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Histochemical reactions of periodontal tissues: a review of the literature
Histochem ical reactions of periodontal tissues: a review of the literature
Fermin A . Carranza, Jr., D .D .S ., and Romulo L . Cabrini,* M .D ., Buenos Aires, Argentina
The literature relating to the histochem istry of the periodontal tissues is reviewed. The
distribution
phosphatase,
of alkaline and acid
mucopolysaccharides
and
glycogen, metachromatic substances, and the disulfide and sulfhydryl groups is described, and their probable role in the histophysiology of the periodontium analyzed.
Histochemical
research
is
may
lead to a better understanding of the mechanism of the biochemical reactions occurring within
the tissues, and may
provide a useful tool in diagnosing patho logical changes.
studies have to be m ade cautiously and very carefully controlled, especially when a hypothesis o f some functional signifi cance is based on them. It is not possible to obtain reliable histochemical data without adequate technical and theoreti cal means; unfortunately, the “ pseudo simplicity” o f some technics has caused them to be employed sometimes under in adequate conditions, leading to results that have to be discarded. In this review (con clu ded M arch 31, 1958) we shall refer to the most im por tant and best-studied histochemical reac tions o f the periodontal tissues and ana lyze briefly their possible physiological role. A L K A L IN E P H O S P H A T A S E
A lthough histochemistry has developed simultaneously with general ■m icroscopi cal technics, its study has been greatly intensified during the last 15 years. T h e methods developed up to the present are, in the majority, qualitative and only a few isolated attempts at quantitative d e termination have been m ade .1 Qualitative chem ical study o f the dis tribution o f a given substance or o f a functional group presents all kinds o f d if ficulties ; for this reason histochemical
This enzyme has been studied in human gingiva by several authors .2-6 A ll o f them agree that by using hydrolysis o f sodium glycerophosphate as a substrate, the en zyme is found in the connective tissue in and around the b lood vessels. A positive reaction in the basal layer o f the epithe lium has been reported when nucleic acid is used as a substrate .5 T h e alkaline phosphatase content o f the oral epithelium o f the rat increases
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during the estrous cycle from a minimum in diestrus to a m axim um in estrus in a way com parable to the variations o f al kaline phosphatase in the vaginal epithe lium o f the rat during the sexual cycle .7,8 T h e phosphatasic activity o f gingival wounds in rats is increased considerably in the superficial layers o f the epithelium adjacent to the incision .9 In view o f these findings the theory has been put forward that the amount o f alkaline phosphatase in the epithelium is related to the process o f keratinization .9,10 In this theory, the gingival epithelium would show very lit tle or no phosphatasic activity probably because, like the skin, it w ould maintain its keratinized state with phosphatasic levels which are not demonstrable histochem ically .9 T h e results o f different investigations on alkaline phosphatase activity in the granules o f keratohyalin d o not agree. Some authors 4,7,9,11>12 have fou n d a positive reaction in these granules; oth ers 5’ 13 have not. Some consider the localization o f alkaline phosphatase in the granules o f keratohyalin to be a d if fusion phenom enon with adsorption by the granules .14 In the gingival connective tissue the enzyme is fou n d especially in the blood vessels. In the larger b lood vessels, co m posed o f several layers, it has been shown that the phosphatasic activity is con cen trated in the endothelium .5 In inflammatory processes the enzyme appears in large quantities and is local ized in the capillaries, in the inflam m a tory infiltrate especially in the polym or phonuclear leukocytes, ánd less notably in the newly form ed collagen fibers .0 In the basal cell layer and at the end o f the papillae of epithelium overlying inflamed connective tissue, alkaline phosphatase has been found, very probably a result of diffusion .15 A study on the alkaline phosphatase activity in the gingiva o f the ferret 16 re vealed a great difference from human gingiva. T h e ferret’ s clinically normal
gingiva contains this enzyme only in the gingival and crevicular epithelia and only slight activity in the lamina propria; in flamed gingiva displays a pronounced increase in alkaline phosphatase activity throughout the gingival and crevicular epithelia, localized in the cytoplasm and also extracellularly associated with the intercellular bridges. T h e connective tissue attachment o f the gingiva contains alkaline phospha tase in the collagen fibers .17 In some in stances the enzymatic activity o f the granulation tissue o f the lateral wall of the pocket blends gradually with this zone. H ow ever, those instances in which there exists an intermediate zone devoid o f inflammatory reaction, prove the in dependence o f the phosphatasic content in both zones .17 In predominantly fibrous gingivae 15 or in diphenylhydantoin sodium hyperplasia where there is a noticeable fibrosis18 there is an increase o f alkaline phospha tase in relation to the form ation o f con nective tissue fibers. Enzymatic activity in the supporting tissues is present in a varying degree. T he periodontal mem brane o f rodents shows a strong reaction fo r alkaline phospha tase in the fibers and in the ground sub stance between them .4 In dogs a strong reaction has been reported in fibers and cells .19,20 In man, a weak and inconstant reaction has been fou n d in the periodon tal m em brane fibers .17 Generally, phos phatase o f the periodontal membrane is associated with a m ore or less intense phosphatasic activity o f the bone and cem entum surfaces. A lveolar bonq has enzyme only in some areas o f its periosteal, endosteal and peri odontal surfaces. In the m ajority o f in stances, the periosteum o f the alveolar crest has an intense enzymatic activity which continues in one or both sides, peri osteal and periodontal .17 In the cem en tum, alkaline phosphatase has been found around Sharpey’s fibers and in the cementoblasts .21
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ACID P H O S P H A T A S E
A cid phosphatase is present in the gin gival epithelium in a greater concentra tion immediately beneath the horny layer, and diminishes toward the basal cell layer. It is absent in the epithelial at tachment and the epithelial lining o f the gingival sulcus .22 In laboratory animals a somewhat d if ferent picture has been fo u n d .23 In the rat and the mouse the gingival epithe lium shows a double reaction: one, stronger, in the layer beneath the horny layer, similar to that fou n d in human gingiva, and a second one, weaker, in the basal cell layer, leaving in between a zone that does not react and that corre sponds, roughly, to the prickle cell layer. This distribution also has been found in the epithelial lining o f the tongue in rat and human specimens. Alveolar bone shows a strong reaction in the areas o f bone resorption, in the osteoclasts and b lood vessel walls a d ja cent to H ow ship’ s lacunae. This confirms previous findings in other regions o f the skeleton .24 M U C O P O L Y S A C C H A R ID E S AND GLYCOGEN
T he periodic acid-Schiff reaction ( PAS) is widely used for the detection o f gingival m ucopolysaccharides .4,6-25-30 T h e con n ec tive tissue o f normal gingiva stains pink with PAS. T h e ground substance covers the collagen fibers, producing an optical effect o f hom ogeneity .26 T h e basement m embrane shows up as an amorphous structure o f light red polor. Treatm ent with ptyalin previous to the staining eliminates the glycogen; com parison with a control section allows the glycogen distribution to be determined am ong the other PAS-positive substances. T h e presence o f glycogen in norm al gingival epithelium is controversial. For some ,6,31 norm al epithelium is devoid o f glycogen ; others 25,29 describe the pres
ence o f glycogen in the prickle cell layer. Glycogen appears in the cellular cyto plasm as small, irregular granulations. T he distribution o f glycogen is h om og enous rather than granular in gingivae fixed by the freezing-drying technic. T h e presence o f glycogen in the prickle cell layer o f the skin epithelium has been related to acanthosis and parakeratinization, or b oth .32 O ther studies33 report glycogen in the skin in relation to para keratosis only, and not to acanthosis ; however, in the latter study, fixation with Formalin was em ployed, which is not the best fo r this technic and reduces the value o f the conclusions .34 In the gingiva a correlation has been reported 27 between glycogen and acan thosis and, indirectly, with inflammation, when this produces a reactional acan thosis. O ther studies6,31 report a corre lation between glycogen and inflam m a tion. A histochemical study 35 o f gingival healing in normal and vitamin C deficient animals reveals that the am ount o f glyco gen increases in hyperplastic areas in d i rect relation to the degree o f inflam m a tion, and is apparently not affected by vitamin C deficiency. T h e author o f a recent study 36 o f the glycogen content of human gingivae in specimens fixed in Formalin, found no correlation between glycogen and age, sex, menstrual cycle, inflammation and degree o f keratinization. It has been shown that with Ritter and Oleson’s technic the basement m em brane takes a different color from the ground substance, which w ould indicate that they are different substances .37 This study also showed no correlation between keratinization and pigmentation and the state o f the basement membrane. Changes or lack o f staining o f the basement mem brane in chronic desqua mative gingivitis as a result o f depoly merization o f the highly polymerized com ponents o f the basement m em brane have been described .25 But other studies6 have found similar changes in the pres
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ence of chronic inflammation, and that both disruption and disappearance o f the basement mem brane occurs in the pres ence o f inflammation, with regeneration in instances o f chronic inflam m ation .37 T h e gingival epithelium usually is de void o f PAS-positive, ptyalin-resistant substances, except for very small amounts in the intercellular spaces and the epi thelial surface. This reaction has been reported to be m uch greater in m ucous membranes than in the skin, suggesting an inverse relationship between the degree o f keratinization o f the epithelium and the am ount o f PAS-positive sub stances.38 Similar behavior has been shown in gingival tissues; that is, a posi tive reaction in parakeratinized areas which disappears when the epithelium attains a com plete keratinic differentia tion .27 A fter treatment with amylase a very mild PAS-positive reaction remains in the intercellular spaces, especially in the surface layers. Small PAS-positive granulations, ir regularly distributed in their scarce cytoplasm, have been observed in the fibrocytes o f the gingival connective tissue.25’ 27 These granulations disappear with amylase and are not preserved p rop erly in material which has been fixed in Formalin or decalcified. This behavior shows that it is glycogen. T h e rem aining cells o f the gingiva show their usual reaction : mast cells react in their granules with irregular intensity; in inflammatory infiltrates the leukocytes are frankly positive but their reaction disappears with amylase; lym ph ocytes and reticulohistiocytes show no reaction; plasma cells show a weak cyto plasmic reaction w hich increases in R us sell’s bodies and does not disappear with amylase. M E T A C H R O M A S IA
T he gingival connective tissue gives a weak m etachrom atic reaction localized
especially in the fibers. T h e mast cells are the only frankly m etachrom atic co m p o nent o f the gingiva .4 Treatm ent with hyaluronidase eliminates the m etachro matic reaction o f the connective tissue but it does not change that o f the mast cell granules .4 Large numbers o f mast cells have been described in the gingiva. In most in stances, a definite distribution o f these cells in the gingiva appears .39 T here are few er mast cells in the connective tissue adjacent to the gingival sulcus; in the central zones o f the gingival corium, especially around the b lood vessels, round and oval mast cells predom inate, whereas in the periphery elongated form s are pre dominant. This distribution o f the d if ferent shapes o f mast cells has suggested a migration o f these cells from the center to the periphery .39 It is not unusual to find mast cells which have penetrated the basal layer o f the epithelium .39,40 T h e disappearance o f mast cells in the presence o f acute inflammation has been described ;41 this has also been reported in instances o f acute necrotizing gingi vitis .39 In chronic gingivitis an increased num ber o f mast cells has been re ported .31,40’ 42 D IS U L F ID E A N D S U L F H Y D R Y L G R O U P S
T h e disulfide and sulfhydryl groups in norm al and inflamed gingivae have been studied using Barnett and Seligm an’s technic .43 T h e concentration o f sulfhydryls was fou n d to be greater in the kera tinized and parakeratinized layers o f the epithelium ; disulfide groups were fou n d to be distributed uniform ly throughout all the epithelial layers. In areas o f gin gival inflammation an increase in the sulfhydryl content o f proliferating epi thelium was also fou n d .43 A histochemical study o f a case o f chronic desquamative gingivitis reports the presence o f sulfhydryl groups through out the epithelial layers with a consider able and unexplained concentration o f
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«
the reaction in the superficial layers; the disulfide bond reaction was negative .44 M IS C E L L A N E O U S
T h e distribution o f nucleic acids has been reported to be similar in both n o r mal and inflamed gingiva except fo r the desoxyribose nucleic acid content o f the nucleus o f inflammatory cells. R ibose nucleic acid appears in large quantities in the cytoplasm o f the basal layers of the gingival epithelium, diminishing toward the surface; it also diminishes in the crevicular epithelium and in zones o f leukocyte infiltration .31 T h e presence o f phospholipids has been reported in gingival epithelium .45 A study o f phosphamidase in oral tissues obtained a negative result in the gingiva .46 D IS C U S S IO N
Alkaline and acid phosphatase are d if ferently distributed and probably have com pletely different physiological roles. Alkaline phosphatase is fou n d mainly in connective tissues (gingival corium , peri odontal membrane, alveolar bone and cem entum surfaces) and is thought to be associated with the metabolism o f co l lagen and with osteogenesis. T h e absence o f alkaline phosphatase in wounds o f scorbutic guinea pigs and its presence in the wounds o f norm al guinea pigs have suggested that it is asso ciated in some way with the form ation of collagen .47 H ow ever, the healing o f wounds is independent from the phosphatasic level o f the b lo o d ; the applica tion o f large quantities o f phosphatase in wounds o f scorbutic animals or the in jec tion o f phosphatase inhibitors into the wounds o f norm al ones, fails to produce any changes .48 This makes the role o f alkaline phosphatase in fibrogenesis hot clear. T h e relation between alkaline phos phatase and epithelial regeneration 49 and keratinization 9,10 also has been suggested.
It has been known for years that a close relationship exists between osteogenesis and the presence o f alkaline phosphatase in large amounts .50 Recently, these b io chem ical data have been fully confirm ed by histochemistry. O n e most recent inves tigation 51 seems to indicate that this en zyme plays a role in the elaboration o f the protein matrix and that its function as a direct agent o f calcium salt precipitation is doubtful. A cid phosphatase, on the other hand, is localized especially in epithelial tissues. T h e existence o f large amounts o f this enzyme in the epithelial layers during keratinization leads to the belief that it is closely related to this process. Similar findings have been shown in the skin .52,53 Connective tissue ( gingival corium and periodontal mem brane) appears to have no enzyme; bone surfaces and h em ato poietic m arrow spaces are rich in acid phosphatase. This distribution in the bone surfaces, almost exclusively in the osteo clasts, suggests that this enzyme plays an active role in bone resorption .24 M ucopolysaccharides are distributed in the epithelium and corium as well as in the alveolar bone. T h e gingival epithe lium has only vestiges o f m ucopolysac charides located in the intercellular spaces and in some bands o f parakeratinized material. It w ould appear that variations in this PAS-positive, ptyalin-resistant sub stance in the epithelium are o f little im portance, and its use is restricted to the detection o f parakeratinized areas and their differentiation from truly ker atinized ones .32 T h e gingival corium has a distribution com m on to connective tissue o f the skin, with a condensation o f PAS-positive m a terial in the basement membrane. A t this time it is premature to think that changes or m odifications o f the reaction o f the basement m em brane may have a signifi cance useful fo r the diagnosis o f gingival changes. Glycogen reacts easily to PAS technic and is identified by means of ptyalin; but
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it is difficult to fix in the tissues, and only data obtained under irreproachable co n ditions o f fixation should be taken into account. A lcoh olic mixtures o f picric acid, or better still, the Altmann-Gersh m ethod, should be used. W ith adequate fixation it is possible to find glycogen in the prickle cell layer o f the epithelium and an increase in acanthosis and hydro pic degeneration. Although these varia tions apparently are constant, their usefulness as a means o f diagnosis has to be discarded because o f the lability o f glycogen during fixation. A bundant mast cells have been found in gingival tissues stained with m etachromatic technics. T h e histophysiological role o f these cells has not yet been ex plained conclusively. It is believed that the cells contain heparin or histamine or some precursor substance o f hyaluronic acid. T here is a tendency today to connect mast cells with the maintenance and repair o f connective tissues,54 which w ould explain their presence in large amounts in the gingiva. Disulfide and sulfhydryl groups have been associated with the process o f ker atinization, the most im portant reaction o f w hich w ould be the transformation o f sulfhydryl groups into disulfide ones .85 R ecent studies56 have shown in-vitro blister form ation due to rupture o f the disulfide bonds which appear to play an im portant part in the integrity o f the intercellular bridges and the tonofibrils. This might need further study in relation to chronic desquamative gingivitis. SUM M ARY
T h e most significant contributions to the field o f histochemistry o f the periodontal tissues have been reviewed, including the follow ing substances: alkaline and acid phosphatase, m ucopolysaccharides and glycogen, m etachrom atic substances, d i sulfide and sulfhydryl groups. T h e distri bution o f these substances is described and their probable role in the histophysiology
o f the periodontium briefly analyzed. H istochem ical research done up to the present opens up a new field w hich will give a better understanding o f the m ech anism o f the biochem ical reactions occu r ring within the tissues, and which may also provide a useful tool fo r the m icro scopical diagnosis o f pathological changes.
* D e p a r f m e n t o f p a t h o lo g y , H o s p i t a l R a m o s M e [ ia . 1. G lic k , D. U s e o f m ic r o c h e m ic a l m e t h o d s f o r q u a n t i t a t iv e lo c a liz a t io n in h is to c h e m is tr y . J . H is t o c h e m . 5:539 N o v . 1957. 2. Z a n d e r , Hè A . D is t r ib u t io n o f p h o s p h a t a s e g i v a l tissu e . J. D . Res. 20:347 A u g . 1941.
in q in -
3. V a llo t t o n , C . E. E tu d e b io - h i s t o lo g iq u e d e la p h o s p h a t a s e . d a n s la g e n c iv e h u m a in e n o r m a le et d a n s les g i n g iv i t e s . 'S c h w e i z . M s c h r . Z a h n h k . 52:512 J u n e 1942. 4. W is l o c k i, G . B., a n d S o g n n a e s , R. F. H is t o c h e m ic a l r e a c t io n s o f n o r m a l te e th . A m . J . A n a t . 87:239 S e p t. 1950. 5. C a b r in i , R. L., a n d C a r r a n z a , F. A . , J r. H i s t o c h e m ic a l st u d y o n a lk a lin e p h o s p h a t a s e in n o r m a l q in g iv a e , v a r y in g the p H a n d th e s u b st r a t e . J . D . Res. 30:28 F e b . 1951.
6
. T u resky, S.; G lic k m a n , I., a n d L itw in , T. H i s t o c h e m ic a l e v a lu a t io n o f n o r m a l a n d in f la m e d h u m a n g i n g iv a e . J. D. Res. 30:792 D e c. 1951. 7. R in g , J . R. C h a n g e s in a lk a lin e p h o s p h a t a s e a c t iv it y o f ra t v a g in a l e p it h e liu m d u r i n g th e e st r o u s c y c le . A n a t . Ree. 107:121 J u n e 1950. . R in g , J . R., a n d L e vy, B. C h a n g e s in a lk a lin e p h o s p h a t a s e a c t iv it y o f ra t o r a l e p it h e liu m d u r i n g the e s t r o u s c y c le a n d in r e s p o n se t o a d m in is t e r e d e s t r o g e n . J . D . Res. 29:817 D e c . 1950.
8
9. R in g , J . R. A lk a l in e p h o s p h a t a s e a c t iv it y o f h e a li n g g i n g i v a l w o u n d s in th e rat. J . D . Res. 31:329 J u n e 1952. 10. F o rd , D . H . S t u d y o f c h a n g e s in v a g in a l a lk a lin e p h o s p h a t a s e a c t iv it y d u r i n g e s t r o u s c y c le in a d u lt a n d in y o u n g " f i r s t e s t r o u s " rats. A n a t . Rèe. 125:261 J u n e 1956. 11. S m ith , C ., a n d P a rk h u rst, H . S t u d ie s o n t h e t h y m u s o f th e m a m m a l; c o m p a r is o n o f ’th e s t a in in g p r o p e r t ie s o f H a s s a l l 's c o r p u s c le s a n d o f t h ic k sk in o f tn e g u in e a p ig . A n a t . Re e. 103:649 A p r i l 1949. 12. L a n s in g , A . I., a n d O p d y k e , D. L. H i s t o l o g i c a l a n d h is t o c h e m ic a l st u d ie s o f th e n ip p le s o f e s t r o g e n t r e a t e d g u in e a p i g s w ith s p e c ia l r e fe re n c e t o k e r a t o h y a lin g r a n u le s . A n a t . Ree. 107:379 A u g . 1950. 13. K o p f, A . W . D is t r ib u t io n o f a lk a lin e p h o s p h a t a s e in n o r m a l a n d p a t h o l o g ic h u m a n skin . A . M . A . A r c h . D e r m a t . 75:1 J a n . 1957. 14. N a d e l, E. M . , a n d W o d in s k y , I. D i s s im i la r it y in a lk a lin e p h o s p h a t a s e s t a in in g r e a c t io n s o f k e r a t o h y a lin e g r a n u le s . J . H is t o c h e m . 3:339 S e p t . 1955. 15. C a r r a n z a , F. A . , J r., a n d C a b r in i , R. L. E s t u d io h is t o q u í m ic o d e la fo s f a t a s a a lc a lin a en e n c ía . Rev. O d o n t . 40:443 N o v . 1952. 16. S t a p le , P. H . A lk a l in e p h o s p h a t a s e a c t iv it y in g i n g i v a o f th e fe rre t. B rit. D . J . 102:305 A p r i l 16, 1957. 17. C a r r a n z a , F. A . , Jr., a n d C a b r in i , R. L. E s t u d io h is t o q u í m ic o d e la fo s fa t a s a a lc a lin a e n lo s t e j id o s perto d o n t a le s . Re v. A . o d o n t., A r g e n t in a 43:206 J u n e 1955. 18. S t a p le , P. H . S o m e t is su e r e a c t io n s a s s o c ia t e d w ith 5:5 d ip h e n y lh y d a n t o in ( " D i l a n t i n " ) s o d iu m t h e r a p y . Brit. D . J . 95:289 D e c . 15, 1953. 19. Z e ro s i, C ., a n d B a ra tie ri, A . R ic e r c e is t o c h ìm ìc h e s u lle lo c a liz z a z io n e d e lla f o s fa t a s i a lc a l in a n e lla p o lp a d e n t a r ia (c o n r ife r im e n t o a lla fo r m a z io n e d e l la d e n t in a ) nel p e r i o d o n t o e nel c e m e n t o . A t t i S o c . l o m b a r d a Se. m e d . b io l. 9:4, 1954. 20. B a ra tie ri, A . R ic e r c e is t o c h ìm ìc h e s u lle lo c a liz z a z io n i d e lla fo s fa t a s i a lc a lin a _ n e g li e le m e n ti d e l p e r i o d o n t o e d e l c e m e n t o . Riv. ita l. S t o m a t . 10:212 M a r c h 1955.
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21. L o r b e r , M . S t u d y o f th e h is t o c h e m ic a l r e a c t io n s o f d e n t a l c e m e n t u m a n d a lv e o l a r b o n e . A n a t . R e c. 111:129 O c t . 1951. 22. C a b r in i , R. L., a n d C a r r a n z a , F. A . , J r. H i s t o c h e m ic a l d is t r ib u t io n o f a c i d p h o s p h a t a s e in h u m a n g i n g i v a . J . P e r io d o n t . 29:34 J a n . 1958. 23. C a r r a n z a , F. A ., Jr., a n d C a b r in i , R. L. E s t u d io h is t o q u í m ic o d e la fo s fa t a s a a c i d a en la m u c o s a o r a l d e ra ta , r a t ó n y h o m b r e . Rev. A . o d o n t. A r g e n t in a , 46:219 J u ly 1958. 24. S c h a jo w ic z , F., a n d C a b r in i , R. L. H is t o c h e m ic a l lo c a liz a t io n o f a c id p h o s p h a t a s e in b o n e tissu e . S c ie n c e 127:1447 J u n e 20, 1958. 25. E n g e l, M . B.; Ray, H . S . , a n d O r b a n , B. P a t h o g e n e s is o f d e s q u a m a t iv e g i n g iv i t i s : d is t u r b a n c e o f c o n n e c tiv e tissu e g r o u n d su b s t a n c e . J . D. Res. 29:410 A u q . 1950. 26. E n g e l, M . B. W a t e r - s o lu b le m u c o p r o t e in s g i n g i v a . J . D . Res. 32:779 D e c . 1953.
o f the
27. C a b r in i , R. L., a n d C a r r a n z a , F. A . , J r. E s t u d io h is t o q u í m ic o d e la s s u b s t a n c ia s " P A S " p o s it iv a s , m e ta c r o m á t ic a s y b a s ó f ila s e n lo s t e j id o s p e r io d o n t a le s . Re v. A . o d o n t . A r g e n t in a 44:309 A u g . 1956. 28. M o r í , M ., a n d O g a t a , Y. H is t o c h e m ic a l s t u d y on fh e p o l y s a c c h a r i d e in g i n g i v a ¡n p a t ie n t s w ith p e r i o d o n t a l d is e a s e . D . A b s t . 2:300 M a y 1957. 29. W e is s , M . B., a n d W e in m a n n , J . P. G l y c o g e n d i s t r ib u t io n In h u m a n g i n g iv a . ( A b s t . ) I. A . D . R . 35:37, 1957. 30. R o s e n b e r g , C . J . D e t e r m in a c ió n d e lo s á c i d o s m u c o p o l is a c é r i d o s e n la s d is t in t a s f o r m a s d e p a r a d e n c io p a t ía s en el c o r io n g i n g iv a l . Re v. d e n t . C h i le 47:149 A o r i l 1957. 31. D e w a r, M . R. O b s e r v a t i o n s o n th e c o m p o s it io n a n d m e t a b o lis m o f n o r m a l a n d in fla m e d g i n g iv a e . J . P e r io d o n t . 26:29 J a n . 1955. 32. B ra u n - F a lc o , O . H is t o c h e m is c h e u n d m o r p h o l o g isc h e Stu d ie n a n n o r m a le r u n d p a t h o l o g is c h v e r ä n d e r t e r H a u t . A r c h . D e r m a t . u. S y p h . 198:111, 1954. 33. S te in e r, K. H is t o c h e m ic a l st u d y o f e p id e r m a l g l y c o g e n in sk in d is e a se s. J. In v e st. D e r m a t . 24:599 J u n e 1955. 34. S c h a jo w ic z , F., a n d C a b r in i , R. L. H is t o c h e m ic a l st u d ie s o n g l y c o g e n in n o r m a l o s s ific a t io n a n d c a l c i f i c a t io n . J . B o n e & J o in t S u r g . 4 0 -A : 1081' O c t . 1958. 35. T u resky, S. S., a n d G lic k m a n , I. H is t o c h e m ic a l e v a lu a t io n o f g i n g i v a l h e a lin g in e x p e r im e n t a l a n im a ls on a d e q u a t e a n d v it a m in C d e f ic ie n t d ie ts . J. D. Res. 33:273 A p r i l 1954.
p e r i o d i c a c id - S c h ilf f m e t h o d . A n a t . R e c. 110:359 J u ly 1951. 39. C a r r a n z a , F. A . , J r., a n d C a b r in i , R. L. M a s t c e lls in h u m a n g i n g iv a . O r a l S u r g ., O r a l M e d . & O r a l P ath . 8:1093 O c t . 1955. 40. S te lla , A . S o b r e Jos m a st z e lle n d e la e n c ia H u m a n a . A n . F a c . o d o n t . M o n t e v i d e o 1:125, 1955. 41. S y lv e n , B. C a r t i l a g e a n d cho ’n d r o it in su lfa t e ; c h o n d r o it in s u lfa t e a n d p h y s i o l o g ic a l o s s if ic a t io n o f c a r t ila g e . J. B o n e & J o in t S u r g . 29:973 O c t . 1947. 42. D e w a r, M . R. M a s t P e r lo d o n t . 29:67 J a n . 1958.
c e lls
in
g in g iv a l
tissu e .
J.
43. T u re sky, S .; C r o w le y , J., a n d G lic k m a n , I. H i s t o c h e m ic a l s t u d y o f p r o t e in - b o u n d su lfh y d r y l a n d d is u lf id e g r o u p s in n o r m a l a n d in fla m e d h u m a n g i n g iv a . J. D. Res. 36:255 A p r i l 1957. 44. S o g n n a e s , R. F.; W e is b e r g e r , D., a n d A lb r i g h t , J . T. P a t h o l o g ic d e s q u a m a t io n o f o r a l e p it h e liu m e x a m in e d b y e le c t r o n m i c r o s c o p y a n d h is to c h e m is tr y . J. N a t . C a n c e r In st. 17:329 S e p t . 1956. 45. M e r o n i, C . M . L o s f o s f o l i p i d o s g l n g iv a le s . Re v. A . o d o n t., A r g e n t in a 45:123 A p r il , 45:159 M a y 1957. 46. M e y e r , J., a n d W e in m a n n , J . P. P h o s p h a m id a s e c o n te n t o f n o r m a l a n d p a t h o l o g ic t is su e s o f t h e o ra l c a v ity . J . H is t o c h e m . 1:305 S e p t . 1953. 47. D a n ie llij J . F.; F e ll, H . B., a n d K o d ic e k , E. Enzymes^ o f h e a lin g w o u n d s ; e ffe c t o f d iffe r e n t d e g r e e s of v it a m in C - d e f ic ie n c y o n p h o s p h a t a s e a c t iv it y in e x p e r i m e n ta l w o u n d s in g u in e a p ig . B rit. J . E x p e r. P ath . 23:367 D e c. 1945. 48. G o u ld , B. S., a n d G o l d , N . I. S t u d ie s o n " a l k a l i n e " p h o s p h a t a s e a s s o c ia t e d w ith r e g e n e r a t in g c o n n e c tiv e tis su e fib e r s. A . M . A . A r c h . P ath . 52:413 N o v . 1951. 4 9 .^ Fre n ch , J . E., a n d B e n d itt, E. P. O b s e r v a t i o n s on lo c a liz a t io n o f a lk a lin e p h o s p h a t a s e in h e a lin g w o u n d s. A . M . A . A r c h . P ath . 57:352 A p r i l 1954. 50. R o b is o n , R. P o s s ib le s ig n if ic a n c e o f h e x o se p h o s p h o r ic e st e rs in o s s ific a t io n . J . B io c h e m . 17:286, 1923. 51. G u t m a n , A . B. C u r r e n t t h e o r ie s o f b o n e s a lt f o r m a tio n , w ith s p e c ia l r e fe re n c e to e n z y m e m e c h a n is m s in e n d o c h o n d r a l c a lc if ic a t io n . B u ll. H o s p . J o i n t D is. 12:74 A p r i l 1951. 52. B ejd l, W . D ie s a u r e P h o s p h a t a s e in H a u t u nd V a g i n a d e s M e n s c h e n u n d ih r e B e d e u t u n g fu r d ie V erh o r n u n g . Z t s c h r . Z e llf o r s c h . 40:389, 1954. 53. M o r e t t i, G ., a n d M e s c o n , H . H is t o c h e m ic a l d i s t r ib u t io n o f a c i d p h o s p h a t a s e s in n o r m a l h u m a n skin . J . In v e st . D e r m a t . 26:347 M a y 1956.
36. T ro tt, J . R. A n i n v e s t ig a t io n in t o th e g ly c o g e n c o n te n t o f th e g i n g iv a e . D . P r a c t it io n e r 7:234 A p r i l 1957.
54. Rile y, J . F. T h e r id d le o f the m a st c e lls : t r ib u t e to P aul E h rlic h . L a n c e t 1:841 A p r i l 24, 1954.
37. Linz, A . , M . O b s e r v a t io n s o n the b a s e m e n t m e m b r a n e o f th e g i n g iv a e . O r a l S u r g ., O r a l M e d . & O r a l Path . 7:763 J u l y 1954.
55. R o th m a n , S. P h y s io lo g y a n d b io c h e m is t r y o f the skin . C h i c a g o , U n iv e r s it y o f C h i c a g o P ress, 1954, c h a p t e r 16.
38. W is l o c k i, G . B.; Fa w c e tt, D . W . , a n d D e m p se y , E. W . S t a i n i n g o f s t r a tifie d s q u a m o u s e p it h e liu m o f m u c o u s m e m b r a n e s a n d sk in o f m a n a n d m o n k e y b y the
56. S t o u g h t o n , R. B., a n d N o v a k , N . D is r u p t io n o f t o n o f lb r ils a n d in t e r c e llu la r b r id g e s b y d is u lf id e - s p lit t in q a g e n t s . J . In v e st. D e r m a t . 26:127 F e b . 1956.
Cancer A m ong M en • Cancer can no longer be regarded as “ a w om an’ s disease.” O f every 100 persons w ho die o f cancer, 54 are men and 46 are wom en. O ne reason for the rising death rate from cancer am ong men is due to cancer o f the lung. It killed 30,000 men last year and 5,000 women— or five times as many as 20 years ago. Am erican Cancer Society, 1960.
Fermin A . Carranza, Jr., D .D .S ., and Romulo L . Cabrini,* M .D ., Buenos Aires, Argentina
The literature relating to the histochem istry of the periodontal tissues is reviewed. The
distribution
phosphatase,
of alkaline and acid
mucopolysaccharides
and
glycogen, metachromatic substances, and the disulfide and sulfhydryl groups is described, and their probable role in the histophysiology of the periodontium analyzed.
Histochemical
research
is
may
lead to a better understanding of the mechanism of the biochemical reactions occurring within
the tissues, and may
provide a useful tool in diagnosing patho logical changes.
studies have to be m ade cautiously and very carefully controlled, especially when a hypothesis o f some functional signifi cance is based on them. It is not possible to obtain reliable histochemical data without adequate technical and theoreti cal means; unfortunately, the “ pseudo simplicity” o f some technics has caused them to be employed sometimes under in adequate conditions, leading to results that have to be discarded. In this review (con clu ded M arch 31, 1958) we shall refer to the most im por tant and best-studied histochemical reac tions o f the periodontal tissues and ana lyze briefly their possible physiological role. A L K A L IN E P H O S P H A T A S E
A lthough histochemistry has developed simultaneously with general ■m icroscopi cal technics, its study has been greatly intensified during the last 15 years. T h e methods developed up to the present are, in the majority, qualitative and only a few isolated attempts at quantitative d e termination have been m ade .1 Qualitative chem ical study o f the dis tribution o f a given substance or o f a functional group presents all kinds o f d if ficulties ; for this reason histochemical
This enzyme has been studied in human gingiva by several authors .2-6 A ll o f them agree that by using hydrolysis o f sodium glycerophosphate as a substrate, the en zyme is found in the connective tissue in and around the b lood vessels. A positive reaction in the basal layer o f the epithe lium has been reported when nucleic acid is used as a substrate .5 T h e alkaline phosphatase content o f the oral epithelium o f the rat increases
C A R R A N Z A — C A B R IN I . . . V O L U M E 60, A P R IL I960 • 49/445
during the estrous cycle from a minimum in diestrus to a m axim um in estrus in a way com parable to the variations o f al kaline phosphatase in the vaginal epithe lium o f the rat during the sexual cycle .7,8 T h e phosphatasic activity o f gingival wounds in rats is increased considerably in the superficial layers o f the epithelium adjacent to the incision .9 In view o f these findings the theory has been put forward that the amount o f alkaline phosphatase in the epithelium is related to the process o f keratinization .9,10 In this theory, the gingival epithelium would show very lit tle or no phosphatasic activity probably because, like the skin, it w ould maintain its keratinized state with phosphatasic levels which are not demonstrable histochem ically .9 T h e results o f different investigations on alkaline phosphatase activity in the granules o f keratohyalin d o not agree. Some authors 4,7,9,11>12 have fou n d a positive reaction in these granules; oth ers 5’ 13 have not. Some consider the localization o f alkaline phosphatase in the granules o f keratohyalin to be a d if fusion phenom enon with adsorption by the granules .14 In the gingival connective tissue the enzyme is fou n d especially in the blood vessels. In the larger b lood vessels, co m posed o f several layers, it has been shown that the phosphatasic activity is con cen trated in the endothelium .5 In inflammatory processes the enzyme appears in large quantities and is local ized in the capillaries, in the inflam m a tory infiltrate especially in the polym or phonuclear leukocytes, ánd less notably in the newly form ed collagen fibers .0 In the basal cell layer and at the end o f the papillae of epithelium overlying inflamed connective tissue, alkaline phosphatase has been found, very probably a result of diffusion .15 A study on the alkaline phosphatase activity in the gingiva o f the ferret 16 re vealed a great difference from human gingiva. T h e ferret’ s clinically normal
gingiva contains this enzyme only in the gingival and crevicular epithelia and only slight activity in the lamina propria; in flamed gingiva displays a pronounced increase in alkaline phosphatase activity throughout the gingival and crevicular epithelia, localized in the cytoplasm and also extracellularly associated with the intercellular bridges. T h e connective tissue attachment o f the gingiva contains alkaline phospha tase in the collagen fibers .17 In some in stances the enzymatic activity o f the granulation tissue o f the lateral wall of the pocket blends gradually with this zone. H ow ever, those instances in which there exists an intermediate zone devoid o f inflammatory reaction, prove the in dependence o f the phosphatasic content in both zones .17 In predominantly fibrous gingivae 15 or in diphenylhydantoin sodium hyperplasia where there is a noticeable fibrosis18 there is an increase o f alkaline phospha tase in relation to the form ation o f con nective tissue fibers. Enzymatic activity in the supporting tissues is present in a varying degree. T he periodontal mem brane o f rodents shows a strong reaction fo r alkaline phospha tase in the fibers and in the ground sub stance between them .4 In dogs a strong reaction has been reported in fibers and cells .19,20 In man, a weak and inconstant reaction has been fou n d in the periodon tal m em brane fibers .17 Generally, phos phatase o f the periodontal membrane is associated with a m ore or less intense phosphatasic activity o f the bone and cem entum surfaces. A lveolar bonq has enzyme only in some areas o f its periosteal, endosteal and peri odontal surfaces. In the m ajority o f in stances, the periosteum o f the alveolar crest has an intense enzymatic activity which continues in one or both sides, peri osteal and periodontal .17 In the cem en tum, alkaline phosphatase has been found around Sharpey’s fibers and in the cementoblasts .21
50/446 • THE J O U R N A L O F THE A M E R IC A N DENTAL A S S O C IA T IO N
ACID P H O S P H A T A S E
A cid phosphatase is present in the gin gival epithelium in a greater concentra tion immediately beneath the horny layer, and diminishes toward the basal cell layer. It is absent in the epithelial at tachment and the epithelial lining o f the gingival sulcus .22 In laboratory animals a somewhat d if ferent picture has been fo u n d .23 In the rat and the mouse the gingival epithe lium shows a double reaction: one, stronger, in the layer beneath the horny layer, similar to that fou n d in human gingiva, and a second one, weaker, in the basal cell layer, leaving in between a zone that does not react and that corre sponds, roughly, to the prickle cell layer. This distribution also has been found in the epithelial lining o f the tongue in rat and human specimens. Alveolar bone shows a strong reaction in the areas o f bone resorption, in the osteoclasts and b lood vessel walls a d ja cent to H ow ship’ s lacunae. This confirms previous findings in other regions o f the skeleton .24 M U C O P O L Y S A C C H A R ID E S AND GLYCOGEN
T he periodic acid-Schiff reaction ( PAS) is widely used for the detection o f gingival m ucopolysaccharides .4,6-25-30 T h e con n ec tive tissue o f normal gingiva stains pink with PAS. T h e ground substance covers the collagen fibers, producing an optical effect o f hom ogeneity .26 T h e basement m embrane shows up as an amorphous structure o f light red polor. Treatm ent with ptyalin previous to the staining eliminates the glycogen; com parison with a control section allows the glycogen distribution to be determined am ong the other PAS-positive substances. T h e presence o f glycogen in norm al gingival epithelium is controversial. For some ,6,31 norm al epithelium is devoid o f glycogen ; others 25,29 describe the pres
ence o f glycogen in the prickle cell layer. Glycogen appears in the cellular cyto plasm as small, irregular granulations. T he distribution o f glycogen is h om og enous rather than granular in gingivae fixed by the freezing-drying technic. T h e presence o f glycogen in the prickle cell layer o f the skin epithelium has been related to acanthosis and parakeratinization, or b oth .32 O ther studies33 report glycogen in the skin in relation to para keratosis only, and not to acanthosis ; however, in the latter study, fixation with Formalin was em ployed, which is not the best fo r this technic and reduces the value o f the conclusions .34 In the gingiva a correlation has been reported 27 between glycogen and acan thosis and, indirectly, with inflammation, when this produces a reactional acan thosis. O ther studies6,31 report a corre lation between glycogen and inflam m a tion. A histochemical study 35 o f gingival healing in normal and vitamin C deficient animals reveals that the am ount o f glyco gen increases in hyperplastic areas in d i rect relation to the degree o f inflam m a tion, and is apparently not affected by vitamin C deficiency. T h e author o f a recent study 36 o f the glycogen content of human gingivae in specimens fixed in Formalin, found no correlation between glycogen and age, sex, menstrual cycle, inflammation and degree o f keratinization. It has been shown that with Ritter and Oleson’s technic the basement m em brane takes a different color from the ground substance, which w ould indicate that they are different substances .37 This study also showed no correlation between keratinization and pigmentation and the state o f the basement membrane. Changes or lack o f staining o f the basement mem brane in chronic desqua mative gingivitis as a result o f depoly merization o f the highly polymerized com ponents o f the basement m em brane have been described .25 But other studies6 have found similar changes in the pres
C A R R A N Z A — C A B R IN I . . . V O L U M E 60, A P R IL I960 • 51/467
ence of chronic inflammation, and that both disruption and disappearance o f the basement mem brane occurs in the pres ence o f inflammation, with regeneration in instances o f chronic inflam m ation .37 T h e gingival epithelium usually is de void o f PAS-positive, ptyalin-resistant substances, except for very small amounts in the intercellular spaces and the epi thelial surface. This reaction has been reported to be m uch greater in m ucous membranes than in the skin, suggesting an inverse relationship between the degree o f keratinization o f the epithelium and the am ount o f PAS-positive sub stances.38 Similar behavior has been shown in gingival tissues; that is, a posi tive reaction in parakeratinized areas which disappears when the epithelium attains a com plete keratinic differentia tion .27 A fter treatment with amylase a very mild PAS-positive reaction remains in the intercellular spaces, especially in the surface layers. Small PAS-positive granulations, ir regularly distributed in their scarce cytoplasm, have been observed in the fibrocytes o f the gingival connective tissue.25’ 27 These granulations disappear with amylase and are not preserved p rop erly in material which has been fixed in Formalin or decalcified. This behavior shows that it is glycogen. T h e rem aining cells o f the gingiva show their usual reaction : mast cells react in their granules with irregular intensity; in inflammatory infiltrates the leukocytes are frankly positive but their reaction disappears with amylase; lym ph ocytes and reticulohistiocytes show no reaction; plasma cells show a weak cyto plasmic reaction w hich increases in R us sell’s bodies and does not disappear with amylase. M E T A C H R O M A S IA
T he gingival connective tissue gives a weak m etachrom atic reaction localized
especially in the fibers. T h e mast cells are the only frankly m etachrom atic co m p o nent o f the gingiva .4 Treatm ent with hyaluronidase eliminates the m etachro matic reaction o f the connective tissue but it does not change that o f the mast cell granules .4 Large numbers o f mast cells have been described in the gingiva. In most in stances, a definite distribution o f these cells in the gingiva appears .39 T here are few er mast cells in the connective tissue adjacent to the gingival sulcus; in the central zones o f the gingival corium, especially around the b lood vessels, round and oval mast cells predom inate, whereas in the periphery elongated form s are pre dominant. This distribution o f the d if ferent shapes o f mast cells has suggested a migration o f these cells from the center to the periphery .39 It is not unusual to find mast cells which have penetrated the basal layer o f the epithelium .39,40 T h e disappearance o f mast cells in the presence o f acute inflammation has been described ;41 this has also been reported in instances o f acute necrotizing gingi vitis .39 In chronic gingivitis an increased num ber o f mast cells has been re ported .31,40’ 42 D IS U L F ID E A N D S U L F H Y D R Y L G R O U P S
T h e disulfide and sulfhydryl groups in norm al and inflamed gingivae have been studied using Barnett and Seligm an’s technic .43 T h e concentration o f sulfhydryls was fou n d to be greater in the kera tinized and parakeratinized layers o f the epithelium ; disulfide groups were fou n d to be distributed uniform ly throughout all the epithelial layers. In areas o f gin gival inflammation an increase in the sulfhydryl content o f proliferating epi thelium was also fou n d .43 A histochemical study o f a case o f chronic desquamative gingivitis reports the presence o f sulfhydryl groups through out the epithelial layers with a consider able and unexplained concentration o f
52/468 • THE J O U R N A L O F THE A M E R IC A N DEN T A L A S S O C IA T IO N
«
the reaction in the superficial layers; the disulfide bond reaction was negative .44 M IS C E L L A N E O U S
T h e distribution o f nucleic acids has been reported to be similar in both n o r mal and inflamed gingiva except fo r the desoxyribose nucleic acid content o f the nucleus o f inflammatory cells. R ibose nucleic acid appears in large quantities in the cytoplasm o f the basal layers of the gingival epithelium, diminishing toward the surface; it also diminishes in the crevicular epithelium and in zones o f leukocyte infiltration .31 T h e presence o f phospholipids has been reported in gingival epithelium .45 A study o f phosphamidase in oral tissues obtained a negative result in the gingiva .46 D IS C U S S IO N
Alkaline and acid phosphatase are d if ferently distributed and probably have com pletely different physiological roles. Alkaline phosphatase is fou n d mainly in connective tissues (gingival corium , peri odontal membrane, alveolar bone and cem entum surfaces) and is thought to be associated with the metabolism o f co l lagen and with osteogenesis. T h e absence o f alkaline phosphatase in wounds o f scorbutic guinea pigs and its presence in the wounds o f norm al guinea pigs have suggested that it is asso ciated in some way with the form ation of collagen .47 H ow ever, the healing o f wounds is independent from the phosphatasic level o f the b lo o d ; the applica tion o f large quantities o f phosphatase in wounds o f scorbutic animals or the in jec tion o f phosphatase inhibitors into the wounds o f norm al ones, fails to produce any changes .48 This makes the role o f alkaline phosphatase in fibrogenesis hot clear. T h e relation between alkaline phos phatase and epithelial regeneration 49 and keratinization 9,10 also has been suggested.
It has been known for years that a close relationship exists between osteogenesis and the presence o f alkaline phosphatase in large amounts .50 Recently, these b io chem ical data have been fully confirm ed by histochemistry. O n e most recent inves tigation 51 seems to indicate that this en zyme plays a role in the elaboration o f the protein matrix and that its function as a direct agent o f calcium salt precipitation is doubtful. A cid phosphatase, on the other hand, is localized especially in epithelial tissues. T h e existence o f large amounts o f this enzyme in the epithelial layers during keratinization leads to the belief that it is closely related to this process. Similar findings have been shown in the skin .52,53 Connective tissue ( gingival corium and periodontal mem brane) appears to have no enzyme; bone surfaces and h em ato poietic m arrow spaces are rich in acid phosphatase. This distribution in the bone surfaces, almost exclusively in the osteo clasts, suggests that this enzyme plays an active role in bone resorption .24 M ucopolysaccharides are distributed in the epithelium and corium as well as in the alveolar bone. T h e gingival epithe lium has only vestiges o f m ucopolysac charides located in the intercellular spaces and in some bands o f parakeratinized material. It w ould appear that variations in this PAS-positive, ptyalin-resistant sub stance in the epithelium are o f little im portance, and its use is restricted to the detection o f parakeratinized areas and their differentiation from truly ker atinized ones .32 T h e gingival corium has a distribution com m on to connective tissue o f the skin, with a condensation o f PAS-positive m a terial in the basement membrane. A t this time it is premature to think that changes or m odifications o f the reaction o f the basement m em brane may have a signifi cance useful fo r the diagnosis o f gingival changes. Glycogen reacts easily to PAS technic and is identified by means of ptyalin; but
C A R R A N Z A — C A B R IN I . . . V O L U M E 60, A P R IL I960 • 53/44»
it is difficult to fix in the tissues, and only data obtained under irreproachable co n ditions o f fixation should be taken into account. A lcoh olic mixtures o f picric acid, or better still, the Altmann-Gersh m ethod, should be used. W ith adequate fixation it is possible to find glycogen in the prickle cell layer o f the epithelium and an increase in acanthosis and hydro pic degeneration. Although these varia tions apparently are constant, their usefulness as a means o f diagnosis has to be discarded because o f the lability o f glycogen during fixation. A bundant mast cells have been found in gingival tissues stained with m etachromatic technics. T h e histophysiological role o f these cells has not yet been ex plained conclusively. It is believed that the cells contain heparin or histamine or some precursor substance o f hyaluronic acid. T here is a tendency today to connect mast cells with the maintenance and repair o f connective tissues,54 which w ould explain their presence in large amounts in the gingiva. Disulfide and sulfhydryl groups have been associated with the process o f ker atinization, the most im portant reaction o f w hich w ould be the transformation o f sulfhydryl groups into disulfide ones .85 R ecent studies56 have shown in-vitro blister form ation due to rupture o f the disulfide bonds which appear to play an im portant part in the integrity o f the intercellular bridges and the tonofibrils. This might need further study in relation to chronic desquamative gingivitis. SUM M ARY
T h e most significant contributions to the field o f histochemistry o f the periodontal tissues have been reviewed, including the follow ing substances: alkaline and acid phosphatase, m ucopolysaccharides and glycogen, m etachrom atic substances, d i sulfide and sulfhydryl groups. T h e distri bution o f these substances is described and their probable role in the histophysiology
o f the periodontium briefly analyzed. H istochem ical research done up to the present opens up a new field w hich will give a better understanding o f the m ech anism o f the biochem ical reactions occu r ring within the tissues, and which may also provide a useful tool fo r the m icro scopical diagnosis o f pathological changes.
* D e p a r f m e n t o f p a t h o lo g y , H o s p i t a l R a m o s M e [ ia . 1. G lic k , D. U s e o f m ic r o c h e m ic a l m e t h o d s f o r q u a n t i t a t iv e lo c a liz a t io n in h is to c h e m is tr y . J . H is t o c h e m . 5:539 N o v . 1957. 2. Z a n d e r , Hè A . D is t r ib u t io n o f p h o s p h a t a s e g i v a l tissu e . J. D . Res. 20:347 A u g . 1941.
in q in -
3. V a llo t t o n , C . E. E tu d e b io - h i s t o lo g iq u e d e la p h o s p h a t a s e . d a n s la g e n c iv e h u m a in e n o r m a le et d a n s les g i n g iv i t e s . 'S c h w e i z . M s c h r . Z a h n h k . 52:512 J u n e 1942. 4. W is l o c k i, G . B., a n d S o g n n a e s , R. F. H is t o c h e m ic a l r e a c t io n s o f n o r m a l te e th . A m . J . A n a t . 87:239 S e p t. 1950. 5. C a b r in i , R. L., a n d C a r r a n z a , F. A . , J r. H i s t o c h e m ic a l st u d y o n a lk a lin e p h o s p h a t a s e in n o r m a l q in g iv a e , v a r y in g the p H a n d th e s u b st r a t e . J . D . Res. 30:28 F e b . 1951.
6
. T u resky, S.; G lic k m a n , I., a n d L itw in , T. H i s t o c h e m ic a l e v a lu a t io n o f n o r m a l a n d in f la m e d h u m a n g i n g iv a e . J. D. Res. 30:792 D e c. 1951. 7. R in g , J . R. C h a n g e s in a lk a lin e p h o s p h a t a s e a c t iv it y o f ra t v a g in a l e p it h e liu m d u r i n g th e e st r o u s c y c le . A n a t . Ree. 107:121 J u n e 1950. . R in g , J . R., a n d L e vy, B. C h a n g e s in a lk a lin e p h o s p h a t a s e a c t iv it y o f ra t o r a l e p it h e liu m d u r i n g the e s t r o u s c y c le a n d in r e s p o n se t o a d m in is t e r e d e s t r o g e n . J . D . Res. 29:817 D e c . 1950.
8
9. R in g , J . R. A lk a l in e p h o s p h a t a s e a c t iv it y o f h e a li n g g i n g i v a l w o u n d s in th e rat. J . D . Res. 31:329 J u n e 1952. 10. F o rd , D . H . S t u d y o f c h a n g e s in v a g in a l a lk a lin e p h o s p h a t a s e a c t iv it y d u r i n g e s t r o u s c y c le in a d u lt a n d in y o u n g " f i r s t e s t r o u s " rats. A n a t . Rèe. 125:261 J u n e 1956. 11. S m ith , C ., a n d P a rk h u rst, H . S t u d ie s o n t h e t h y m u s o f th e m a m m a l; c o m p a r is o n o f ’th e s t a in in g p r o p e r t ie s o f H a s s a l l 's c o r p u s c le s a n d o f t h ic k sk in o f tn e g u in e a p ig . A n a t . Re e. 103:649 A p r i l 1949. 12. L a n s in g , A . I., a n d O p d y k e , D. L. H i s t o l o g i c a l a n d h is t o c h e m ic a l st u d ie s o f th e n ip p le s o f e s t r o g e n t r e a t e d g u in e a p i g s w ith s p e c ia l r e fe re n c e t o k e r a t o h y a lin g r a n u le s . A n a t . Ree. 107:379 A u g . 1950. 13. K o p f, A . W . D is t r ib u t io n o f a lk a lin e p h o s p h a t a s e in n o r m a l a n d p a t h o l o g ic h u m a n skin . A . M . A . A r c h . D e r m a t . 75:1 J a n . 1957. 14. N a d e l, E. M . , a n d W o d in s k y , I. D i s s im i la r it y in a lk a lin e p h o s p h a t a s e s t a in in g r e a c t io n s o f k e r a t o h y a lin e g r a n u le s . J . H is t o c h e m . 3:339 S e p t . 1955. 15. C a r r a n z a , F. A . , J r., a n d C a b r in i , R. L. E s t u d io h is t o q u í m ic o d e la fo s f a t a s a a lc a lin a en e n c ía . Rev. O d o n t . 40:443 N o v . 1952. 16. S t a p le , P. H . A lk a l in e p h o s p h a t a s e a c t iv it y in g i n g i v a o f th e fe rre t. B rit. D . J . 102:305 A p r i l 16, 1957. 17. C a r r a n z a , F. A . , Jr., a n d C a b r in i , R. L. E s t u d io h is t o q u í m ic o d e la fo s fa t a s a a lc a lin a e n lo s t e j id o s perto d o n t a le s . Re v. A . o d o n t., A r g e n t in a 43:206 J u n e 1955. 18. S t a p le , P. H . S o m e t is su e r e a c t io n s a s s o c ia t e d w ith 5:5 d ip h e n y lh y d a n t o in ( " D i l a n t i n " ) s o d iu m t h e r a p y . Brit. D . J . 95:289 D e c . 15, 1953. 19. Z e ro s i, C ., a n d B a ra tie ri, A . R ic e r c e is t o c h ìm ìc h e s u lle lo c a liz z a z io n e d e lla f o s fa t a s i a lc a l in a n e lla p o lp a d e n t a r ia (c o n r ife r im e n t o a lla fo r m a z io n e d e l la d e n t in a ) nel p e r i o d o n t o e nel c e m e n t o . A t t i S o c . l o m b a r d a Se. m e d . b io l. 9:4, 1954. 20. B a ra tie ri, A . R ic e r c e is t o c h ìm ìc h e s u lle lo c a liz z a z io n i d e lla fo s fa t a s i a lc a lin a _ n e g li e le m e n ti d e l p e r i o d o n t o e d e l c e m e n t o . Riv. ita l. S t o m a t . 10:212 M a r c h 1955.
54/470 * THE J O U R N A L O F THE A M E R IC A N D EN T A L A S S O C IA T IO N
21. L o r b e r , M . S t u d y o f th e h is t o c h e m ic a l r e a c t io n s o f d e n t a l c e m e n t u m a n d a lv e o l a r b o n e . A n a t . R e c. 111:129 O c t . 1951. 22. C a b r in i , R. L., a n d C a r r a n z a , F. A . , J r. H i s t o c h e m ic a l d is t r ib u t io n o f a c i d p h o s p h a t a s e in h u m a n g i n g i v a . J . P e r io d o n t . 29:34 J a n . 1958. 23. C a r r a n z a , F. A ., Jr., a n d C a b r in i , R. L. E s t u d io h is t o q u í m ic o d e la fo s fa t a s a a c i d a en la m u c o s a o r a l d e ra ta , r a t ó n y h o m b r e . Rev. A . o d o n t. A r g e n t in a , 46:219 J u ly 1958. 24. S c h a jo w ic z , F., a n d C a b r in i , R. L. H is t o c h e m ic a l lo c a liz a t io n o f a c id p h o s p h a t a s e in b o n e tissu e . S c ie n c e 127:1447 J u n e 20, 1958. 25. E n g e l, M . B.; Ray, H . S . , a n d O r b a n , B. P a t h o g e n e s is o f d e s q u a m a t iv e g i n g iv i t i s : d is t u r b a n c e o f c o n n e c tiv e tissu e g r o u n d su b s t a n c e . J . D. Res. 29:410 A u q . 1950. 26. E n g e l, M . B. W a t e r - s o lu b le m u c o p r o t e in s g i n g i v a . J . D . Res. 32:779 D e c . 1953.
o f the
27. C a b r in i , R. L., a n d C a r r a n z a , F. A . , J r. E s t u d io h is t o q u í m ic o d e la s s u b s t a n c ia s " P A S " p o s it iv a s , m e ta c r o m á t ic a s y b a s ó f ila s e n lo s t e j id o s p e r io d o n t a le s . Re v. A . o d o n t . A r g e n t in a 44:309 A u g . 1956. 28. M o r í , M ., a n d O g a t a , Y. H is t o c h e m ic a l s t u d y on fh e p o l y s a c c h a r i d e in g i n g i v a ¡n p a t ie n t s w ith p e r i o d o n t a l d is e a s e . D . A b s t . 2:300 M a y 1957. 29. W e is s , M . B., a n d W e in m a n n , J . P. G l y c o g e n d i s t r ib u t io n In h u m a n g i n g iv a . ( A b s t . ) I. A . D . R . 35:37, 1957. 30. R o s e n b e r g , C . J . D e t e r m in a c ió n d e lo s á c i d o s m u c o p o l is a c é r i d o s e n la s d is t in t a s f o r m a s d e p a r a d e n c io p a t ía s en el c o r io n g i n g iv a l . Re v. d e n t . C h i le 47:149 A o r i l 1957. 31. D e w a r, M . R. O b s e r v a t i o n s o n th e c o m p o s it io n a n d m e t a b o lis m o f n o r m a l a n d in fla m e d g i n g iv a e . J . P e r io d o n t . 26:29 J a n . 1955. 32. B ra u n - F a lc o , O . H is t o c h e m is c h e u n d m o r p h o l o g isc h e Stu d ie n a n n o r m a le r u n d p a t h o l o g is c h v e r ä n d e r t e r H a u t . A r c h . D e r m a t . u. S y p h . 198:111, 1954. 33. S te in e r, K. H is t o c h e m ic a l st u d y o f e p id e r m a l g l y c o g e n in sk in d is e a se s. J. In v e st. D e r m a t . 24:599 J u n e 1955. 34. S c h a jo w ic z , F., a n d C a b r in i , R. L. H is t o c h e m ic a l st u d ie s o n g l y c o g e n in n o r m a l o s s ific a t io n a n d c a l c i f i c a t io n . J . B o n e & J o in t S u r g . 4 0 -A : 1081' O c t . 1958. 35. T u resky, S. S., a n d G lic k m a n , I. H is t o c h e m ic a l e v a lu a t io n o f g i n g i v a l h e a lin g in e x p e r im e n t a l a n im a ls on a d e q u a t e a n d v it a m in C d e f ic ie n t d ie ts . J. D. Res. 33:273 A p r i l 1954.
p e r i o d i c a c id - S c h ilf f m e t h o d . A n a t . R e c. 110:359 J u ly 1951. 39. C a r r a n z a , F. A . , J r., a n d C a b r in i , R. L. M a s t c e lls in h u m a n g i n g iv a . O r a l S u r g ., O r a l M e d . & O r a l P ath . 8:1093 O c t . 1955. 40. S te lla , A . S o b r e Jos m a st z e lle n d e la e n c ia H u m a n a . A n . F a c . o d o n t . M o n t e v i d e o 1:125, 1955. 41. S y lv e n , B. C a r t i l a g e a n d cho ’n d r o it in su lfa t e ; c h o n d r o it in s u lfa t e a n d p h y s i o l o g ic a l o s s if ic a t io n o f c a r t ila g e . J. B o n e & J o in t S u r g . 29:973 O c t . 1947. 42. D e w a r, M . R. M a s t P e r lo d o n t . 29:67 J a n . 1958.
c e lls
in
g in g iv a l
tissu e .
J.
43. T u re sky, S .; C r o w le y , J., a n d G lic k m a n , I. H i s t o c h e m ic a l s t u d y o f p r o t e in - b o u n d su lfh y d r y l a n d d is u lf id e g r o u p s in n o r m a l a n d in fla m e d h u m a n g i n g iv a . J. D. Res. 36:255 A p r i l 1957. 44. S o g n n a e s , R. F.; W e is b e r g e r , D., a n d A lb r i g h t , J . T. P a t h o l o g ic d e s q u a m a t io n o f o r a l e p it h e liu m e x a m in e d b y e le c t r o n m i c r o s c o p y a n d h is to c h e m is tr y . J. N a t . C a n c e r In st. 17:329 S e p t . 1956. 45. M e r o n i, C . M . L o s f o s f o l i p i d o s g l n g iv a le s . Re v. A . o d o n t., A r g e n t in a 45:123 A p r il , 45:159 M a y 1957. 46. M e y e r , J., a n d W e in m a n n , J . P. P h o s p h a m id a s e c o n te n t o f n o r m a l a n d p a t h o l o g ic t is su e s o f t h e o ra l c a v ity . J . H is t o c h e m . 1:305 S e p t . 1953. 47. D a n ie llij J . F.; F e ll, H . B., a n d K o d ic e k , E. Enzymes^ o f h e a lin g w o u n d s ; e ffe c t o f d iffe r e n t d e g r e e s of v it a m in C - d e f ic ie n c y o n p h o s p h a t a s e a c t iv it y in e x p e r i m e n ta l w o u n d s in g u in e a p ig . B rit. J . E x p e r. P ath . 23:367 D e c. 1945. 48. G o u ld , B. S., a n d G o l d , N . I. S t u d ie s o n " a l k a l i n e " p h o s p h a t a s e a s s o c ia t e d w ith r e g e n e r a t in g c o n n e c tiv e tis su e fib e r s. A . M . A . A r c h . P ath . 52:413 N o v . 1951. 4 9 .^ Fre n ch , J . E., a n d B e n d itt, E. P. O b s e r v a t i o n s on lo c a liz a t io n o f a lk a lin e p h o s p h a t a s e in h e a lin g w o u n d s. A . M . A . A r c h . P ath . 57:352 A p r i l 1954. 50. R o b is o n , R. P o s s ib le s ig n if ic a n c e o f h e x o se p h o s p h o r ic e st e rs in o s s ific a t io n . J . B io c h e m . 17:286, 1923. 51. G u t m a n , A . B. C u r r e n t t h e o r ie s o f b o n e s a lt f o r m a tio n , w ith s p e c ia l r e fe re n c e to e n z y m e m e c h a n is m s in e n d o c h o n d r a l c a lc if ic a t io n . B u ll. H o s p . J o i n t D is. 12:74 A p r i l 1951. 52. B ejd l, W . D ie s a u r e P h o s p h a t a s e in H a u t u nd V a g i n a d e s M e n s c h e n u n d ih r e B e d e u t u n g fu r d ie V erh o r n u n g . Z t s c h r . Z e llf o r s c h . 40:389, 1954. 53. M o r e t t i, G ., a n d M e s c o n , H . H is t o c h e m ic a l d i s t r ib u t io n o f a c i d p h o s p h a t a s e s in n o r m a l h u m a n skin . J . In v e st . D e r m a t . 26:347 M a y 1956.
36. T ro tt, J . R. A n i n v e s t ig a t io n in t o th e g ly c o g e n c o n te n t o f th e g i n g iv a e . D . P r a c t it io n e r 7:234 A p r i l 1957.
54. Rile y, J . F. T h e r id d le o f the m a st c e lls : t r ib u t e to P aul E h rlic h . L a n c e t 1:841 A p r i l 24, 1954.
37. Linz, A . , M . O b s e r v a t io n s o n the b a s e m e n t m e m b r a n e o f th e g i n g iv a e . O r a l S u r g ., O r a l M e d . & O r a l Path . 7:763 J u l y 1954.
55. R o th m a n , S. P h y s io lo g y a n d b io c h e m is t r y o f the skin . C h i c a g o , U n iv e r s it y o f C h i c a g o P ress, 1954, c h a p t e r 16.
38. W is l o c k i, G . B.; Fa w c e tt, D . W . , a n d D e m p se y , E. W . S t a i n i n g o f s t r a tifie d s q u a m o u s e p it h e liu m o f m u c o u s m e m b r a n e s a n d sk in o f m a n a n d m o n k e y b y the
56. S t o u g h t o n , R. B., a n d N o v a k , N . D is r u p t io n o f t o n o f lb r ils a n d in t e r c e llu la r b r id g e s b y d is u lf id e - s p lit t in q a g e n t s . J . In v e st. D e r m a t . 26:127 F e b . 1956.
Cancer A m ong M en • Cancer can no longer be regarded as “ a w om an’ s disease.” O f every 100 persons w ho die o f cancer, 54 are men and 46 are wom en. O ne reason for the rising death rate from cancer am ong men is due to cancer o f the lung. It killed 30,000 men last year and 5,000 women— or five times as many as 20 years ago. Am erican Cancer Society, 1960.