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Intercellular mucopolysaccharides in human gingival epithelium
Arch.oralNo/. Vo1.9, pp.751-752, 1964.Pergamon
Press Ltd.
Printed in Ct. Britain.
SHORT COMMUNICATION INTERCELLULAR MUCOPOLYSACCHARIDES HUMAN GINGIVAL EPITHELIUM
IN
G. CIMA~ONIand A. J. HELD Dental School, University of Geneva, 30, rue Lombard, Geneva, Switzerland A COMPREHENSIVE review of the pertinent literature on the intercellular cementing substance (ICS) of gingival epithelium has recently been presented by THONARDand SHERP(1962). These authors showed periodic acid-Schiff (PAS) and Hale positive intercellular material in biopsy specimens of human gingiva, thus confirming the previous findings of BRAUN-FALCO(1958). They also demonstrated such a material by metachromasia at the pH of 6 and 2-5. The use of multiple enzyme preparations was reported to have no effect on the ICS, with the exception of a chondroitinase preparation which diminished to a considerable extent the metachromatic reaction. Alcian blue staining was not employed. The identification of ICS is of practical importance: this has been shown by investigators who have produced inflammatory lesions by testicular hyaluronidase, either by injecting the enzyme in monkey gingiva (AISENBERG and AISENBERG,1951) or by introducing it in human gingival crevices (SCHULTZ-HAUDT,1958). Along these lines, the recent work of CIMASONI,FIOREand HELD (1963) seems significant: these authors have reported an important amount of PAS, Hale and Alcian Blue positive material along the insertion zone of human gingival epithelium reattached to an artificial dentinal groove. The Alcian Blue positive substance was removed by treatment with testicular hyaluronidase. The present report further illustrates the existence of acid mucopolysaccharides in the intercellular spaces of human gingival epithelium. It indicates how the ICS can be shown by PAS, Hale, toluidine blue and also by the Alcian blue technique. Alcian blue is a specific staining for acid mucopolysaccharides and has never been used previously in this field; it therefore deserves the following comments. The staining is based on a salt linkage, at a low pH, between a blue water-soluble pigment and the acidic groups of acid mucopolysaccharides and nucleic acids. Similarly to the dialyzed iron reaction (Hale), the specificity of the Alcian blue lies in the pH of the solution: at a low pH, only the tissue components having a lower pK are able to dissociate and therefore bind the pigment. Before reaching satisfactory results, technical difficulties were encountered, particularly the loss of sections from the slides at the moment of staining. Slide fixation in a mixture of 10 % formalin and 80% alcohol, or freeze drying of sections mounted on slides did not help in overcoming this 751
152
G. C~MASONI and A. J. HELD
obstacle. Minimal loss and constantly positive results were, however, obtained on sections picked up on warm slides and stained without further manipulations after being stored for indefinite lengths of time in the cryostat (Kryotom-Gefrieranlage WKF). Biopsy specimens from five adult patients showed deposits of Alcian blue in the intercellular epithelial spaces and nuclei (Fig. 1, c), more evident in the stratum germinativum and stratum spinosum. In all cases, the ICS revealed by the Hale and the Alcian blue techniques (Fig. 1, b, c) or by metachromasia (Fig. 1, d) was granular in character, whereas in sections treated for the PAS reaction (Fig. 1, a) this material had an homogeneous appearance. Treatment of sections with CCamylase and two commercial preparations of testicular hylauronidase had no effect on the staining properties of the ICS. The PAS positiveness indicates that the acid mucopolysaccharides can be bound to a protein moiety (BRADEN, 1955). Acknowledgement-This Foundation (grant 2478).
study was supported
by the Swiss National
Science
REFERENCES AISENBERG,M. S. and AISENBERG,A. S. 1951. Hyaluronidase in periodontal disease. Oral Surg. 4,317-320. BRADEN,A. W. H. 1955. The reactions of isolated mucopolysaccharides to several histochemical tests. Stain Technol. 30,19-26. BIUUN-FALCO, 0. 1958. The histochemistry of psoriasis. Ann. N. Y. Acad. Sci. 73,936-976. CIMASONI,G., FIORE-DONNO,G. and HELD, A. J. 1963. Mucopolysaccharides in human epithelial reattachment. Helv. odont. acta 7, 60-67. SCHULTZ-HAUDT,S. D. 1958. Observations on the acid mucopolysaccharides of human gingiva. Odont. Tia!skr. 66, 1-98. THONARD,J. C. and SHERP,H. W. 1962. Histochemical demonstration of acid mucopolysaccharides in human gingival epithelial intercellular spaces. Arch. oral Biol. 7, 125-136.
INTERCELLULAR
FIG.
1. Normal
MUCOPOLYSACCHARIDES
human
gingival
IN HUMAN
GINGIVAL
EPlTHELlUiM
epithelium
(a) PAS staining: the ICS and some intracellular substance (glycogen) arc heavily stained. (b) Hale (Rinehard) technique: notice deposits of dialysed iron in the intercellular spaces (indicating acid mucopolysaccharides) and in the nuclei (nucleic acids). (c) Alcian blue: the general appearance is similar to that of Fig. (b). The nuclei seem to be less stained than with the Hale technique. (d) Metachromasia (toluidine hlue, pH = 6). Fresh frozen sections, magnification: x 600. PI 4TI
I
Press Ltd.
Printed in Ct. Britain.
SHORT COMMUNICATION INTERCELLULAR MUCOPOLYSACCHARIDES HUMAN GINGIVAL EPITHELIUM
IN
G. CIMA~ONIand A. J. HELD Dental School, University of Geneva, 30, rue Lombard, Geneva, Switzerland A COMPREHENSIVE review of the pertinent literature on the intercellular cementing substance (ICS) of gingival epithelium has recently been presented by THONARDand SHERP(1962). These authors showed periodic acid-Schiff (PAS) and Hale positive intercellular material in biopsy specimens of human gingiva, thus confirming the previous findings of BRAUN-FALCO(1958). They also demonstrated such a material by metachromasia at the pH of 6 and 2-5. The use of multiple enzyme preparations was reported to have no effect on the ICS, with the exception of a chondroitinase preparation which diminished to a considerable extent the metachromatic reaction. Alcian blue staining was not employed. The identification of ICS is of practical importance: this has been shown by investigators who have produced inflammatory lesions by testicular hyaluronidase, either by injecting the enzyme in monkey gingiva (AISENBERG and AISENBERG,1951) or by introducing it in human gingival crevices (SCHULTZ-HAUDT,1958). Along these lines, the recent work of CIMASONI,FIOREand HELD (1963) seems significant: these authors have reported an important amount of PAS, Hale and Alcian Blue positive material along the insertion zone of human gingival epithelium reattached to an artificial dentinal groove. The Alcian Blue positive substance was removed by treatment with testicular hyaluronidase. The present report further illustrates the existence of acid mucopolysaccharides in the intercellular spaces of human gingival epithelium. It indicates how the ICS can be shown by PAS, Hale, toluidine blue and also by the Alcian blue technique. Alcian blue is a specific staining for acid mucopolysaccharides and has never been used previously in this field; it therefore deserves the following comments. The staining is based on a salt linkage, at a low pH, between a blue water-soluble pigment and the acidic groups of acid mucopolysaccharides and nucleic acids. Similarly to the dialyzed iron reaction (Hale), the specificity of the Alcian blue lies in the pH of the solution: at a low pH, only the tissue components having a lower pK are able to dissociate and therefore bind the pigment. Before reaching satisfactory results, technical difficulties were encountered, particularly the loss of sections from the slides at the moment of staining. Slide fixation in a mixture of 10 % formalin and 80% alcohol, or freeze drying of sections mounted on slides did not help in overcoming this 751
152
G. C~MASONI and A. J. HELD
obstacle. Minimal loss and constantly positive results were, however, obtained on sections picked up on warm slides and stained without further manipulations after being stored for indefinite lengths of time in the cryostat (Kryotom-Gefrieranlage WKF). Biopsy specimens from five adult patients showed deposits of Alcian blue in the intercellular epithelial spaces and nuclei (Fig. 1, c), more evident in the stratum germinativum and stratum spinosum. In all cases, the ICS revealed by the Hale and the Alcian blue techniques (Fig. 1, b, c) or by metachromasia (Fig. 1, d) was granular in character, whereas in sections treated for the PAS reaction (Fig. 1, a) this material had an homogeneous appearance. Treatment of sections with CCamylase and two commercial preparations of testicular hylauronidase had no effect on the staining properties of the ICS. The PAS positiveness indicates that the acid mucopolysaccharides can be bound to a protein moiety (BRADEN, 1955). Acknowledgement-This Foundation (grant 2478).
study was supported
by the Swiss National
Science
REFERENCES AISENBERG,M. S. and AISENBERG,A. S. 1951. Hyaluronidase in periodontal disease. Oral Surg. 4,317-320. BRADEN,A. W. H. 1955. The reactions of isolated mucopolysaccharides to several histochemical tests. Stain Technol. 30,19-26. BIUUN-FALCO, 0. 1958. The histochemistry of psoriasis. Ann. N. Y. Acad. Sci. 73,936-976. CIMASONI,G., FIORE-DONNO,G. and HELD, A. J. 1963. Mucopolysaccharides in human epithelial reattachment. Helv. odont. acta 7, 60-67. SCHULTZ-HAUDT,S. D. 1958. Observations on the acid mucopolysaccharides of human gingiva. Odont. Tia!skr. 66, 1-98. THONARD,J. C. and SHERP,H. W. 1962. Histochemical demonstration of acid mucopolysaccharides in human gingival epithelial intercellular spaces. Arch. oral Biol. 7, 125-136.
INTERCELLULAR
FIG.
1. Normal
MUCOPOLYSACCHARIDES
human
gingival
IN HUMAN
GINGIVAL
EPlTHELlUiM
epithelium
(a) PAS staining: the ICS and some intracellular substance (glycogen) arc heavily stained. (b) Hale (Rinehard) technique: notice deposits of dialysed iron in the intercellular spaces (indicating acid mucopolysaccharides) and in the nuclei (nucleic acids). (c) Alcian blue: the general appearance is similar to that of Fig. (b). The nuclei seem to be less stained than with the Hale technique. (d) Metachromasia (toluidine hlue, pH = 6). Fresh frozen sections, magnification: x 600. PI 4TI
I