6 86 12.0 3.8 102 11.5 3.4 188 15.5 36.9 346 34.8 48.9 306 28.7 36.8 178 16.1 9.4 89 9.1 4.80 49 6.8 0.4 38 4.3 0.1 0.0 0.0 0.0 0.0 0.0 0.1 1.0 0.1 0.5 0.0 0.0 0.3 0.0 0.0 0.2 0.0 0.0 0.0 0.0 0.0 0.1 37 3.0 4.0 48 4.0 1.4 18 1.8 0.8 14 1.3 1.4 34 3.1 1.2 24 2.4 1.4 13 1.8 1.2 14 1.6 0.6 40 3.3 1.5 27 2.7 1.4 22 2.1 2.2 16 1.4 2.2 6 0.6 0.8 7 1.0 0.3 7 0.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.3 0.0 0.0 0.1 0.0 0.0 0.3 0.0 0.0 21 1.7 1.7 19 1.9 3.0 45 4.2 4.8 174 15.7 17.2 149 15.2 15.6 71 9.9 11.7 93 10.5 7 0.7 0.9 89 8.4 25.1 326 29.5 52.9 524 53.5 68.5 491 68.5 82.3 631 71.3 84.8 0.0 Total 1210 994 1065 1106 980 717 885 11.o 'Number of silver grains in 8-APN-treated cells. 'Percentage of total grains at each time period in 8-APN-treated cells. 3Percentage of total grains at each time period in normal cells (Cho and Garant, 1981~) delayed movement of labeled collagen pre- tained in normal cells (3.4% and 0.1%) 24 h cursors from RER and Golgi complex was after 3H-proline injection. The collagenous matrix adjacent to the ficlearly evident at 1h, for 34.2% and 16.1% of all silver grains were still located over the broblasts consisted of 640-banded fibrils of RER and Golgi complex. By 24 h the number uniform width plus a n amorphous material of silver grains over RER and the Golgi com- with fine filaments (Figs. 11,12). At 1h most plex fell to a minimal value of 11.5% and silver grains were located over the banded 4.3%, respectively (Table 1). These values collagen fibrils (Fig. 111, while at 4 h both were still higher, however, than those ob- banded fibrils and amorphous material were labeled (Fig. 12). DISCUSSION Fig. 1. Li ht microscopic radioautograph 10 min after injection of H-proline. The majority of silver grains are located at the periphery of cell, while few grains are found in the Golgi area (g). x 1,200. P Fig. 2. Lifht microscopic radioautograph 30 min after injection of H-proline. In addition to localization of silver grains at the periphery of cell, numerous grains are now observed in the Golgi area (g). Most of the grains which appear to be over the extracellular matrix are observed to be associated with cell processes upon close examination (arrows). x 1,100, Fig. 3. Light microscopic radioautograph 8 h after injection of 3H-proline. The majority of silver grains are located in the extracellular matrix close to cell processes (arrows). A few silver grains are associated with the Golgi area (9) and very few are located between cells. x 1,350. Of the mesenchymal collagen secreting cells, the odontoblasts and osteoblasts are clearly polarized with all collagen secretion granules being released from that portion of the cell in contact with predentin and osteoid, respectively. We recently showed that PDL fibroblasts are also highly polarized and that microtubules play a n important role in collagen secretion from the distal end of the cell, i.e., the end of the cell furthest away from the nuclear pole (Cho and Garant, 1981b,c). We suggested that the distal cell processes of normal PDL fibroblasts were specialized for secretion of collagen precursors on the basis of the following morphologic observations: 1) secretory granules labeled with 3H-proline are formed in the Golgi com- Fig. 4. Light microscopic radioautograph 2 days after administration of 3H-proline. Silver grains deposited as a loosely arranged band (arrows) are now pushed away from the cell processes. Only a few silver grains are observed over the Golgi area (g) and other portions of cell. x 1,300. Fig. 5. Light microscopic radioautograph 4 days after administration of 3H-proline. Bands of silver grains (arrows) are pushed farther away as new unlabeled extra- cellular collagen matrix (*) is formed between cell processes and the silver grains. The cell body, including the Golgi area (g),is free of silver grains. X 1,300. Fig. 6. Light microscopic radioauto aphy of normal H-proline. ' Silver fibroblasts, after administration of P grains are evenly distributed over the extracellular collagen matrix among the cells. Heavy labeling appears to be associated with thin cytoplasmic processes (arrowheads) of the fibroblasts. x 1,280. COLLAGEN SECRETION FROM 8-APN-TREATED FIBROBLASTS Fig. 7. Electron microscopic radioautograph 3 min after injection of 3H-proline. Most silver grains are located over RER, while the Golgi area (G) remains unlabeled. RBC, red blood cell. X12,600. 47 Fig. 8. Electron microscopic radioautograph 10 min after administration of 3H-proline. The majority of silver grains are still present over RER but labeling of the Gold area (GI begins. Extravasation of red blood cells (RBC) was a common finding in the PDL of 8-APNtreated animals. ~ 8 , 4 0 0 . 48 M.-I. CHO AND P.R. GARANT COLLAGEN SECRETION FROM b-APN-TREATED FIBROBLASTS plex and translocated in association with microtubules to the distal processes; 2) secretory granules are found in higher concentration in the distal processes than elsewhere in the cell; 3) fusion of secretory granules with the cell membrane of the distal cell processes was occasionally observed; and 4) tritiated proline-labeled collagen is visualized in secretory granules in distal cell processes and the adjacent extracellular matrix a t 30 to 60 min after intravascular injection of the radioisotope. However, because of the apparent random distribution of fibroblasts in the normal PDL and the ramification of the distal cell processes, it was not possible to observe in radioautographs an “oriented” flow of secretory product from the distal end of the cell (see Fig. 6). The palisading of polarized fibroblasts in the PDL of 0-APN-treated animals provided a situation where the deposition of new collagen could be observed as a “band” of radiolabeled matrix after injection of 3Hproline intravascularly. The most distinct “bands” were located adjacent to the distal cytoplasm at 24 h and were subsequently pushed away during a 3-day observation period, as new collagen matrix continued to be deposited from the distal ends of the cells. This process appears similar to the formation of osteoid in bone. Thus, the PDL fibroblasts are functionally, as well as morphologically, polarized. These results suggest that PDL fibroblasts function like the odontoblasts and osteoblasts, in that collagen precursors are released from a specialized region of the cell. Fig. 9. Electron microscopic radioautograph 20 min after administration of 3H-proline. Increased labeling over the Golgi area is observed. However, RER still demonstrates heavy labeling. ~21,000. Fig. 10. Electron microscopicradioautography 30 min after injection of ‘H-proline. Numerous silver grains are located in the Golgi area (Gj as well as over the rer. Some silver grains are now associated with secretory granules (psg; inset: high magnification of Golgi complex labelled with arrow), secretory granules (sg), cell processes, and extracellular collagen fibrils (colj near the cell processes. However, intercellular spaces (*) remain relatively unlabeled. ~ 7 , 2 0 0inset, ; x 17,680. 49 That this process is difficult to visualize in normal PDL is due to the way in which the cells are distributed in the matrix, the variation in the number and size of the distal cell processes, and may be further obscured by cell migration. In comparing the radioautographs from normal animals (Fig. 6) with those obtained from the P-APN-treated animals (Figs. 3-5), one may also conclude that the apparent random distribution of labeled newly secreted collagen in normal PDL is not due to the free diffusion of procollagen prior to its polymerization as collagen, but due to the underlying organization of the cells. If procollagen was able to diffuse freely within the extracellular matrix of the PDL, one would not expect to obtain the bands of newly labeled collagen as shown in Figures 3-5. Furthermore, in PAPN-treated animals with a lesser potential for cross-linking, one would expect higher mobility of procollagen. That new collagen polymerized a s a band adjacent to the distal end of the fibroblast in /3-APN-treated animals is highly suggestive that new collagen is also polymerized close to the secretory pole of fibroblasts in the normal PDL. This observation supports our previous conclusion that collagen fibrillogenesis occurs rapidly at the distal cell process (Garant and Cho, 1979a, Cho and Garant, 1981b,c). This concept is also in agreement with the observations of Trelstad and Hayashi (1975, 1979) that the conversion of procollagen to collagen with elongation of collagen fibrils occurs near the fibroblastic cell surface, perhaps within a deep cytoplasmic recess. Comparison of the results of a previous study of 3H-proline utilization by normal PDL fibroblasts (Cho and Garant, 1981~) with those obtained in this study (Table 1) revealed a pattern of delayed translocation of newly synthesized collagen through the 0APN-treated fibroblasts. Just as in normal fibroblasts, the radiolabeled secretory product in @-APN-treated fibroblasts moved sequentially from RER to Golgi, to secretory granule, to distal processes, and to the extracellular space. However, there was a slower decline in labeling for each cytoplasmic compartment in the P-APN-treated group (Fig. 13). At 30 min after 3H-proline injection, 55.2% and 28.7% of all silver grains were superimposed over RER and Golgi in the 0-APNtreated group, while in normal cells the RER 50 M.-I. CHO AND P.R. GARANT Fig. 11. Electron microscopic radioautograph 1 h after administration of 'H-proline. Silver grains are observed over 640 A banded extracellular collagen fibrils located adjacent to cytoplasmic processes (arrows). The labeled bundles of collagen fibrils are short in length and few in number. G, Golgi area; M, mitochondria. X 13,500. Fig. 12. Electron microscopic radioautograph 4 h after administration of 'H-proline. Silver grains are still located over collagen fibrils adjacent to cell processes (arrows). However, labeled collagen fibrils are much shorter in length and surrounded by disorganized abnormal collagen matrix (*I, which is also labeled. sg, Secretory granules. x 13,500. COLLAGEN SECRETION FROM (3-APN-TREATEDFIBROBLASTS 51 Fig. 13. Graphic representation of silver grain distribution over the normal and the P-APN-treated periodontal ligament at various time periods after injection of 3H-proline. ECC: extracellular matrix collagen fibrils; Golgi: Golgi complex; RER rough endoplasmic reticulum. and Golgi counts were 29.2% and 36.8%. These results indicate that 0-APN caused slower release and translocation of secretory product from the RER to the Golgi. These observations reinforce our previous impression that fl-APN-treated fibroblasts contain more RER and that their Golgi region contained a greater number of transitional elements of RER origin (Cho and Garant, 1983). The slower translocation of secretory product to the Golgi apparatus was also reflected in the delay of collagen secretion denoted in Figure 13. In normal PDL fibroblasts secretion of labeled, newly synthesized collagen occurs approximately 30 min after injection of 3H-proline (Cho and Garant, 1981~).Although some labeled collagen was secreted from fl-APN-treated fibroblasts a t 30 min after injection of the radioisotope, there was much less. In fact, the analysis of radioautographs showed a 50% reduction of collagen secretion at 1h in /3-APN-treated fibroblasts. 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