Microtubule and microfilament populations of cell processes in the dental pulp.код для вставкиСкачать
THE ANATOMICAL RECORD 198:421-426 (1980) Microtubule and Microfilament Populations of Cell Processes in the Dental Pulp (2. R. HOLLAND Departments of Anatomy and Oral Biology, Colleges of Medicine and Dentistry, llniuersity of Manitoba, Winnipeg, Manitoba R3E OW3 ABSTRACT An attempt has been made to characterize the nature of the unidentified cell processes participating in gap junctions in the odontoblast layer. In peripheral and pulpal nerves, there is a strong relationship between axon caliber and microfilament and microtubule populations. This characteristic, together with the ratio of microtubules to microfilaments, has been measured and compared for four types of cell processes found in the dental pulp, including those participating in gap junctions. The processes taking part in the gap junctions cannot be distinguished from pulpal axons on the basis of microtubuleto-microfilament ratio nor on the relationship between microtubule and microfilament population and process caliber. While these findings do not prove that the "gap members" are nerve fibers, it does support the hypothesis that the processes taking part in gap junctions in the peripheral dental pulp are nerve fibers. It has been suggested that some nerve fibers in the dental pulp are electrically coupled (Matthews and Holland, '75). Electrophysiological experiments strongly support this hypothesis (Matthews, '771, but, as yet, the anatomical basis of this coupling has not been established. Gap junctions that occur predominantly in the odontoblast layer often involve cell processes which may be axons (Holland, '75, '76, '77). These gap junctions may couple axons directly or through some intermediate cell, possibly a n odontoblast. The principal problem in testing this hypothesis has been the difficulty in identifying, with certainty, axons t h a t have lost their Schwann cell sheath, a difficulty that also occurs in other tissues (Whitear, '60; Munger, '66; Fillenz and Woods, '70). Friede and Samorjaski ('70) showed that in the sciatic nerve of rats and mice, there was a definite relationship between axon caliber and neurofilament and microtubule populations. Bueltman et al., ('72) showed a similar relationship for the pulpal axons in the marmoset. It may be that this relationship is characteristic of axons. This paper reports an examination of the possibility of using this relationship to establish the axonal or other nature of the cell processes taking part in gap junctions in the peripheral pulp. To this end, the microfilament and microtubule populations of three groups of cells, one definitely 0003-276)3180/1983-0421$01.400 1980 ALAN R. LISS, INC. axonal and the others of connective tissue cells, have been compared with the same feature of the unidentified cell process taking part in the gap junctions. MATERIALS AND METHODS Three cats 6-8 months of age were anesthetized with sodium pentobarbitone and perfused via the left ventricle with a solution of 2.5% glutaraldehyde in 0.08 M phosphate buffer for 1 hour. The canine teeth were removed and a disc 1 mm thick was cut from the crown 4-5 mm from the tip of the tooth. These discs were then washed in 0.08 M phosphate buffer and postfixed in 2% osmium tetroxide in distilled water for 2 hours. Following a wash in distilled water, the tissue blocks were stained en bloc with 2% aqueous uranyl acetate for 1hour. The blocks were dehydrated in alcohols to which WOuranyl acetate was added, except the absolute alcohol, and then embedded in araldite resin. Ultrathin sections of the odontoblast layer and central pulp were cut and collected on formvar-supported slot grids and stained with saturated aqueous urany1 acetate and lead citrate. The sections were examined in the electron microscope at ~10,000.Micrographs were printed at ~30,000 of four types of cell processes: 1) Processes in the odontoblast layer which Received September 11, 1979 accepted February 29, 1980. 422 G. R. HOLLAND participate in gap junctions (Fig. 1). 2) Odontoblastic processes in the predentin (Fig. 2). 3 ) Non-myelinated axons in the sub-odontoblastic region (Fig. 3 ) . 4) Processes in the central pulp, presumably fibroblastic (Fig. 4). Many processes from each group were photographed from each specimen, with no attempt a t selection. After printing, those processes which were outside the caliber range of non-myelinated axons in the pulp established by Beasley and Holland ('78) were eliminated, as were those processes in which the microtubule and microfilaments could not be counted due to obliquity. The profiles of the axons and cell processes were traced on acetate sheets and their area measured on a Quantimet Image Analysis System. The number of microtubules and microfilaments within each axon and process was counted. The two features that showed the strongest correlation in Friede and Samorjaski's ('70) study were the microfilament-to-microtubule ratio and the relationship between process caliber and total population of microfilaments and microtubules. These features were compared for all four groups of processes. STATISTICAL TREATMENT OF DATA The ratios of microtubules to microfilaments for each group were compared with the others. The homogeneity of the variances was first tested for each pair of samples using Bartlett's test (Brownlee, '65). If the variances were homogeneous, the comparison was made using the student t-test. If the variances were not homogeneous, Welch's approximation was applied and a modified student t-test used (Brownlee, '65). The relationships of microfilaments and microtubule population to process area for each group were also compared with each of the others. These relationships were estimated by regression lines. If the variances proved homogeneous by Bartlett's test, a n analysis of covariance (Dunn and Clark, '74) was performed and the slopes of the regression lines compared. If the variances were not homogeneous, Welch's approximation was employed, combining the variances, and the modified ttest used to compare the slopes. If slopes were not significantly different, intercepts were also tested for differences. RESULTS 1) Microtubule-to-microfilament ratios These are shown in Table 1. Table 2 shows the result of the comparison of the different groups of processes. 2 ) Microtubule and microfilament population related to process caliber Slopes, intercepts, and correlation co-efficients of this relationship are shown in Table 3 . The regression lines themselves are shown in Fig. 5. Tables 4 and 5 show the comparison of these regression lines. DISCUSSION The relationship between axon caliber and tubule and filament numbers is as strong for pulpal axons (correlation coefficient 0.70) as Friede and Samorjaski ('70) (c.c. 0.78) demonstrated for axons in the sciatic nerve. The slope on the regression lines is, however, different from that for non-myelinated axons in the sciatic nerve (Friede and Samorjaski, '70) but is surprisingly similar to that for myelinated nerves. Similarly, the microtubule: microfilament ratio for unmyelinated axons in the pulp is almost identical to that for myelinated axons in the sciatic nerve. The reason for this similarity is that the pulpal axons are probably the preterminal branches of myelinated axons and have retained their characteristics. Few non-myelinated axons are found near the apex of the tooth, often fewer than 400 (unpublished observation),yet at the midcrown level, several thousand are found (Beasley and Holland, '78). Other workers have also reported the preponderance of microtubules over microfilaments in non-myelinated pulpal axons (Stockinger and Pritz, '71; Bueltman et al., '72). There is also a good correlation between process caliber and microtubule and microfilament population for the processes taking part in the gap junctions (correlation coefficient 0.71) and for the fibroblast process of the central pulp (correlation coefficient 0.84). The correlation is low (correlation coefficient 0.28) for odontoblastic processes in the predentin. When the four types of processes are compared, it is not possible to distinguish between pulpal axons and the processes taking part in gap junctions on the basis of their microtubule to microfilament ratios. It is, however, possible to distinguish between the "gap members" CELLPROCESSESOFTHEDENTALPULP Fig. 1. Fig. 2. Fig. 3. Fig. 4. A typical gap junction from the odontoblast layer. x 45,000. An odontoblast process from the predentine. x 45,000. A pulpal axon ensheathed in a Schwann cell. X 45,000. A fibroblast process from the central pulp. x 45,000. 423 G.R. HOLLAND 424 TABLE 1 . Microtubule-bmicrofilament ratio for cell processes in the dental pulp and predentin. No. of orocesses Mean microtubule-tomicrofilament ratio S.D. 22 0.22 0.28 14 21 9 0.24 0.10 1.70 0.18 0.13 0.91 Processes participating i n gap junctions Pulpal axons Fibroblast processes Odontoblast processes TABLE 2. Comparison of microtubule and micmfilament ratios for cell processes in the dental pulp and predentin. Gap members v. axons Odontoblast pr. v. axons Fibroblast pr. v. axons Odontoblast pr. v. gap members Fibroblast pr. v. gap members Fibroblast pr. v. odontoblast pr . D.F. t Significance level 35 8.404 33 8.637 -0.74 -4.689 3.10 -4.787 N.S. < 0.01 < 0.01 < 0.01 1.75 < 0.05 -5.239 < 0.01 41 8.14 TABLE 3. Regression analysis of microtubule and microfilament counts with pmcess caliber. Slope of regression Process participating in gap junctions Pulpal axons Fibroblast processes Odontoblast processes Intercept Correlation coefficient 221.436 86.8638 0.71 352.895 215.86 49.24 -0.0754 29.4846 119.029 0.70 0.84 0.28 TABLE 4. Comparison of regression lines. Slope F. -. Gap members vs. pulpal axons Gap members vs. fibroblast pr . Pulpal axons vs. fibroblast ur. - (D.F.) Intercept ~~ Signif. F. . . ~ _ _ _ _ _ . _ - - (D.F.) Signif. ~~ 1.265 (1,321 NS 2.503 (1,33) NS 0.009 (1,39) NS 9.935 (1,40) 0.01 1.94 (1,31) NS 1.214 (1,321 NS TABLE 5. Comparison of regression lines by t-test after Welch's approximation. _ ~- __ Gap members vs. odontoblast processes Pulpal axons vs. odontoblast processes Fibroblast pr. vs. cdontoblast Drocesses _ T _____ Slopes _ _ _ ~ ~ (D.F.) Significance -7.3 (12.83) < 0.01 -8.69 (20.977) < 0.01 (9.814) < 0.01 -7.491 Intercepts _ ~ _ . . ~ _ _ . _ _ _ _ _ Comparison not valid, as slopes differ. 425 CELL PROCESSES OF THE DENTAL PULP S I N ~ W V ~ I ~ O U ~oI W s m a n i o u 3 i w ON ivioi v) :: t s n. SIN3WVlIJOU3IW 9 S31flQn10131W ON l V l O l 426 G. R. HOLLAND and odontoblast and fibroblastic processes. When the same comparisons are made using the caliber-to-population relationships, both the slopes and intercepts of the gap members and pulpal axons are not significantly different. The gap members do differ significantly from both the fibroblast and odontoblastic processes on this basis, although using this feature, it is not possible to recognize pulpal axons and fibroblasts as different populations. The lack of difference between pulpal axons and fibroblastic processes may be important. It may, however, be due to the “contamination” of the fibroblastic population with unsheathed axons. The fact that regression lines, except in the case of the pulpal axons, do not pass through zero is interesting. In the case of the gap members, the odontoblast processes, and the fibroblast processes, it presumably indicates that there is a minimal number of these structures always present, even in the narrowest of processes. In the case of the pulpal axons, the intercept is close to zero, but statistically, this was not significantly different from the intercept for the gap members. The principal conclusion from the findings must be that the processes taking part in the gap junctions in the peripheral dental pulp cannot be distinguished from pulpal axons on the basis of their microtubule and microfilament populations. Several earlier workers have attempted to identify structures involved in specialized junctions in the pulp and dentin as nerves (Stockinger and Pritz, ’70; Vacek and Palckova, ’59; Arwill, ’68; Frank, ’66a, b, ’68a, b). They used only qualitative morphological criteria, and their conclusions have not achieved widespread acceptance. While the current findings do not prove that the “gap members” are nerve fibers, it does support the hypothesis quantitatively and suggests that other techniques be pursued to test the hypothesis further. ACKNOWLEDGMENTS The data reported in this paper was collected while the author was a member of the Department of Endodontics and Dows Institute, College of Dentistry, University of Iowa. I would like to thank Doug Holmes, who helped with microscopy, and Barry Rittman, who carried out the measurements on the Quantimet System. The statistical analysis was conducted by Mary Cheang of the Computer Department for Health Sciences, University of Manitoba. This work was supported by USPHS grant 5-501-5313 and grant Number 321-3113-04 from the Medical Research Council of Canada. LITERATURE CITED Arwill, T. (1968) The ultrastructure of the pulpo-dentina1 border zone. 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