Archs oral Bid.
Vol. 35, Suppl., pp. 2233-22X Printed in Great Britain
THE ELASTIC
0003-9969190 $3.00 + 0.00 Pergamon Press plc
1990
SYSTEM FIBRES IN HEALTHY GINGIVA
HUMAN
C. CHAVRIER
Facultt d’Odontologie de Lyon, Laboratoire d’Histo-Physiologie et de Pathologie des Tissus Parodontaux, rue Guillaume Paradin, 69372 Lyon Cedex 8. France Summary-In human gingiva, the elastic system fibres, namely oxytalan, elaunin and elastic fibres, are distributed in the upper, medium and deep layers of gingival connective tissue, respectively. They are formed by a microfibrillar and an amorphous component characterized as elastin. In the gingival connective tissue fibroblastic cells are likely to be the main source of production of elastin in the extracellular matrix. Elastin is secreted as a soluble precursor (tropo-elastin), which spontaneously forms insoluble aggregates of elastin. Elastin is then laid down at the surface of the microfibrillar component, which could serve as a site for deposition of elastin during elastogenesis, and subsequently be incorporated in an amorphous area to form elaunin and elastic fibres. Key words: extra-cellular matrix, elastin, immunolabelling,
INTRODUCTION The extracellular matrix of gingival connective tissue is made up of collagen, elastin, proteoglycans and non-collagenous structural glycoproteins. Collagen and elastic system fibres are the two main structural and fibrillar glycoproteins of the extracellular matrix of healthy human gingiva. If collagenous fibres ac-
count for 60% of the total tissue protein, the elastic system fibres account for less than 6% (Page, 1972; Schluger, Yuodelis and Page, 1977). Elastic fibres play an important role in rendering the gingiva pliable to mechanical stresses of distension and twisting. The elastic system fibres are composed of 3 different types, namely oxytalan, elaunin and elastic fibres, as described by Cotta-Pereira, Rodrigo and Bittencourt-Sampaio (1976). Ultrastructurally the oxytalan fibres are formed by bundles of thin microfibrils with an average diameter of about 11 nm. The elastic fibres are formed by an abundant homogeneous core of amorphous material surrounded by a few similar microfilaments. The elaunin fibres have an intermediate structure characterized by patches of amorphous material intermingled with the tubular microfibrils. MATERIALS AND
monoclonal antibody, elastogenesis, gingival
and with 0.01 M sodium azide. Sections were then washed and placed overnight in 1% bovine serum albumin at 4°C. After further washing, the sections were incubated overnight at 4°C in purified antielastin antibody (one-tenth dilution), washed and then reacted with peroxidase-conjugated antiserum diluted 1: 50. The bound peroxidase complexes were visualized by treatment with 3,3’-diaminobenzidine, according to Graham and Karnovsky (1966). Sections were then fixed in 1% osmium tetroxide, dehydrated and flat-embedded in Epon. Ultra-thin sections were prepared and observed with no further staining using a Philips 300 electron microscope. Control sections were incubated with 0.1 M phosphate buffer without immune serum or with peroxidase-conjugated antiserum alone. In some cases, cryostat sections were treated before immunolabelling by elastase digestion (type III; Sigma, 1.1 mg/ml in 0.2 M tris, pH 8.8) for 20 min at 37°C.
METHODS
Five dental students (23 yr-of-age) with healthy gingiva were selected for the experiments after having given their informed consent. A 9mm3 segment of attached gingiva was removed under local anesthesia from the upper incisor of each subject. Samples were then prepared for indirect immunolabelling. For indirect immunolabelling using the peroxidase procedure, the blocks were immediately fixed with 4% paraformaldehyde-O.1 M sodium cacodylate buffer (pH 7.4) for 8 h at 4”C, washed and frozen. Cryostat sections (10 pm) were cut and treated with 0.3% hyaluronidase (bovine testis type I; Sigma, St Louis, MO, U.S.A.) for 30 min at room temperature 223s
RESULTS
In human gingiva (Fig. 1) oxytalan, elaunin and elastic fibres are distributed in the upper, medium and deep layers of gingival connective tissue respectively (Chavrier et al., 1988). The application of anti-elastin antibody clearly demonstrated the presence of elastin in the amorphous and microbrillar components. However, in control sections pre-treated with elastase, specific labelling was abolished in the digested amorphous component and in the microfibrils of oxytalan, elaunin and elastic fibres. DISCUSSION Previous biochemical studies clearly demonstrated that microfibrils were comprised of high molecularweight glycoproteins (Sear, Grant and Jackson, 1981) while the amorphous component was elastin. However, after pre-treatment of the sections with
2245
C. CHAVRIER
Eplthellum
Oxytalan subepitheliol
Glngivol connective tissue
Elounin medium
,;:; :., -II)
fibres layei
fibres
loyer
,..y...? .. .
;:
;I.:
Elastic fi bres deep layer
:...
Al veolor bone
Fig. I. Distribution of the elastic system fibres in healthy human gingival connective
pancreatic elastase, specific labelling was abolished not only in the digested amorphous component but also in the undigested fibrillar component of the oxytalan, elaunin and elastic fibres (Chavrier et al., 1988). This suggests that elastin is only associated with the surface of the microfibrils, which could serve as a site for deposition of elastin during elastogenesis. Recent studies using different anti-elastin antibodies have confirmed the presence of elastin at the surface of the microfibrillar components in different tissues (Fukuda and Ferrans, 1984; Fukuda, Ferrans and Crystal, 1984; Schwartz and Fleishmajer, 1986). Oxytalan, elaunin and elastic fibres represent the consectutive stages of elastogenesis (Gawlik, 1965; Cotta-Pereira et al., 1976; Cotta-Pereira,
REFERENCES
OF
EF
Guerra-Rodrigo and David-Ferreira, 1978). In gingival connective tissue it seems likely that the main source of production of elastin is the fibroblast (Fig. 2). This cell synthesizes elastin as a soluble precursor molecule termed tropo-elastin. The newly synthesized tropo-elastin spontaneously forms insoluble aggregates that are then cross-linked in a series of reactions ‘initiated by lysyl-oxidase. The polymerization of elastin requires the formation of two unique lysine-derived cross-links, namely desmosine and isodesmosine (Gosline and Rosenbloom, 1984). The elastin is deposited at the surface of microfibrils and incorporated in the amorphous area to form elaunin and elastic fibres (Chavrier et al., 1988).
4 FIBROBLAST
Fig. 2. Hypothetical pathway of elastogenesis in human gingival connective tissue: TE, tropoelastin; E, elastin; OF, oxytalan fibres; eF, elaunin fibres; EF, elastic fibres.
Chavrier C., Hartmann D. J., Couble M. L. and Herbage D. (1988) Distribution and organization of the elastic system fibres in healthy human gingiva: ultrastructural and immunohistochemical study. Histochem. J. 89, 47-52. Cotta-Pereira G., Rodrigo F. G. and Bittencourt-Sampaio S. (1976) Oxytalan, elaunin and elastic fibres in human skin. J. Invesf. Dermat. 66, 143-148. Cotta-Pereira G., Guerra-Rodrigo F. G. and DavidFerreira J. F. (1978) Comparative study between the elastic system fibres in human thin and thick skin. Biol. Cell 31, 291-302. Fukuda Y. and Ferrans V. J. (1984) The electron microscopic immunohistochemistry of elastase-treated aorta and nuchal ligament of fetal and postnatal sheep. J. Histochem. Cytochem. 32, 741-756.
Elastin fibres in gingiva Fukuda Y., Ferrans V. J. and Crystal R. G. (1984) Development of elastic fibres of nuchal ligament, aorta and lung of fetal and postnatal sheep: an ultrastructural and electron microscopic immunohistochemical study. Am. J. Anat. 170, 597-629.
Gawlik Z. (1965) Morphological and morphochemical properties of the elastic system in the motor organ of man. Folia Histochem. Cytochem. 3, 233-25
1.
Gosline J. M. and Rosenbloom J. (1984) Elastin in extracellular matrix. In: Biochemistry (Edited by Piez K. A. and Reddi A. H.) pp. 191-227. Elsevier, New York. Graham R. C. and Karnovsky M. J. (1966) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cyto-
225s
chemistry by a new technique. J. Histochem. Cytochem. 14, 291-302.
Page R. C. (1972) In: Developmental Aspects of Oral Biology (Edited by Slavkin H. C. and Baretta L. A.) Vol. I, pp. 291-324. Academic Press, New York. Schluger S., Yuodelis R. A. and Page R. C. (1977) In: Periodontal Disease, pp. 8-37. Lea & Febiger, Philadelphia. Schwartz E. and Fleischmajer R. (1986) Association of elastin with oxytalan fibers of the dermis and with extracellular microfibrils of cultured skin fibroblasts. J. Histochem. Cytochem. 8, 1063-1068.
Sear C. H. J., Grant M. E. and Jackson J. (1981) The nature of the microfibrillar glycoproteins of elastic fibres: a biosynthetic study. Biochem. J. 194, 587-598.
Vol. 35, Suppl., pp. 2233-22X Printed in Great Britain
THE ELASTIC
0003-9969190 $3.00 + 0.00 Pergamon Press plc
1990
SYSTEM FIBRES IN HEALTHY GINGIVA
HUMAN
C. CHAVRIER
Facultt d’Odontologie de Lyon, Laboratoire d’Histo-Physiologie et de Pathologie des Tissus Parodontaux, rue Guillaume Paradin, 69372 Lyon Cedex 8. France Summary-In human gingiva, the elastic system fibres, namely oxytalan, elaunin and elastic fibres, are distributed in the upper, medium and deep layers of gingival connective tissue, respectively. They are formed by a microfibrillar and an amorphous component characterized as elastin. In the gingival connective tissue fibroblastic cells are likely to be the main source of production of elastin in the extracellular matrix. Elastin is secreted as a soluble precursor (tropo-elastin), which spontaneously forms insoluble aggregates of elastin. Elastin is then laid down at the surface of the microfibrillar component, which could serve as a site for deposition of elastin during elastogenesis, and subsequently be incorporated in an amorphous area to form elaunin and elastic fibres. Key words: extra-cellular matrix, elastin, immunolabelling,
INTRODUCTION The extracellular matrix of gingival connective tissue is made up of collagen, elastin, proteoglycans and non-collagenous structural glycoproteins. Collagen and elastic system fibres are the two main structural and fibrillar glycoproteins of the extracellular matrix of healthy human gingiva. If collagenous fibres ac-
count for 60% of the total tissue protein, the elastic system fibres account for less than 6% (Page, 1972; Schluger, Yuodelis and Page, 1977). Elastic fibres play an important role in rendering the gingiva pliable to mechanical stresses of distension and twisting. The elastic system fibres are composed of 3 different types, namely oxytalan, elaunin and elastic fibres, as described by Cotta-Pereira, Rodrigo and Bittencourt-Sampaio (1976). Ultrastructurally the oxytalan fibres are formed by bundles of thin microfibrils with an average diameter of about 11 nm. The elastic fibres are formed by an abundant homogeneous core of amorphous material surrounded by a few similar microfilaments. The elaunin fibres have an intermediate structure characterized by patches of amorphous material intermingled with the tubular microfibrils. MATERIALS AND
monoclonal antibody, elastogenesis, gingival
and with 0.01 M sodium azide. Sections were then washed and placed overnight in 1% bovine serum albumin at 4°C. After further washing, the sections were incubated overnight at 4°C in purified antielastin antibody (one-tenth dilution), washed and then reacted with peroxidase-conjugated antiserum diluted 1: 50. The bound peroxidase complexes were visualized by treatment with 3,3’-diaminobenzidine, according to Graham and Karnovsky (1966). Sections were then fixed in 1% osmium tetroxide, dehydrated and flat-embedded in Epon. Ultra-thin sections were prepared and observed with no further staining using a Philips 300 electron microscope. Control sections were incubated with 0.1 M phosphate buffer without immune serum or with peroxidase-conjugated antiserum alone. In some cases, cryostat sections were treated before immunolabelling by elastase digestion (type III; Sigma, 1.1 mg/ml in 0.2 M tris, pH 8.8) for 20 min at 37°C.
METHODS
Five dental students (23 yr-of-age) with healthy gingiva were selected for the experiments after having given their informed consent. A 9mm3 segment of attached gingiva was removed under local anesthesia from the upper incisor of each subject. Samples were then prepared for indirect immunolabelling. For indirect immunolabelling using the peroxidase procedure, the blocks were immediately fixed with 4% paraformaldehyde-O.1 M sodium cacodylate buffer (pH 7.4) for 8 h at 4”C, washed and frozen. Cryostat sections (10 pm) were cut and treated with 0.3% hyaluronidase (bovine testis type I; Sigma, St Louis, MO, U.S.A.) for 30 min at room temperature 223s
RESULTS
In human gingiva (Fig. 1) oxytalan, elaunin and elastic fibres are distributed in the upper, medium and deep layers of gingival connective tissue respectively (Chavrier et al., 1988). The application of anti-elastin antibody clearly demonstrated the presence of elastin in the amorphous and microbrillar components. However, in control sections pre-treated with elastase, specific labelling was abolished in the digested amorphous component and in the microfibrils of oxytalan, elaunin and elastic fibres. DISCUSSION Previous biochemical studies clearly demonstrated that microfibrils were comprised of high molecularweight glycoproteins (Sear, Grant and Jackson, 1981) while the amorphous component was elastin. However, after pre-treatment of the sections with
2245
C. CHAVRIER
Eplthellum
Oxytalan subepitheliol
Glngivol connective tissue
Elounin medium
,;:; :., -II)
fibres layei
fibres
loyer
,..y...? .. .
;:
;I.:
Elastic fi bres deep layer
:...
Al veolor bone
Fig. I. Distribution of the elastic system fibres in healthy human gingival connective
pancreatic elastase, specific labelling was abolished not only in the digested amorphous component but also in the undigested fibrillar component of the oxytalan, elaunin and elastic fibres (Chavrier et al., 1988). This suggests that elastin is only associated with the surface of the microfibrils, which could serve as a site for deposition of elastin during elastogenesis. Recent studies using different anti-elastin antibodies have confirmed the presence of elastin at the surface of the microfibrillar components in different tissues (Fukuda and Ferrans, 1984; Fukuda, Ferrans and Crystal, 1984; Schwartz and Fleishmajer, 1986). Oxytalan, elaunin and elastic fibres represent the consectutive stages of elastogenesis (Gawlik, 1965; Cotta-Pereira et al., 1976; Cotta-Pereira,
REFERENCES
OF
EF
Guerra-Rodrigo and David-Ferreira, 1978). In gingival connective tissue it seems likely that the main source of production of elastin is the fibroblast (Fig. 2). This cell synthesizes elastin as a soluble precursor molecule termed tropo-elastin. The newly synthesized tropo-elastin spontaneously forms insoluble aggregates that are then cross-linked in a series of reactions ‘initiated by lysyl-oxidase. The polymerization of elastin requires the formation of two unique lysine-derived cross-links, namely desmosine and isodesmosine (Gosline and Rosenbloom, 1984). The elastin is deposited at the surface of microfibrils and incorporated in the amorphous area to form elaunin and elastic fibres (Chavrier et al., 1988).
4 FIBROBLAST
Fig. 2. Hypothetical pathway of elastogenesis in human gingival connective tissue: TE, tropoelastin; E, elastin; OF, oxytalan fibres; eF, elaunin fibres; EF, elastic fibres.
Chavrier C., Hartmann D. J., Couble M. L. and Herbage D. (1988) Distribution and organization of the elastic system fibres in healthy human gingiva: ultrastructural and immunohistochemical study. Histochem. J. 89, 47-52. Cotta-Pereira G., Rodrigo F. G. and Bittencourt-Sampaio S. (1976) Oxytalan, elaunin and elastic fibres in human skin. J. Invesf. Dermat. 66, 143-148. Cotta-Pereira G., Guerra-Rodrigo F. G. and DavidFerreira J. F. (1978) Comparative study between the elastic system fibres in human thin and thick skin. Biol. Cell 31, 291-302. Fukuda Y. and Ferrans V. J. (1984) The electron microscopic immunohistochemistry of elastase-treated aorta and nuchal ligament of fetal and postnatal sheep. J. Histochem. Cytochem. 32, 741-756.
Elastin fibres in gingiva Fukuda Y., Ferrans V. J. and Crystal R. G. (1984) Development of elastic fibres of nuchal ligament, aorta and lung of fetal and postnatal sheep: an ultrastructural and electron microscopic immunohistochemical study. Am. J. Anat. 170, 597-629.
Gawlik Z. (1965) Morphological and morphochemical properties of the elastic system in the motor organ of man. Folia Histochem. Cytochem. 3, 233-25
1.
Gosline J. M. and Rosenbloom J. (1984) Elastin in extracellular matrix. In: Biochemistry (Edited by Piez K. A. and Reddi A. H.) pp. 191-227. Elsevier, New York. Graham R. C. and Karnovsky M. J. (1966) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cyto-
225s
chemistry by a new technique. J. Histochem. Cytochem. 14, 291-302.
Page R. C. (1972) In: Developmental Aspects of Oral Biology (Edited by Slavkin H. C. and Baretta L. A.) Vol. I, pp. 291-324. Academic Press, New York. Schluger S., Yuodelis R. A. and Page R. C. (1977) In: Periodontal Disease, pp. 8-37. Lea & Febiger, Philadelphia. Schwartz E. and Fleischmajer R. (1986) Association of elastin with oxytalan fibers of the dermis and with extracellular microfibrils of cultured skin fibroblasts. J. Histochem. Cytochem. 8, 1063-1068.
Sear C. H. J., Grant M. E. and Jackson J. (1981) The nature of the microfibrillar glycoproteins of elastic fibres: a biosynthetic study. Biochem. J. 194, 587-598.
