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Histochemical study of the connective tissue of the dental pulp
Arch.
oral
Bid.
Vol.9,
pp.l49-162,
1964.
Pergamon Press Ltd. Printed inCt.Britain.
HISTOCHEMICAL STUDY OF THE CONNECTlVE TISSUE OF THE DENTAL PULP E. ZERLOTTI Faculdade
de Farmacia e Odontologia de Aracatuba, Estado de Sao Paulo, Brazil
Summary-This study was concerned with the reactivity and organization of the connective tissue of the dental pulp and with the changes of the tissue colloids during ageing. Newer methods of preparation of the specimens and certain specific histochemical procedures were used. The results indicate that the extracellular matrix of the connective tissue of the dental pulp contains glycoproteins, sialic acid, acid mucopolysaccharides and proteins bearing reactive c-amino groups of lysine-hydroxylysine. The fibres and the ground substance are considered to interact forming coacervates. The extracellular matrix is organized as a heterogeneous colloid containing soluble and insoluble fractions. In young pulps the soluble fraction is large and the tissues are markedly affected by buffer solutions or enzymes. In ageing thereis a decrease of the soluble fraction and a change in the aggregation of the tissue colloids. These changes are signalled by the high resistance to buffer solutions and proteolytic enzymes and by the changes in staining of the tissues. The alteration in the internal organization of the proteins probably induces a redistribution of ions and is related to pulp stone formation.
INTRODUCTION EXCELLENT morphological studies of the dental pulp have been made in the past (ORBAN, 1929, 1957; SCHOUR, 1953; LANGELAND, 1957). However, newer concepts about the organization and behaviour of connective tissues (GERSH and CATCHPOLE, 1949, 1960; JOSEPH,ENGEL and CATCHPOLE, 1954; JOSEPHet al., 1959; ENGEL et d., 1960) now permit the study of the dental pulp from a different point of view. We have utilized these concepts and some newer histochemical methods (ZERLOTTI and ENGEL, 1962) to investigate the organization and reactivity of some protein components of this tissue. In this way it was also possible to discern changes in the tissue colloids during ageing. The extracellular matrix of the connective tissue of the dental pulp was shown to contain protein bearing appreciable amount of lysine-hydroxylysine, glycoproteins, some of them containing sialic acid and acid mucopolysaccharides. The matrix is organized as a heterogeneous colloid composed of soluble and insoluble phases. With ageing there appears to be a shift in the relation between the two fractions as the tissues lose water. This is accompanied by a decrease in the reactivity of ground substance and of fibres as determined by histochemical means. 149
150
E. ZERLO~I
MATERIALS AND METHODS Pulps from non-carious teeth with normal periodontal tissues were utilized. A total of 55 teeth were used, including: I: 25 premolars from patients 11-15 years old, extracted prior to orthodontic treatment (young group); II: 25 premolars and molars from patients 40-55 years old (mature group); III: 5 premolars from patients older than 70 years (aged group). Teeth of patients in groups II and III were extracted to facilitate prosthetic treatment. Wherever possible the extractions were performed under block anaesthesia Pulps were removed from the teeth by the method described by ENGSTR~~M and OHMAN(1960) and immediately fixed by the freeze-drying method (GERSH, 1932, 1948). Tissues were frozen in isopentane chilled to - 150°C immersed in liquid nitrogen, and then dehydrated in vacua for a period of 4-6 days at -30 to -35°C. The specimens were embedded in paraffin wax (m.p. 57-59°C) in vacua for lo-15 min. Serial sections were cut at 6-10 p and mounted on slides using a minimal amount of albumin. The frozen-dried sections were denatured in absolute ethanol overnight at 25°C to precipitate proteins, thereby minimizing loss of water-soluble tissue components. Histochemical and histological methods
Sections of all specimens were stained with Harris’ haematoxylin and eosin for morphological studies. The periodic acid-Schiff (PAS) method of HoTcHKrss (1948) and MCMANUS(1948) was used to localize glycogen and other carbohydrate or carbohydrate-containing proteins bearing free 1,Zglycol groups. To enhance the specificity of the method, various enzymes were employed prior to staining. Glycogen was removed by incubating tissue sections in a-amylase (Nutritional Blochemicals) (1 mg/l ml of Sorensen’s 0.1 M phosphate buffer, pH 6.0) for 30 min at 37°C. Sialic acid was digested by Neuraminidase (Behrimwerke) (100 units of sialidase extracted from Vibrio cholerae culture in 1 ml of 0.05 M sodium acetate buffer pH 5.5). Some sections were incubated in Collagenase (Agricultural Biologicals) (0.1 mg/l ml of Sorensen’s 0.1 M phosphate buffer, pH 7.0) at 37” C for 3 hr. Control sections were incubated in the buffers alone. Free amino groups of tissue proteins were localized by means of the 2,4-dinitrofluorobenzene (DNFB) reagent of SANGER(1945) using the method described by ZERLOTTIand ENGEL (1962). The sections were examined with monochromatic light at a wavelength of 410-420 rnp, using a Zeiss interference wedge, where the extinction of the dinitrophenyl derivatives is high. Contrast and resolution were greatly improved by this means, especially when a high energy light source (HBO 200 Osram) was used. Acid mucopolysaccharides were identified by their metachromatic staining with toluidine blue. During the present study, sections were stained with a 16 x 10e3 M solution of toluidine blue at pH 7.0 for 15 min. The excess of dye was blotted with filter paper and the sections mounted in water and examined immediately. In some experiments hyaluronic acid and chondroitin sulphate A and C were removed
HISTOCHEMICAL
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151
before staining by treating representative sections with testicular hyaluronidase (Wydase, Wythe Pharmaceutical Co.) at a concentration of 150 T.R.U./l ml of Sorensen’s 0.1 M phosphate buffer, pH 6.0, for 2 hr at 37°C. Control sections were incubated in the buffer alone. The gallocyanin-chrome alum technique was used for the localization of total nucleic acid (EINARSON,1932, 1951). Ribonucleic acid was removed by incubating the post-fixed sections with ribonuclease prior to staining. Slides were incubated for 4 hr at 37°C in a solution of ribonuclease (Worthington) in distilled water (5 mg/l ml), pH 5.5. Deoxyribonucleic acid was digested by treating the denatured sections for 4 hr at 37°C and pH 4.3 with deoxyribonuclease (Worthington) dissolved at a final concentration of 2 mg/l ml in a solution which contained 0.01 per cent gelatin and 0.01 M magnesium sulphate. Control sections were incubated in water. The Feulgen reaction was used to stain deoxyribonucleic acid. The silver impregnation method of GOMORI(1937) and the acid fuchsin-picric acid staining of Van Gieson (LILLIE, 1954) were used to localize and to differentiate between reticular and collagen fibres. Some sections were incubated in collagenase solution at 37°C for 3 hr and stained with the silver impregnation. Extraction of soluble components Soluble elements of the dental pulp were extracted by treating the sections with two different buffer solutions (JACKSONand WILLIAMS,1956; JACKSON,1957) prior to staining: (1) An “acid citrate soluble fraction” was removed by incubating undenatured sections in 0.1 M citrate buffer (0.1 M citric acid, 75 ml and 0.1 sodium citrate, 25 ml), pH 3.5, for 4 hr at 37°C; (2) A “neutral salt soluble fraction” was extracted with 0.1 M phosphate buffer (0.1 M Na,HPO,, 60 ml and 0.1 M NaH,POp, 40 ml), pH 7.0, under the same conditions. The extracted sections were then stained with the periodic acid-Schiff method, 2,4-dinitrofluorobenzene and silver impregnation. FINDINGS During the removal of the pulps from young teeth the odontoblastic. layer generally could not be readily detached from the dentine; occasionally small groups of cells were removed. For this reason more emphasis is given to the other structures of the connective tissue of the dental pulp and the discussion of odontoblasts is incomplete. Localization of the PAS-positive material The nuclei of the fibroblasts were unreactive with PAS, but small granules (< 1 CL) could be detected in their cytoplasm (Fig. 1). The cells containing PAS-positive granules were located mostly in the pulp chamber and there was no evidence of their association with any particular structure. Most of these intracellular inclusions resisted digestion by amylase although some of them were removed by the enzyme. They were only occasionally found in pulp cells of aged patients. Similar material could not be observed in the cytoplasm of the odontoblasts, but in one case PASpositive granules were found in the extracellular substance between odontoblasts of young pulps. It was not possible to determine if these granules were glycogen or amylase-resistant glycoproteins.
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The PAS-positive ground substance was slightly more reactive in pulps of young patients than in mature or aged pulps (Table 1). The argyrophilic fibres also stained with PAS and could be better differentiated from the ground substance in mature pulps. In specimens of aged patients the collagen fibres which composed most of TABLE 1. REACTIVITYOF THE~~ROUNDSLJBSTANCEOFTHEDENTAL
Periodic acid Schiff (PAS)
2,4-dinitrofluorobenzene (DNFB)
PULP
Toluidine blue (Metachromasia)
_
Young pulps (11-15 years) Mature pulps (40-55 years) Aged pulps (more than 70 years)
/
1
+ -t
I~
I i
-t
i’F
I
!+I
i
I ! I
-I -I 4.
i
.I !
-; -1.
t -I-
+
+ i_
i
T
+
+
I 1 1. ~1. -t-t / -I-
‘f
+
-t
f
‘F
the extracellular matrix were not intensely stained. The ground substance in these pulps also showed a low reactivity with PAS. The basement membranes of blood In young pulps they were slightly vessels were distinctly visualized in all pulps. fainter and thinner than those of mature or aged patients (Figs 2, 3 and 4). The ground substance was more intensely stained around the pulp nodules which, with exception of their borders, were PAS-negative. In sections treated with amylase an overall decrease of staining intensity was observed. This was probably due to some proteolytic activity of the enzyme preparation. Glycogen was found in nerve fibres in the form of coarse and numerous granules. The fibres and the ground substance of young pulps were severely affected by collagenase (Table 2); in mature pulps they were somewhat more resistant and the collagen fibre bundles of aged tissues were not affected by the enzyme. The short incubation period of the tissues with the enzyme prevented the complete digestion of the fibres and permitted the differentiation between elements having varying degrees of resistance to collagenase. The reactivity of the dental pulp with PAS was not affected by incubating the sections with sialidase. Localization of amino groups Nuclei of fibroblasts, odontoblasts, endothelial cells and defense cells stained more intensely than their cytoplasm. In the fibroblasts the nucleoli and the granular In the very weakly stained cytoplasm of chromatin appeared as darker structures.
HISTOCHEMICALSTIJDY
TABLE
2.
EFFECTSOFBUFFERS
OFTHECoNNECTlVt71SSUtOPTHE
AND ENZYMESON
Argyrophilic Reticular fibres of deep pulp Phosphate buffer pH 7.0 4 hr at 37°C
Removes argyrophilia
Citrate buffer pH 3.5 4 hr at 37°C
Removes argyrophilia Disrupts fibres
C’ollagenese (0.1 mg/ml)
Digested
DENTAL
THE FIBRESOF THEDENTAL
fibres ___ .__~~ Korff’s fibrcs
153
PULP
PULP
Collagen libres
No effect
No effect
No etlect
No etfcct
No etfect
3 hr at 37°C -
fibroblasts, small DNFB-positive granules were observed in cells of young and mature pulps. This intracellular material could not be found in aged pulps. The cytoplasm of the odontoblasts was more reactive than that of other pulp cells, but no granules were observed. The argyrophilic fibres and the ground substance reacted with DNFB (Table I). The bundles of collagen fibres which formed most of the extracellular matrix of aged pulps were less reactive than the fine argyrophilic fibres. The ground substance also stained faintly in these pulps. The calcified areas of the dental pulp were unstained. Only the borders of the nodules reacted with DNFB (Fig. 5). These observations were consistent with the findings that immature bone and predentine reacted with DNFB, but the fully calcified tissues were unstained (ZERLOTTI and ENGEL, 1962). The ground substance surrounding the nodules was more reactive. Localization
of acid mucopolysaccharides
The pulps of young patients gave a strong metachromatic reaction. The metachromasia observed in the nuclei of the pulp cells was probably due to nucleic acids. The cytoplasm of some fibroblasts contained small metachromatic granules which resembled those observed in sections stained with PAS or DNFB. In the odontoblasts, the metachromatic material was dispersed throughout the cytoplasm and no granules were seen. Following exposure to hyaluronidase the intracellular material found in the fibroblasts behaved differently from that observed in the odontoblasts. The cytoplasmic granules of the former were completely digested but the metachromasia of the odontoblasts was only partially removed by the enzyme. It is possible that the hyaluronidase-resistant materials of the cytoplasm of these cells is mainly ribonucleic acid. The metachromasia was less intense in fibroblasts and odontoblasts of mature teeth and absent in the cells of aged group. The ground substance of young pulps was also metachromatic. No differences could be observed between the staining of the Weil’s zone and deeper regions, as reported by SASSO and CASTRO (1957).
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The basement membranes of the blood vessels stained more intensely than the rest of ground substance. In mature pulps the ground substance was less reactive, but the argyrophilic fibres were well stained. Aged pulps were weakly metachromatic mainly due to the great quantity of collagen present. The ground substance was only faintly stained (Table 1). The calcified nodules were unstained or orthochromatic, but their borders, the ground substance and fibres surrounding them stained metachromatically. When the sections were incubated with hyaluronidase most of the metachromasia observed in the extracellular matrix was removed. The remaining metachromatic material could be sialic acid, which resists hyaluronidase, stains metachromatically and has been shown to be present in the dental pulp by analytical methods (AMICI and GRAZIANO,1959; CASTELLANI et al., 1959). To test this possibility, some sections were incubated with sialidase. The results showed a decreased metachromasia of the ground substance. However, Alcian blue and Azure A, used according to the methods described by SPICERand WARREN(1959), failed to demonstrate the presence of sialic acid in the pulps, perhaps due to shortcomings of this technique. Localization of nucleic acids Ribonucleic acid. The cytoplasm of fibroblasts and defense cells of young, mature
and aged pulps was weakly stained by the gallocyanin-chrome alum technique. In the odontoblasts of young and mature pulps, however, the cytoplasm was more intensely stained (Fig. 6). The material was distributed throughout the cytoplasm and was completely removed by incubation in ribonuclease (Fig. 7). Cytoplasmic staining was not observed in odontoblasts of aged pulps. Deoxyribonucleic acid. In sections digested with deoxyribonuclease and stained with gallocyanin-chrome alum, the nuclei were very faintly coloured. The chromatin was barely visible and the nucleoli appeared more prominent. Nuclear deoxyribonucleic acid was also demonstrated by staining with the Feulgen method (Fig. 8). Ej&ects of treatment with buglers Neutral phosphate bu&r. Soluble components
of the cells and the extracellular matrix were extracted when the sections were incubated in phosphate buffer at pH 7.0 (Tables 1 and 2). PAS-staining material of the ground substance of young and mature pulps was almost completely removed by the buffer solution (Fig. 9). In some young pulps the interfibrillar space seemed to be empty and the fibres were very weakly stained. The effect on aged pulps was less marked. The PAS staining of the basement membranes was less intense than that observed in sections of untreated tissues, but they were less affected by the buffer than the extracellular matrix of the rest of the pulp. Similar results could be observed in the argyrophilic fibres of sections stained with the silver impregnation technique. The fibres, which in untreated sections stained black, lost their ability to react with silver and stained pink. The orientation of the fibres was not affected and the delicate network could be seen. The KorR’s fibres, however, were not affected by the phosphate buffer and continued to stain black.
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The pulp nodules, which in untreated sections were unstained, reacted weakly with PAS and DNFB after treatment with the buffer. The PAS and DNFB-positive cytoplasmic granules described before were completely removed by the buffer, but some glycogen still remained in the nerve fibres after incubation. Nucleoproteins, which were stained with DNFB, were partially removed. salt soluble fraction” was The results obtained suggested that the “neutral primarily composed of extrafibrillar, water-soluble glycoprotein. Soluble collagen (JACKSON, 1957), nucleoproteins and electrolytes also would be included in the extracted fraction. Acid citrate buffer. The citrate buffer, pH 3.5, appeared to remove the water soluble components of the tissues and also affected their structure by changing the states of organization and aggregation of proteins (Table 1). In sections stained with silver impregnation technique the argyrophilic fibres appeared disrupted and lost most of their capacity to stain (Figs 10 and 11). The Korff’s fibres, however, were not affected, retaining their structure and their black staining. In young and mature pulps treated with the PAS the ground substance was irregularly stained (Fig. 12); in some areas it was unstained and in others it was as reactive as that of control sections. It was not possible to determine whether the stained extrafibrillar structure was ground substance which partially resisted the action of the buffer, or if it represented the degradation products of the fibres. The extracellular matrix of aged pulps was more resistant to the action of the buffer than the younger tissues. Similar effects on the extracellular matrix were observed in sections which were reacted with DNFB (Table 2). The calcified nodules, which were unstained in the control sections, stained intensely with PAS and DNFB (Fig. 13) after incubation in acid citrate buffer. The cytoplasmic granules were completely dissolved and the DNFB-positive nucleoproteins were partially removed. The cytoplasm of the fibroblasts, which was weakly stained in untreated sections, stained more intensely with DNFB.
DISCUSSION
Composition and organization of the connective tissue of the dental pulp The extracellular matrix of the dental pulp is characterized by the presence of glycoproteins, acid mucopolysaccharides and proteins containing appreciable amounts of lysine-hydroxylysine. The composition of the glycoproteins, which are mostly present in the ground substance, is not known, although it seems that some of them contain sialic acid, as suggested by the decrease of staining of the sections treated with sialidase. The proteins containing lysine-hydroxylysine are important constituents of both the fibres and the ground substance. On the basis of histochemical findings the matrix is shown to contain at least two fractions: one readily soluble in water and saline, the other insoluble and resistant to mild reagents. These two moieties correspond respectively to the waterrich and the colloid-rich phases described by JOSEPH et al. (1952). These two phases
156
E. ZERLOTTI
are considered to coexist in equilibrium and their composition is thought to remain constant. Their relative amounts can vary in different tissues or in the same tissue in various physiological and pathological conditions (JOSEPHet al., 1952; ENGEL et al., 1960; GERSHand CATCHPOLE,1960). These phases appear to have a morphologic counterpart at the submicroscopic level as shown by electron microscopic studies of various connective tissues fixed by freeze-drying (BONDAREFF, 1957; DENNIS, 1959). These concepts appear to be related also to the findings of HALDI, WYNN and CULPEPPER(1961) who extracted a “dental pulp fluid” from dogs’ teeth by drilling a hole into the pulp chambers. This “pulp fluid” seems to correspond to the warerrich phase of the extracellular matrix. It is conceivable that a part of the soluble proteins contained in this phase could be loosely bound to the colloid, explaining the low protein content of the “pulp fluid”. The colloids of the dental pulp and of all connective tissues have amphoteric properties. The carboxyl groups of collagen, the glycoproteins, and the acid mucopolysaccharides confer on the extracellular matrix a net negative charge at physiological pH. As a consequence of the amphoteric properties and of the charge, certain selective interactions with anions, cations and polar molecules (including various stains) could be anticipated (JOSEPHet al., 1952, 1954 and 1959; ENGELer al., 1961). The basement membranes of the blood vessels of the dental pulp are particularly rich in glycoproteins as shown by their deep staining with the PAS method. Their ground substance is more resistant to the action of buffers and enzymes than the adjacent ground substance, suggesting the predominance of the colloid-rich phase in these areas. The reticular fibres of the basement membranes are more densely packed around the blood vessels, but they are affected in the same way as the contiguous argyrophilic fibrous network. The amino acid composition of collagen and reticulin is similar (WINDRUM,KENT and EASTOE,1955). Perhaps they differ in the tertiary structure of their proteins and certainly in the amount of carbohydrates associated with the fibres. The capacity of the reticular fibres to react with silver seems to be related to the carbohydrate bound to the surface (SCHWARZ,1957). The extraction of the neutral salt soluble fractions does affect the glycoproteins. This produces the loss of the argyrophilia of the reticular fibres of the deep pulp. Treatment with acid citrate buffer or incubation with collagenase disrupts these fibres. In collagen fibres the absence of argyrophilia is a characteristic of these elements and has been related to their maturation when they lose a part of their carbohydrate fraction and become more cross-linked (SCHWARZ, 1957). This is consistent with our findings; that collagen fibres resist the action of buffers and of collagenase during a short incubation period. The exact chemical composition of the Korff’s fibres is unknown. Histochemically, however, they are characterized by their reactivity with silver and their resistance to neutral and acid buffers. These special properties are probably related to the role of these fibres in the formation of the dentine matrix. In the cases of all three fibres they could be considered as interacting with the contiguous ground substance to form coacervates.
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Odontoblasts which are involved in the formation of the dentine show a large amount of ribonucleic acid in their cytoplasm which suggests intense activity of these cells in protein synthesis. The low content of ribonucleic acid found in the cytoplasm of fibroblasts could be due to the inactivity of these cells or to the low sensitivity of the histochemical method used. The former hypothesis does not seem feasible, since granules containing proteins and carbohydrates can be found in their cytoplasm. Changes in ageing of connective tissues Certain general changes which occur in the extracellular matrix of other connective tissues during aging are observed also in the dental pulp. These are enumerated below: 1. Decrease in the ratio of ground substance to collagen. For example, the hexosamine:collagen ratio of dermis decreases from 0.040 in persons 23-35 years old to 0.021 in old (72-77 years) patients (KORENCHEVSKY, 1961). In aged pulps the extracellular matrix is formed mostly of collagenous bundles. Collagen fibres replace to a large extent the argyrophilic fibres and the ground substance. Analytical studies would be required to confirm this observation. 2. Increase in the resistance to proteolytic enzymes. KEECH (1954) observed that the amount of collagen hydrolysed by collagenase decreases in the ratio of 11.6:g.l for individuals l-10 years and 71-80 years old. This can also be observed in dental pulps. The reticular fibres and ground substance of young pulps are readily digested by some enzymes, but the collagen fibres and the extracellular substance of ageo tissues are more resistant to their actions. 3. Decrease in the solubility. The most soluble fraction of collagen exists in the ratio of 50: 15: 4 in individuals less than I year, l-10 years and more than 30 years respectively (BANFIELD, 1952). These findings are in agreement with our observation that old dental pulps are more resistant to the action of buffer solutions. 4. Decrease in water content. YOON et al. (1960) observed that the water content of the dental pulps is lower in aged tissues. 5. Decrease in the chemical reactivity. Collagen loses its capacity to react with silver in maturation and the extracellular matrix of the dental pulp stains less intensely with PAS, DNFB and toluidine blue. All these factors suggest that in old age there is a change in the aggregation ot the tissue colloids and a restriction of the water-rich phase. An increase in crosslinking (hydrogen bonds and salt linkages) seems to be a characteristic physicochemical phenomenon in ageing (VERZAR, 1957). This would increase the resistance of the colloids to disruptive agents. The low chemical reactivity of the ground substance and fibres of the dental pulp could result from the masking of reactive (s-amino groups of lysine and hydroxylysine, glycol and nucleophilic groups of carbohydrates) groups through cross-linkages during ageing. These changes in water content and macromolecular structure of the pulps would be expected to be accompanied by changes in electrolyte distribution. For example, a loss of sodium and an increase in potassium would be anticipated (JOSEPH e/ c/l.,
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1961). Calcium and fluoride are found to be in higher concentration in old pulps (YOON et al., 1960). These elements may be present in the tissues partially bound to the proteins and partially incorporated in the apatite crystals of pulp nodules. The high occurrence of calcification in aged pulps could also be related to the changes of the tissue colloids. The reorganization in the internal structures would release the calcium bound to negatively charged tissue colloids. The increase of the local concentration of this cation in a closed system would induce apatite formation (ENGEL, JOSEPHand LASKIN, 1962). The organization of cellular proteins also appears to change during ageing. RUZICKA(1924) stated that “in living organisms a continuous process of solidification of cellular proteins probably causes the main changes in old age”. It is difficult to study these alterations in the cytoplasm of the pulp cells histochemically, since these proteins are normally highly aggregated, as can be observed by the low reactivity to DNFB. This suggests that the e-amino groups of lysine and hydroxylysine are involved in forming tertiary structures. These amino groups, however, are exposed to DNFB by treatment with acid citrate buffer which alters the organization of the proteins. The only evidence for the changes of the cellular colloids in old age found during this study was the reduced number or even the absence of cytoplasmic inclusions in the fibroblasts in aged pulps. The results of this study confirm that the pulpal connective tissue is similar in composition, organization and histochemical reactivity to other connective tissues. Certain unique properties are probably due to the anatomical and physiological peculiarities of teeth. It is nevertheless reasonable to infer that the biological reactivity of the dental pulp in response to changes in internal milieu would parallel that of the other connective tissues. Acknowledgement-The ENGEL
author wishes to express gratitude to Dr. MILTON B.
for his guidance.
This paper is based on the thesis submitted by the author as partial fulfilment of the requirements for the degree of Master of Science in Histology in the Graduate College, University of Illinois, Chicago, 1962. Rksum&Cette Etude a pour objet I’Ctudedes propriktks histochimiques et l’organisation du tissu conjonctif pulpaire ainsi que les changements des colloides tissulaires en fonction du vieillissement. Des mkthodes plus rkentes de prdparation des specimens et certaines colorations histochimiques spkcifiques ont BtB utilisks. 11apparait ainsi que la matrice extra-cellulaire du tissu conjonctif pulpaire contient des glycoprotkines, de l’acide sialique, des mucopolysaccharides et des prottines pourvues de groupements actifs E-ammts de lysine-hydroxylysine. Les fibres et la substance fondamentale semblent Btroitement li6es et forment des agglomkrats. La matrice extra-cellulaire a une structure colloldale hetbrogkne contenant des fractions soluble et insoluble. Au niveau des pulpes jeunes, la fraction soluble est importante et les tissus sont t&s sensibles & l’action de solutions tampons et d’enzymes. Avec le vieillissement, la fraction soluble dkcroit et l’on note un changement dans le groupement des colloides tissulaires. Ces transformations se traduisent par une rksistance Clevke aux solutions tampons, aux enzymes prottolytiques et par des
HISTOCHEMICAL STUDYOF THE CONNECTIVETISSUEOF THE DENTALPULP
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diffkrences de coloration des tissus. L’altkration de l’organisation interne des proteines provoquent vraisemblablement une redistribution des ions et elle est 1iQ B la formation de pulpolithes. Zusammenfassung-Diese Untersuchung hat die histochemische Wirksamkeit, die Gestaltung des Zahnpulpabindegewebes so wie die Vertinderungen innerhalb der Gewebskolloiden wlhrend des Altems zum Gegenstand. Moderne Pr¶tsmethoden und verschiedene spezifische histochemische FLrbungsmethoden sind dabei beniitz t worden. Das Ergebnis ist, dass die extrazellulire Zahnpulpasbindegewebematrix Glycoproteinen, Sialische SBure, MucopolysaccharidensBure, und Proteinen enthglt welche mit reagierenden c-amino Gruppen der Lysine-Hydroxylysine versehen sind. Die Fasern und die Grundsubstanz sind eng verbunden und bilden Komplexe. Die extrazellullre Matrix hat eine ungleichmlssige Kolloidstruktur, welche liisliche und unltisliche Gruppen enthtilt. Bei jungen Pulpas sind die l&lichen Gruppen sehr reichhaltig und die Gewebe sind stark empfindlich fiir PufferlGsungen und Enzymen. WBhrend des Altems wird eine Verminderung der liislichen Gruppe sowie eine Vertinderung in der Anordnung der Gewebskolloiden beobachtet. Diese Vertinderungen Hussern sich durch eine htihere Widerstandsfahigkeit gegeniiber PufferlGsungen und proteolytischen Enzymen sowie durch Anderungen in der F&bung der Gewebe. Die VerPnderungen in der inneren Proteinanordnung veranlassen wahrscheinlich eine Wiederverteilung der Ionen und stehen im Zusammenhang mit der Dentikelbildung. REFERENCES AMICI, G.
and GRAZIANO, V. 1959. Sulla presenza di acido sialico nella polpa e nella dentina di bue. Rass. Trim. Odontoiat. 40, 343-348. BANFIELD, W. G. 1952. The solubility and swelling of collagen in dilute acid with age variations in man. hat. Rec. 114, 157-171. BONDAREFF,W. 1957. Submicroscopic morphology of connective tissue ground substance with particular reference to fibrillogenesis and aging. Gerontologia 1, 222-233. CASTELLANI,A. A., FERRI, G., BOLOGNANI, L. and GRAZIANO,V. 1959. Contenuto di acido sialico in alcuni tessuti connetivi. Boll. Sot. ital. Biol. sper. 35, 2145-2148. DENNIS, J. B. 1959. Effects of various factors on the distribution of ferrocyanide in ground substance. A.M.A. Arch. Path. 67, 533-549. EINARSON, L. 1932. A method for progressive selective staining for nissl and nuclear substance of nerve cells. Amer. J. Path. 8, 295-305. EINARSON,L. 1951. On the theory of gdllocyanin-chromalum staining and its implication for quantitative estimation of basophilia. Acta path. micro&ok stand. 26, 82-102. ENGEL, M. B., JOSEPH,N. R., LASKIN,D. M. and CATCHPOLE,H. R. 1960. A theory of connective tissue behavior: Its implication in periodontal disease. Ann. N. Y. Acad. Sci. 85, 399420. ENGEL, M. B., JOSEPH,N. R., LASKIN, D. M. and CATCHPOLE,H. R. 1961. Binding of anions by connective tissue: dermis and cartilage. Amer. J. Physiol. 201, 621-627. ENCJEL,M. B., JOSEPH,N. R. and LASKIN,D. M. 1962. Morphologic and physicochemical events in calcification of turkey tendon. In: Printed abstracts, International Association for Dental Research, 40th General Meeting, p. 29. ENGSTR~M,H. and OHMAN, A. 1960. Studies on the innervation of human teeth. J. dent. R~.~. 39, 799-809. GERSH, I. 1932. The Altman technique for fixation by drying while freezing. Anar. Rec. 53, 309-337. GERSH, I. 1948. Application in pathology of the method of fixation by freezing and drying of tissues. Bull. int. Ass. med. Mus. 28, 179-185. GERSH, I. and CATCHPOLE, H. R. 1949. The organization of ground substance and basement membranes and its significance in tissue injury, disease and growth. Amer. J. Anat. g&457-521. GERSH, I. and CATCHPOLE, H. R. 1960. The nature of ground substance of connective tissue. Persp. Biol. Med. 3, 282-319. GOMORI, G. 1937. Silver impregnation of reticulin in paraffin sections. Amer. J. Path. 13, 993-1001. HALDI, J., WYNN, W. and CULPEPPER,W. D. 1961. Dental pulp fluid. Arch. oral Biol. 3,201-206.
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HOXHKI~~, R. D. 1948. A microbiochemical reaction resulting in the staining of polysaccharide structures in fixed tissue preparations. Arch. Biochem. 10, 131-141. JACKSON,D. S. 1957. Connective tissue growth stimulated by caragenin. Biochem. J. 65,277-284. JACKSON,D. S. and WILLIAMS,G. 1956. Nature of reticulin. Nature, Land. 178, 915-916. JOSEPH, N. R., CATCHPOLE,H. R., LASKIN, D. M. and ENGEL, M. B. 1959. Titration curves of colloidal surfaces-II. Connective tissues. Arch. biochem. biophys. 84, 224-242. JOSEPH, N. R., ENGEL, M. B. and CATCHPOLE,H. R. 1952. Interactions of ions and connective tissue. Biochim. Biophys. Acfa 8, 575-587. JOSEPH,N. R., ENGEL, M. B. and CATCHPOLE,H. R. 1954. Homeostasia of connective tissues-II. Potassium-sodium equilibrium. A.M.A. Arch. Path. 58, 40-58. JOSEPH,N. R., ENGEL, M. B. and CATCHPOLE,H. R. 1961. Distribution of sodium and potassium in certain cells and tissues. Nature, Land. 191, 1175-l 178. KEECH, M. K. 1954. The effect of collagenase on human skin collagen. Comparison of different age-groups and of cases with and without collagen disease. Yale J. biol. Med. 23,295-306. KORENCHEVSKY, V. 1961. Physiological and Pathological Aging. Hafner, New York. LANGELAND,K. 1957. Tissue changes in the dental pulp. Odont. Tidskr. 65, 239-386. LILLIE, R. D. 1954. Histoputhologic Technique und Practical Histochemistry (2nd Ed.). The Blakiston Div., McGraw-Hill, New York. MCMANIJS, J. F. A. 1948. Histological and histochemical uses of periodic acid. Stain Technol. 23,99-108. ORBAN, B. J. 1929. Contribution to the histology of the dental-pulp and periodontal membrane, with special reference to the cells of defense of these tissues. J. Amer. dent. Ass. 16, 965-996. ORBAN, B. J. 1957. Oral Histology and Embryology. C. V. Mosby, St. Louis. RUZICKA, V. 1924. Beitrage zum Studium der Poroplasmahysteresis und der histerischen Vorgange. Arch. mikr. Anat. 101, 459482. SANGER,I. 1945. The free amino groups of insulin. Eiochem. J. 39, 507-515. SASSO, W. S. and CASTRO,N. M. 1957. Histochemical study of amelogenesis and dentinogenesis. Oral Surg. 10, 1323-1329. SCHOUR,I. 1953. Noyes’ Oral Histology and Embryology (7th Ed.). Lea and Febiger, Philadelphia. SCHWARZ,W. 1957. Morphology and differentiation of the connective tissue fibres. In: Connective Tissue (Ed. by TUNBRIDGE,R. F.), p. 144. Thomas, Springfield. SPICER, S. S. and WARREN, L. 1959. The histochemistry of sialic acid containing mucoproteins. J. Histochem. Cytochem. 8, 135-137. VERZAR,F. 1957. Studies on adaptation as a method of gerontological research. CIBA Fundation Colloquia on Aging, Vol. 111,pp. 60-68. Little, Brown, Boston. YOON, S. H., BRUDEVOLD,F., SMITH, F. A. and GARDNER, D. E. 1960. Fluoride and calcium concentrations in human dental pulps. J. dent. Res. 39, 671-672. WINDRUM,G. M., KENT, P. W. and EASTOE,J. E. 1955. The constitution of human renal reticulin. Brit. J. exp. Path. 36, 49-56. ZERLOTTI,E. and ENGEL, M. B. 1962. The reactivity of proteins of some connective tissues and epithelial structures with 2:4_dinitrofluorobenzene. J. Histochem. Cytochem. 10, 537-546.
PLATE I All sections were from tissues fixed by freeze-drying. FIG. 1, High power of a young pulp stained with PAS showing granules (arrow) in the cytoplasm of fibroblast. Nucleus of fibroblast (r). x 1860. FIG. 2. Young pulp stained with PAS. Blood vessel (v), basement membrane (b). x 560. FIG. 3. Section of mature pulp stained with PAS. The basement membrane (b) is deeply stained. Blood vessel (v). x 560. FIG. 4. Aged pulp stained with PAS. The basement membrane (b) is strongly reactive. Collagen fibres stain weakly. Blood vessel (v). x 560. FIG. 5. Mature pulp stained with DNFB. The pulp nodule (s) is not stained. The extracellular substance around it (arrow) is more reactive. x 560.
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All
sections were
from tissues fixed by freeze-drying.
FIG. 6. Young pulp stained with gallocyanin-chromalum technique. Note the staining of the cytoplasm (arrow) of odontoblasts (0) and fibroblasts (f). x 560. FIG. 7. Section of the same pulp digested with ribonuclease prior to staining with gallocyanin-chromalum. The reactive material was removed from the cytoplasm of odontoblasts (0) and fibroblasts (f). x 560. FIG. 8. Section of young pulp stained blasts (0) and fibroblasts (f). x 560.
with the Feulgen technique.
Odonto-
FIG. 9. Mature pulp treated with phosphate buffer prior to staining with PAS. The ground substance was removed to a large extent. Blood vessel (v), basement membrane (b) and nerve bundle (n). :i 560.
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PLATE3 All sections were from tissues fixed by freeze-drying. FIG. 10. Section of young pulp stained with the Gomori’s silver impregnation technique. The reticulin fibres are more densely packed around the blood vessels (v). x 560. FIG. 11. Section of the same pulp treated with acid citrate buffer prior to staining with the Gomori’s impregnation technique. The reticulin fibres are completely disrupted. Blood vessel (v). x 560. FIG. 12. Section of mature pulp incubated with acid citrate buffer before staining with PAS. The extracellular matrix is disrupted. Blood vessel (v) and basement membrane (b). x 560. FIG. 13. Section of mature pulp treated with acid citrate buffer prior to staining by DNFB. Pulp nodule (s) is strongly reactive. The extracellular matrix is disrupted and weakly stained. x 560.
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oral
Bid.
Vol.9,
pp.l49-162,
1964.
Pergamon Press Ltd. Printed inCt.Britain.
HISTOCHEMICAL STUDY OF THE CONNECTlVE TISSUE OF THE DENTAL PULP E. ZERLOTTI Faculdade
de Farmacia e Odontologia de Aracatuba, Estado de Sao Paulo, Brazil
Summary-This study was concerned with the reactivity and organization of the connective tissue of the dental pulp and with the changes of the tissue colloids during ageing. Newer methods of preparation of the specimens and certain specific histochemical procedures were used. The results indicate that the extracellular matrix of the connective tissue of the dental pulp contains glycoproteins, sialic acid, acid mucopolysaccharides and proteins bearing reactive c-amino groups of lysine-hydroxylysine. The fibres and the ground substance are considered to interact forming coacervates. The extracellular matrix is organized as a heterogeneous colloid containing soluble and insoluble fractions. In young pulps the soluble fraction is large and the tissues are markedly affected by buffer solutions or enzymes. In ageing thereis a decrease of the soluble fraction and a change in the aggregation of the tissue colloids. These changes are signalled by the high resistance to buffer solutions and proteolytic enzymes and by the changes in staining of the tissues. The alteration in the internal organization of the proteins probably induces a redistribution of ions and is related to pulp stone formation.
INTRODUCTION EXCELLENT morphological studies of the dental pulp have been made in the past (ORBAN, 1929, 1957; SCHOUR, 1953; LANGELAND, 1957). However, newer concepts about the organization and behaviour of connective tissues (GERSH and CATCHPOLE, 1949, 1960; JOSEPH,ENGEL and CATCHPOLE, 1954; JOSEPHet al., 1959; ENGEL et d., 1960) now permit the study of the dental pulp from a different point of view. We have utilized these concepts and some newer histochemical methods (ZERLOTTI and ENGEL, 1962) to investigate the organization and reactivity of some protein components of this tissue. In this way it was also possible to discern changes in the tissue colloids during ageing. The extracellular matrix of the connective tissue of the dental pulp was shown to contain protein bearing appreciable amount of lysine-hydroxylysine, glycoproteins, some of them containing sialic acid and acid mucopolysaccharides. The matrix is organized as a heterogeneous colloid composed of soluble and insoluble phases. With ageing there appears to be a shift in the relation between the two fractions as the tissues lose water. This is accompanied by a decrease in the reactivity of ground substance and of fibres as determined by histochemical means. 149
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MATERIALS AND METHODS Pulps from non-carious teeth with normal periodontal tissues were utilized. A total of 55 teeth were used, including: I: 25 premolars from patients 11-15 years old, extracted prior to orthodontic treatment (young group); II: 25 premolars and molars from patients 40-55 years old (mature group); III: 5 premolars from patients older than 70 years (aged group). Teeth of patients in groups II and III were extracted to facilitate prosthetic treatment. Wherever possible the extractions were performed under block anaesthesia Pulps were removed from the teeth by the method described by ENGSTR~~M and OHMAN(1960) and immediately fixed by the freeze-drying method (GERSH, 1932, 1948). Tissues were frozen in isopentane chilled to - 150°C immersed in liquid nitrogen, and then dehydrated in vacua for a period of 4-6 days at -30 to -35°C. The specimens were embedded in paraffin wax (m.p. 57-59°C) in vacua for lo-15 min. Serial sections were cut at 6-10 p and mounted on slides using a minimal amount of albumin. The frozen-dried sections were denatured in absolute ethanol overnight at 25°C to precipitate proteins, thereby minimizing loss of water-soluble tissue components. Histochemical and histological methods
Sections of all specimens were stained with Harris’ haematoxylin and eosin for morphological studies. The periodic acid-Schiff (PAS) method of HoTcHKrss (1948) and MCMANUS(1948) was used to localize glycogen and other carbohydrate or carbohydrate-containing proteins bearing free 1,Zglycol groups. To enhance the specificity of the method, various enzymes were employed prior to staining. Glycogen was removed by incubating tissue sections in a-amylase (Nutritional Blochemicals) (1 mg/l ml of Sorensen’s 0.1 M phosphate buffer, pH 6.0) for 30 min at 37°C. Sialic acid was digested by Neuraminidase (Behrimwerke) (100 units of sialidase extracted from Vibrio cholerae culture in 1 ml of 0.05 M sodium acetate buffer pH 5.5). Some sections were incubated in Collagenase (Agricultural Biologicals) (0.1 mg/l ml of Sorensen’s 0.1 M phosphate buffer, pH 7.0) at 37” C for 3 hr. Control sections were incubated in the buffers alone. Free amino groups of tissue proteins were localized by means of the 2,4-dinitrofluorobenzene (DNFB) reagent of SANGER(1945) using the method described by ZERLOTTIand ENGEL (1962). The sections were examined with monochromatic light at a wavelength of 410-420 rnp, using a Zeiss interference wedge, where the extinction of the dinitrophenyl derivatives is high. Contrast and resolution were greatly improved by this means, especially when a high energy light source (HBO 200 Osram) was used. Acid mucopolysaccharides were identified by their metachromatic staining with toluidine blue. During the present study, sections were stained with a 16 x 10e3 M solution of toluidine blue at pH 7.0 for 15 min. The excess of dye was blotted with filter paper and the sections mounted in water and examined immediately. In some experiments hyaluronic acid and chondroitin sulphate A and C were removed
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before staining by treating representative sections with testicular hyaluronidase (Wydase, Wythe Pharmaceutical Co.) at a concentration of 150 T.R.U./l ml of Sorensen’s 0.1 M phosphate buffer, pH 6.0, for 2 hr at 37°C. Control sections were incubated in the buffer alone. The gallocyanin-chrome alum technique was used for the localization of total nucleic acid (EINARSON,1932, 1951). Ribonucleic acid was removed by incubating the post-fixed sections with ribonuclease prior to staining. Slides were incubated for 4 hr at 37°C in a solution of ribonuclease (Worthington) in distilled water (5 mg/l ml), pH 5.5. Deoxyribonucleic acid was digested by treating the denatured sections for 4 hr at 37°C and pH 4.3 with deoxyribonuclease (Worthington) dissolved at a final concentration of 2 mg/l ml in a solution which contained 0.01 per cent gelatin and 0.01 M magnesium sulphate. Control sections were incubated in water. The Feulgen reaction was used to stain deoxyribonucleic acid. The silver impregnation method of GOMORI(1937) and the acid fuchsin-picric acid staining of Van Gieson (LILLIE, 1954) were used to localize and to differentiate between reticular and collagen fibres. Some sections were incubated in collagenase solution at 37°C for 3 hr and stained with the silver impregnation. Extraction of soluble components Soluble elements of the dental pulp were extracted by treating the sections with two different buffer solutions (JACKSONand WILLIAMS,1956; JACKSON,1957) prior to staining: (1) An “acid citrate soluble fraction” was removed by incubating undenatured sections in 0.1 M citrate buffer (0.1 M citric acid, 75 ml and 0.1 sodium citrate, 25 ml), pH 3.5, for 4 hr at 37°C; (2) A “neutral salt soluble fraction” was extracted with 0.1 M phosphate buffer (0.1 M Na,HPO,, 60 ml and 0.1 M NaH,POp, 40 ml), pH 7.0, under the same conditions. The extracted sections were then stained with the periodic acid-Schiff method, 2,4-dinitrofluorobenzene and silver impregnation. FINDINGS During the removal of the pulps from young teeth the odontoblastic. layer generally could not be readily detached from the dentine; occasionally small groups of cells were removed. For this reason more emphasis is given to the other structures of the connective tissue of the dental pulp and the discussion of odontoblasts is incomplete. Localization of the PAS-positive material The nuclei of the fibroblasts were unreactive with PAS, but small granules (< 1 CL) could be detected in their cytoplasm (Fig. 1). The cells containing PAS-positive granules were located mostly in the pulp chamber and there was no evidence of their association with any particular structure. Most of these intracellular inclusions resisted digestion by amylase although some of them were removed by the enzyme. They were only occasionally found in pulp cells of aged patients. Similar material could not be observed in the cytoplasm of the odontoblasts, but in one case PASpositive granules were found in the extracellular substance between odontoblasts of young pulps. It was not possible to determine if these granules were glycogen or amylase-resistant glycoproteins.
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The PAS-positive ground substance was slightly more reactive in pulps of young patients than in mature or aged pulps (Table 1). The argyrophilic fibres also stained with PAS and could be better differentiated from the ground substance in mature pulps. In specimens of aged patients the collagen fibres which composed most of TABLE 1. REACTIVITYOF THE~~ROUNDSLJBSTANCEOFTHEDENTAL
Periodic acid Schiff (PAS)
2,4-dinitrofluorobenzene (DNFB)
PULP
Toluidine blue (Metachromasia)
_
Young pulps (11-15 years) Mature pulps (40-55 years) Aged pulps (more than 70 years)
/
1
+ -t
I~
I i
-t
i’F
I
!+I
i
I ! I
-I -I 4.
i
.I !
-; -1.
t -I-
+
+ i_
i
T
+
+
I 1 1. ~1. -t-t / -I-
‘f
+
-t
f
‘F
the extracellular matrix were not intensely stained. The ground substance in these pulps also showed a low reactivity with PAS. The basement membranes of blood In young pulps they were slightly vessels were distinctly visualized in all pulps. fainter and thinner than those of mature or aged patients (Figs 2, 3 and 4). The ground substance was more intensely stained around the pulp nodules which, with exception of their borders, were PAS-negative. In sections treated with amylase an overall decrease of staining intensity was observed. This was probably due to some proteolytic activity of the enzyme preparation. Glycogen was found in nerve fibres in the form of coarse and numerous granules. The fibres and the ground substance of young pulps were severely affected by collagenase (Table 2); in mature pulps they were somewhat more resistant and the collagen fibre bundles of aged tissues were not affected by the enzyme. The short incubation period of the tissues with the enzyme prevented the complete digestion of the fibres and permitted the differentiation between elements having varying degrees of resistance to collagenase. The reactivity of the dental pulp with PAS was not affected by incubating the sections with sialidase. Localization of amino groups Nuclei of fibroblasts, odontoblasts, endothelial cells and defense cells stained more intensely than their cytoplasm. In the fibroblasts the nucleoli and the granular In the very weakly stained cytoplasm of chromatin appeared as darker structures.
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TABLE
2.
EFFECTSOFBUFFERS
OFTHECoNNECTlVt71SSUtOPTHE
AND ENZYMESON
Argyrophilic Reticular fibres of deep pulp Phosphate buffer pH 7.0 4 hr at 37°C
Removes argyrophilia
Citrate buffer pH 3.5 4 hr at 37°C
Removes argyrophilia Disrupts fibres
C’ollagenese (0.1 mg/ml)
Digested
DENTAL
THE FIBRESOF THEDENTAL
fibres ___ .__~~ Korff’s fibrcs
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PULP
PULP
Collagen libres
No effect
No effect
No etlect
No etfcct
No etfect
3 hr at 37°C -
fibroblasts, small DNFB-positive granules were observed in cells of young and mature pulps. This intracellular material could not be found in aged pulps. The cytoplasm of the odontoblasts was more reactive than that of other pulp cells, but no granules were observed. The argyrophilic fibres and the ground substance reacted with DNFB (Table I). The bundles of collagen fibres which formed most of the extracellular matrix of aged pulps were less reactive than the fine argyrophilic fibres. The ground substance also stained faintly in these pulps. The calcified areas of the dental pulp were unstained. Only the borders of the nodules reacted with DNFB (Fig. 5). These observations were consistent with the findings that immature bone and predentine reacted with DNFB, but the fully calcified tissues were unstained (ZERLOTTI and ENGEL, 1962). The ground substance surrounding the nodules was more reactive. Localization
of acid mucopolysaccharides
The pulps of young patients gave a strong metachromatic reaction. The metachromasia observed in the nuclei of the pulp cells was probably due to nucleic acids. The cytoplasm of some fibroblasts contained small metachromatic granules which resembled those observed in sections stained with PAS or DNFB. In the odontoblasts, the metachromatic material was dispersed throughout the cytoplasm and no granules were seen. Following exposure to hyaluronidase the intracellular material found in the fibroblasts behaved differently from that observed in the odontoblasts. The cytoplasmic granules of the former were completely digested but the metachromasia of the odontoblasts was only partially removed by the enzyme. It is possible that the hyaluronidase-resistant materials of the cytoplasm of these cells is mainly ribonucleic acid. The metachromasia was less intense in fibroblasts and odontoblasts of mature teeth and absent in the cells of aged group. The ground substance of young pulps was also metachromatic. No differences could be observed between the staining of the Weil’s zone and deeper regions, as reported by SASSO and CASTRO (1957).
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The basement membranes of the blood vessels stained more intensely than the rest of ground substance. In mature pulps the ground substance was less reactive, but the argyrophilic fibres were well stained. Aged pulps were weakly metachromatic mainly due to the great quantity of collagen present. The ground substance was only faintly stained (Table 1). The calcified nodules were unstained or orthochromatic, but their borders, the ground substance and fibres surrounding them stained metachromatically. When the sections were incubated with hyaluronidase most of the metachromasia observed in the extracellular matrix was removed. The remaining metachromatic material could be sialic acid, which resists hyaluronidase, stains metachromatically and has been shown to be present in the dental pulp by analytical methods (AMICI and GRAZIANO,1959; CASTELLANI et al., 1959). To test this possibility, some sections were incubated with sialidase. The results showed a decreased metachromasia of the ground substance. However, Alcian blue and Azure A, used according to the methods described by SPICERand WARREN(1959), failed to demonstrate the presence of sialic acid in the pulps, perhaps due to shortcomings of this technique. Localization of nucleic acids Ribonucleic acid. The cytoplasm of fibroblasts and defense cells of young, mature
and aged pulps was weakly stained by the gallocyanin-chrome alum technique. In the odontoblasts of young and mature pulps, however, the cytoplasm was more intensely stained (Fig. 6). The material was distributed throughout the cytoplasm and was completely removed by incubation in ribonuclease (Fig. 7). Cytoplasmic staining was not observed in odontoblasts of aged pulps. Deoxyribonucleic acid. In sections digested with deoxyribonuclease and stained with gallocyanin-chrome alum, the nuclei were very faintly coloured. The chromatin was barely visible and the nucleoli appeared more prominent. Nuclear deoxyribonucleic acid was also demonstrated by staining with the Feulgen method (Fig. 8). Ej&ects of treatment with buglers Neutral phosphate bu&r. Soluble components
of the cells and the extracellular matrix were extracted when the sections were incubated in phosphate buffer at pH 7.0 (Tables 1 and 2). PAS-staining material of the ground substance of young and mature pulps was almost completely removed by the buffer solution (Fig. 9). In some young pulps the interfibrillar space seemed to be empty and the fibres were very weakly stained. The effect on aged pulps was less marked. The PAS staining of the basement membranes was less intense than that observed in sections of untreated tissues, but they were less affected by the buffer than the extracellular matrix of the rest of the pulp. Similar results could be observed in the argyrophilic fibres of sections stained with the silver impregnation technique. The fibres, which in untreated sections stained black, lost their ability to react with silver and stained pink. The orientation of the fibres was not affected and the delicate network could be seen. The KorR’s fibres, however, were not affected by the phosphate buffer and continued to stain black.
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The pulp nodules, which in untreated sections were unstained, reacted weakly with PAS and DNFB after treatment with the buffer. The PAS and DNFB-positive cytoplasmic granules described before were completely removed by the buffer, but some glycogen still remained in the nerve fibres after incubation. Nucleoproteins, which were stained with DNFB, were partially removed. salt soluble fraction” was The results obtained suggested that the “neutral primarily composed of extrafibrillar, water-soluble glycoprotein. Soluble collagen (JACKSON, 1957), nucleoproteins and electrolytes also would be included in the extracted fraction. Acid citrate buffer. The citrate buffer, pH 3.5, appeared to remove the water soluble components of the tissues and also affected their structure by changing the states of organization and aggregation of proteins (Table 1). In sections stained with silver impregnation technique the argyrophilic fibres appeared disrupted and lost most of their capacity to stain (Figs 10 and 11). The Korff’s fibres, however, were not affected, retaining their structure and their black staining. In young and mature pulps treated with the PAS the ground substance was irregularly stained (Fig. 12); in some areas it was unstained and in others it was as reactive as that of control sections. It was not possible to determine whether the stained extrafibrillar structure was ground substance which partially resisted the action of the buffer, or if it represented the degradation products of the fibres. The extracellular matrix of aged pulps was more resistant to the action of the buffer than the younger tissues. Similar effects on the extracellular matrix were observed in sections which were reacted with DNFB (Table 2). The calcified nodules, which were unstained in the control sections, stained intensely with PAS and DNFB (Fig. 13) after incubation in acid citrate buffer. The cytoplasmic granules were completely dissolved and the DNFB-positive nucleoproteins were partially removed. The cytoplasm of the fibroblasts, which was weakly stained in untreated sections, stained more intensely with DNFB.
DISCUSSION
Composition and organization of the connective tissue of the dental pulp The extracellular matrix of the dental pulp is characterized by the presence of glycoproteins, acid mucopolysaccharides and proteins containing appreciable amounts of lysine-hydroxylysine. The composition of the glycoproteins, which are mostly present in the ground substance, is not known, although it seems that some of them contain sialic acid, as suggested by the decrease of staining of the sections treated with sialidase. The proteins containing lysine-hydroxylysine are important constituents of both the fibres and the ground substance. On the basis of histochemical findings the matrix is shown to contain at least two fractions: one readily soluble in water and saline, the other insoluble and resistant to mild reagents. These two moieties correspond respectively to the waterrich and the colloid-rich phases described by JOSEPH et al. (1952). These two phases
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are considered to coexist in equilibrium and their composition is thought to remain constant. Their relative amounts can vary in different tissues or in the same tissue in various physiological and pathological conditions (JOSEPHet al., 1952; ENGEL et al., 1960; GERSHand CATCHPOLE,1960). These phases appear to have a morphologic counterpart at the submicroscopic level as shown by electron microscopic studies of various connective tissues fixed by freeze-drying (BONDAREFF, 1957; DENNIS, 1959). These concepts appear to be related also to the findings of HALDI, WYNN and CULPEPPER(1961) who extracted a “dental pulp fluid” from dogs’ teeth by drilling a hole into the pulp chambers. This “pulp fluid” seems to correspond to the warerrich phase of the extracellular matrix. It is conceivable that a part of the soluble proteins contained in this phase could be loosely bound to the colloid, explaining the low protein content of the “pulp fluid”. The colloids of the dental pulp and of all connective tissues have amphoteric properties. The carboxyl groups of collagen, the glycoproteins, and the acid mucopolysaccharides confer on the extracellular matrix a net negative charge at physiological pH. As a consequence of the amphoteric properties and of the charge, certain selective interactions with anions, cations and polar molecules (including various stains) could be anticipated (JOSEPHet al., 1952, 1954 and 1959; ENGELer al., 1961). The basement membranes of the blood vessels of the dental pulp are particularly rich in glycoproteins as shown by their deep staining with the PAS method. Their ground substance is more resistant to the action of buffers and enzymes than the adjacent ground substance, suggesting the predominance of the colloid-rich phase in these areas. The reticular fibres of the basement membranes are more densely packed around the blood vessels, but they are affected in the same way as the contiguous argyrophilic fibrous network. The amino acid composition of collagen and reticulin is similar (WINDRUM,KENT and EASTOE,1955). Perhaps they differ in the tertiary structure of their proteins and certainly in the amount of carbohydrates associated with the fibres. The capacity of the reticular fibres to react with silver seems to be related to the carbohydrate bound to the surface (SCHWARZ,1957). The extraction of the neutral salt soluble fractions does affect the glycoproteins. This produces the loss of the argyrophilia of the reticular fibres of the deep pulp. Treatment with acid citrate buffer or incubation with collagenase disrupts these fibres. In collagen fibres the absence of argyrophilia is a characteristic of these elements and has been related to their maturation when they lose a part of their carbohydrate fraction and become more cross-linked (SCHWARZ, 1957). This is consistent with our findings; that collagen fibres resist the action of buffers and of collagenase during a short incubation period. The exact chemical composition of the Korff’s fibres is unknown. Histochemically, however, they are characterized by their reactivity with silver and their resistance to neutral and acid buffers. These special properties are probably related to the role of these fibres in the formation of the dentine matrix. In the cases of all three fibres they could be considered as interacting with the contiguous ground substance to form coacervates.
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Odontoblasts which are involved in the formation of the dentine show a large amount of ribonucleic acid in their cytoplasm which suggests intense activity of these cells in protein synthesis. The low content of ribonucleic acid found in the cytoplasm of fibroblasts could be due to the inactivity of these cells or to the low sensitivity of the histochemical method used. The former hypothesis does not seem feasible, since granules containing proteins and carbohydrates can be found in their cytoplasm. Changes in ageing of connective tissues Certain general changes which occur in the extracellular matrix of other connective tissues during aging are observed also in the dental pulp. These are enumerated below: 1. Decrease in the ratio of ground substance to collagen. For example, the hexosamine:collagen ratio of dermis decreases from 0.040 in persons 23-35 years old to 0.021 in old (72-77 years) patients (KORENCHEVSKY, 1961). In aged pulps the extracellular matrix is formed mostly of collagenous bundles. Collagen fibres replace to a large extent the argyrophilic fibres and the ground substance. Analytical studies would be required to confirm this observation. 2. Increase in the resistance to proteolytic enzymes. KEECH (1954) observed that the amount of collagen hydrolysed by collagenase decreases in the ratio of 11.6:g.l for individuals l-10 years and 71-80 years old. This can also be observed in dental pulps. The reticular fibres and ground substance of young pulps are readily digested by some enzymes, but the collagen fibres and the extracellular substance of ageo tissues are more resistant to their actions. 3. Decrease in the solubility. The most soluble fraction of collagen exists in the ratio of 50: 15: 4 in individuals less than I year, l-10 years and more than 30 years respectively (BANFIELD, 1952). These findings are in agreement with our observation that old dental pulps are more resistant to the action of buffer solutions. 4. Decrease in water content. YOON et al. (1960) observed that the water content of the dental pulps is lower in aged tissues. 5. Decrease in the chemical reactivity. Collagen loses its capacity to react with silver in maturation and the extracellular matrix of the dental pulp stains less intensely with PAS, DNFB and toluidine blue. All these factors suggest that in old age there is a change in the aggregation ot the tissue colloids and a restriction of the water-rich phase. An increase in crosslinking (hydrogen bonds and salt linkages) seems to be a characteristic physicochemical phenomenon in ageing (VERZAR, 1957). This would increase the resistance of the colloids to disruptive agents. The low chemical reactivity of the ground substance and fibres of the dental pulp could result from the masking of reactive (s-amino groups of lysine and hydroxylysine, glycol and nucleophilic groups of carbohydrates) groups through cross-linkages during ageing. These changes in water content and macromolecular structure of the pulps would be expected to be accompanied by changes in electrolyte distribution. For example, a loss of sodium and an increase in potassium would be anticipated (JOSEPH e/ c/l.,
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1961). Calcium and fluoride are found to be in higher concentration in old pulps (YOON et al., 1960). These elements may be present in the tissues partially bound to the proteins and partially incorporated in the apatite crystals of pulp nodules. The high occurrence of calcification in aged pulps could also be related to the changes of the tissue colloids. The reorganization in the internal structures would release the calcium bound to negatively charged tissue colloids. The increase of the local concentration of this cation in a closed system would induce apatite formation (ENGEL, JOSEPHand LASKIN, 1962). The organization of cellular proteins also appears to change during ageing. RUZICKA(1924) stated that “in living organisms a continuous process of solidification of cellular proteins probably causes the main changes in old age”. It is difficult to study these alterations in the cytoplasm of the pulp cells histochemically, since these proteins are normally highly aggregated, as can be observed by the low reactivity to DNFB. This suggests that the e-amino groups of lysine and hydroxylysine are involved in forming tertiary structures. These amino groups, however, are exposed to DNFB by treatment with acid citrate buffer which alters the organization of the proteins. The only evidence for the changes of the cellular colloids in old age found during this study was the reduced number or even the absence of cytoplasmic inclusions in the fibroblasts in aged pulps. The results of this study confirm that the pulpal connective tissue is similar in composition, organization and histochemical reactivity to other connective tissues. Certain unique properties are probably due to the anatomical and physiological peculiarities of teeth. It is nevertheless reasonable to infer that the biological reactivity of the dental pulp in response to changes in internal milieu would parallel that of the other connective tissues. Acknowledgement-The ENGEL
author wishes to express gratitude to Dr. MILTON B.
for his guidance.
This paper is based on the thesis submitted by the author as partial fulfilment of the requirements for the degree of Master of Science in Histology in the Graduate College, University of Illinois, Chicago, 1962. Rksum&Cette Etude a pour objet I’Ctudedes propriktks histochimiques et l’organisation du tissu conjonctif pulpaire ainsi que les changements des colloides tissulaires en fonction du vieillissement. Des mkthodes plus rkentes de prdparation des specimens et certaines colorations histochimiques spkcifiques ont BtB utilisks. 11apparait ainsi que la matrice extra-cellulaire du tissu conjonctif pulpaire contient des glycoprotkines, de l’acide sialique, des mucopolysaccharides et des prottines pourvues de groupements actifs E-ammts de lysine-hydroxylysine. Les fibres et la substance fondamentale semblent Btroitement li6es et forment des agglomkrats. La matrice extra-cellulaire a une structure colloldale hetbrogkne contenant des fractions soluble et insoluble. Au niveau des pulpes jeunes, la fraction soluble est importante et les tissus sont t&s sensibles & l’action de solutions tampons et d’enzymes. Avec le vieillissement, la fraction soluble dkcroit et l’on note un changement dans le groupement des colloides tissulaires. Ces transformations se traduisent par une rksistance Clevke aux solutions tampons, aux enzymes prottolytiques et par des
HISTOCHEMICAL STUDYOF THE CONNECTIVETISSUEOF THE DENTALPULP
159
diffkrences de coloration des tissus. L’altkration de l’organisation interne des proteines provoquent vraisemblablement une redistribution des ions et elle est 1iQ B la formation de pulpolithes. Zusammenfassung-Diese Untersuchung hat die histochemische Wirksamkeit, die Gestaltung des Zahnpulpabindegewebes so wie die Vertinderungen innerhalb der Gewebskolloiden wlhrend des Altems zum Gegenstand. Moderne Pr¶tsmethoden und verschiedene spezifische histochemische FLrbungsmethoden sind dabei beniitz t worden. Das Ergebnis ist, dass die extrazellulire Zahnpulpasbindegewebematrix Glycoproteinen, Sialische SBure, MucopolysaccharidensBure, und Proteinen enthglt welche mit reagierenden c-amino Gruppen der Lysine-Hydroxylysine versehen sind. Die Fasern und die Grundsubstanz sind eng verbunden und bilden Komplexe. Die extrazellullre Matrix hat eine ungleichmlssige Kolloidstruktur, welche liisliche und unltisliche Gruppen enthtilt. Bei jungen Pulpas sind die l&lichen Gruppen sehr reichhaltig und die Gewebe sind stark empfindlich fiir PufferlGsungen und Enzymen. WBhrend des Altems wird eine Verminderung der liislichen Gruppe sowie eine Vertinderung in der Anordnung der Gewebskolloiden beobachtet. Diese Vertinderungen Hussern sich durch eine htihere Widerstandsfahigkeit gegeniiber PufferlGsungen und proteolytischen Enzymen sowie durch Anderungen in der F&bung der Gewebe. Die VerPnderungen in der inneren Proteinanordnung veranlassen wahrscheinlich eine Wiederverteilung der Ionen und stehen im Zusammenhang mit der Dentikelbildung. REFERENCES AMICI, G.
and GRAZIANO, V. 1959. Sulla presenza di acido sialico nella polpa e nella dentina di bue. Rass. Trim. Odontoiat. 40, 343-348. BANFIELD, W. G. 1952. The solubility and swelling of collagen in dilute acid with age variations in man. hat. Rec. 114, 157-171. BONDAREFF,W. 1957. Submicroscopic morphology of connective tissue ground substance with particular reference to fibrillogenesis and aging. Gerontologia 1, 222-233. CASTELLANI,A. A., FERRI, G., BOLOGNANI, L. and GRAZIANO,V. 1959. Contenuto di acido sialico in alcuni tessuti connetivi. Boll. Sot. ital. Biol. sper. 35, 2145-2148. DENNIS, J. B. 1959. Effects of various factors on the distribution of ferrocyanide in ground substance. A.M.A. Arch. Path. 67, 533-549. EINARSON, L. 1932. A method for progressive selective staining for nissl and nuclear substance of nerve cells. Amer. J. Path. 8, 295-305. EINARSON,L. 1951. On the theory of gdllocyanin-chromalum staining and its implication for quantitative estimation of basophilia. Acta path. micro&ok stand. 26, 82-102. ENGEL, M. B., JOSEPH,N. R., LASKIN,D. M. and CATCHPOLE,H. R. 1960. A theory of connective tissue behavior: Its implication in periodontal disease. Ann. N. Y. Acad. Sci. 85, 399420. ENGEL, M. B., JOSEPH,N. R., LASKIN, D. M. and CATCHPOLE,H. R. 1961. Binding of anions by connective tissue: dermis and cartilage. Amer. J. Physiol. 201, 621-627. ENCJEL,M. B., JOSEPH,N. R. and LASKIN,D. M. 1962. Morphologic and physicochemical events in calcification of turkey tendon. In: Printed abstracts, International Association for Dental Research, 40th General Meeting, p. 29. ENGSTR~M,H. and OHMAN, A. 1960. Studies on the innervation of human teeth. J. dent. R~.~. 39, 799-809. GERSH, I. 1932. The Altman technique for fixation by drying while freezing. Anar. Rec. 53, 309-337. GERSH, I. 1948. Application in pathology of the method of fixation by freezing and drying of tissues. Bull. int. Ass. med. Mus. 28, 179-185. GERSH, I. and CATCHPOLE, H. R. 1949. The organization of ground substance and basement membranes and its significance in tissue injury, disease and growth. Amer. J. Anat. g&457-521. GERSH, I. and CATCHPOLE, H. R. 1960. The nature of ground substance of connective tissue. Persp. Biol. Med. 3, 282-319. GOMORI, G. 1937. Silver impregnation of reticulin in paraffin sections. Amer. J. Path. 13, 993-1001. HALDI, J., WYNN, W. and CULPEPPER,W. D. 1961. Dental pulp fluid. Arch. oral Biol. 3,201-206.
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HOXHKI~~, R. D. 1948. A microbiochemical reaction resulting in the staining of polysaccharide structures in fixed tissue preparations. Arch. Biochem. 10, 131-141. JACKSON,D. S. 1957. Connective tissue growth stimulated by caragenin. Biochem. J. 65,277-284. JACKSON,D. S. and WILLIAMS,G. 1956. Nature of reticulin. Nature, Land. 178, 915-916. JOSEPH, N. R., CATCHPOLE,H. R., LASKIN, D. M. and ENGEL, M. B. 1959. Titration curves of colloidal surfaces-II. Connective tissues. Arch. biochem. biophys. 84, 224-242. JOSEPH, N. R., ENGEL, M. B. and CATCHPOLE,H. R. 1952. Interactions of ions and connective tissue. Biochim. Biophys. Acfa 8, 575-587. JOSEPH,N. R., ENGEL, M. B. and CATCHPOLE,H. R. 1954. Homeostasia of connective tissues-II. Potassium-sodium equilibrium. A.M.A. Arch. Path. 58, 40-58. JOSEPH,N. R., ENGEL, M. B. and CATCHPOLE,H. R. 1961. Distribution of sodium and potassium in certain cells and tissues. Nature, Land. 191, 1175-l 178. KEECH, M. K. 1954. The effect of collagenase on human skin collagen. Comparison of different age-groups and of cases with and without collagen disease. Yale J. biol. Med. 23,295-306. KORENCHEVSKY, V. 1961. Physiological and Pathological Aging. Hafner, New York. LANGELAND,K. 1957. Tissue changes in the dental pulp. Odont. Tidskr. 65, 239-386. LILLIE, R. D. 1954. Histoputhologic Technique und Practical Histochemistry (2nd Ed.). The Blakiston Div., McGraw-Hill, New York. MCMANIJS, J. F. A. 1948. Histological and histochemical uses of periodic acid. Stain Technol. 23,99-108. ORBAN, B. J. 1929. Contribution to the histology of the dental-pulp and periodontal membrane, with special reference to the cells of defense of these tissues. J. Amer. dent. Ass. 16, 965-996. ORBAN, B. J. 1957. Oral Histology and Embryology. C. V. Mosby, St. Louis. RUZICKA, V. 1924. Beitrage zum Studium der Poroplasmahysteresis und der histerischen Vorgange. Arch. mikr. Anat. 101, 459482. SANGER,I. 1945. The free amino groups of insulin. Eiochem. J. 39, 507-515. SASSO, W. S. and CASTRO,N. M. 1957. Histochemical study of amelogenesis and dentinogenesis. Oral Surg. 10, 1323-1329. SCHOUR,I. 1953. Noyes’ Oral Histology and Embryology (7th Ed.). Lea and Febiger, Philadelphia. SCHWARZ,W. 1957. Morphology and differentiation of the connective tissue fibres. In: Connective Tissue (Ed. by TUNBRIDGE,R. F.), p. 144. Thomas, Springfield. SPICER, S. S. and WARREN, L. 1959. The histochemistry of sialic acid containing mucoproteins. J. Histochem. Cytochem. 8, 135-137. VERZAR,F. 1957. Studies on adaptation as a method of gerontological research. CIBA Fundation Colloquia on Aging, Vol. 111,pp. 60-68. Little, Brown, Boston. YOON, S. H., BRUDEVOLD,F., SMITH, F. A. and GARDNER, D. E. 1960. Fluoride and calcium concentrations in human dental pulps. J. dent. Res. 39, 671-672. WINDRUM,G. M., KENT, P. W. and EASTOE,J. E. 1955. The constitution of human renal reticulin. Brit. J. exp. Path. 36, 49-56. ZERLOTTI,E. and ENGEL, M. B. 1962. The reactivity of proteins of some connective tissues and epithelial structures with 2:4_dinitrofluorobenzene. J. Histochem. Cytochem. 10, 537-546.
PLATE I All sections were from tissues fixed by freeze-drying. FIG. 1, High power of a young pulp stained with PAS showing granules (arrow) in the cytoplasm of fibroblast. Nucleus of fibroblast (r). x 1860. FIG. 2. Young pulp stained with PAS. Blood vessel (v), basement membrane (b). x 560. FIG. 3. Section of mature pulp stained with PAS. The basement membrane (b) is deeply stained. Blood vessel (v). x 560. FIG. 4. Aged pulp stained with PAS. The basement membrane (b) is strongly reactive. Collagen fibres stain weakly. Blood vessel (v). x 560. FIG. 5. Mature pulp stained with DNFB. The pulp nodule (s) is not stained. The extracellular substance around it (arrow) is more reactive. x 560.
HISTOCHEMICAL
STUDY
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1
PLATER
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OF THE DENTALPULP
PLATE 2
All
sections were
from tissues fixed by freeze-drying.
FIG. 6. Young pulp stained with gallocyanin-chromalum technique. Note the staining of the cytoplasm (arrow) of odontoblasts (0) and fibroblasts (f). x 560. FIG. 7. Section of the same pulp digested with ribonuclease prior to staining with gallocyanin-chromalum. The reactive material was removed from the cytoplasm of odontoblasts (0) and fibroblasts (f). x 560. FIG. 8. Section of young pulp stained blasts (0) and fibroblasts (f). x 560.
with the Feulgen technique.
Odonto-
FIG. 9. Mature pulp treated with phosphate buffer prior to staining with PAS. The ground substance was removed to a large extent. Blood vessel (v), basement membrane (b) and nerve bundle (n). :i 560.
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E. ZERLOTTI
PLATE3 All sections were from tissues fixed by freeze-drying. FIG. 10. Section of young pulp stained with the Gomori’s silver impregnation technique. The reticulin fibres are more densely packed around the blood vessels (v). x 560. FIG. 11. Section of the same pulp treated with acid citrate buffer prior to staining with the Gomori’s impregnation technique. The reticulin fibres are completely disrupted. Blood vessel (v). x 560. FIG. 12. Section of mature pulp incubated with acid citrate buffer before staining with PAS. The extracellular matrix is disrupted. Blood vessel (v) and basement membrane (b). x 560. FIG. 13. Section of mature pulp treated with acid citrate buffer prior to staining by DNFB. Pulp nodule (s) is strongly reactive. The extracellular matrix is disrupted and weakly stained. x 560.
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