/. Periodonlal Res. 13'. 513-524. 1978
Experimental cellular immune reactions in the gingiva of beagledogs H. RYLANDER, R. ATTSTROM, J. LINDHE AND N . NOBREUS
Departments of Periodontology, Universities of Goteborg and Lund, Sweden and Royal Dental College, Aarhus, Denmark Four Beagle dogs were sensitized to l-dinitro-2.4 chlorobenzene (DTSCB) during a preexperimental period of three months. The animals were fed a hard diet and subjected to plaque removal three times weekly throughout the study. During the experimental period of 75 days DNCB was applied to the gingiva three times weekly on one side of the jaws and the vehicle (Orabase) was applied on the other. Gingivai biopsies were taken on day 75 of the experiment. Semithin sections were subjected to light microscopic analysis using morphometric point counting procedures. The morphometric data showed that the gingiva on the experimental (DNCB) side contained leukocyte infiltrates dominated by mononuclear inflammatory cells, in the marginal oral epithelium, junctions] epithelium as well as in the connective tissue immediately below these epithelia. The oral epithelium was the site of severe alterations such as intercellular edema and lack of a stratum corneum. In the diseased portion of the cormective tissue the establishment of a lymphocyte-mactophage dominated infiltrate was secured simultaneously with a drastic reduction of the collagen content. The results of the study indicate that the reaction elicited in the tissues was related to delayed hypersensitivity and that the establishment of an experimental cellular immune reaction caused damage to the gingival structures. (Accepted for publication April 20, 1977)
Introduction
In the early plaque induced gingival lesion, lymphocytes predominate in the inflammatory cell infiltrate which is iocated in the connective tissue subjacent to the junctional epithelium (Zachrisson 1968, Listgarten & Ellegaard 1973, Payne et al. 1975, Schroeder et al. 1973a). It has heen suggested that lymphocytes within the gingival infiltrate are involved in delayed hypersenitivity reactions and that such T-cell-delendent reactions may play an important 'ole in connective tissue destruction in gin;;ivitis and periodontitis (Schroeder et al.
1973a, Schroeder & Page 1972. Horton et al. 1974, Page & Schroeder 1976). Indeed, in vitro studies hy Ivanyi & Lehneh (1970, 1971), Ivanyi et al. (1972), Horton, Oppenheim & Mergenhagen (1973, 1974), Lehner et al. (1974), Lang (1975) and others have shown that cell-mediated imnaunity may play a role in the initiation and further development of periodontitis in humans. In dogs with plaque-dependent gingivitis, short-term immunosuppression by antithymocyte serum gave some alterations of the cellular composition of the connective tissue infiltrate, but did not influence the clinical intensity of gingivitis as measured
514
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by amounts of crevicular leukocytes and gingival fluid (Nobreus. Attstrom & Egelberg 1974a, b). These results were interpreted to indicate that cellular immunity plays a minor role in the pathogenesis of vascular and cellular changes in gingivitis. Recent experiments, however, have revealed that the number of crevicular leukocytes and the amount of gingival exudate not only indicate the presence (and size) of an infiltrate in the gingival connective tissue but also reflect the presence of chemotactic and permeability-inducing factors in the gingival sulcus (Hellden & Lindhe 1973, Hellden & Kahnberg 1973, Kahnberg et al. 1976). Short-term experiments in dogs have shown that experimental contact hypersensitivity reactions elicited in normal gingiva of sensitized animals gave rise to symptoms of gingivitis (Nobreus & Attstrom 1974). Immuno-suppression by anti-thymocyte serum decreased the intensity of these experimental gingival lesions both clinically and histopathoiogically. Contact hypersensitivity reactions in the gingiva during long time may cause a tissue damage comparable to lesions in plaqueinduced gingivites and periodontitis. The aim of the present study was to characterize histopathoiogically the gingival lesion following prolonged gingival challenge with DNCB in sensitized Beagle dogs with healthy gingiva. Material and Methods Animals: Four one-year-old Beagle dogs were used. Pre-experimental period (Day -90 to Day 0), On day -90 the teeth were thoroughly scaled and polished and the animals were from this day and onward given a hard diet {Egelberg 1965) which did not allow gross plaque formation. In addition the teeth were clean-
ET
A L .
ed three times weekly with rubber cups and pumice. The dogs were sensitized against l-chloro-2.4-dinitrochIoro benzene (DNCB) as described previously (Nobreus et al. 1974c). Contact hypersensitivity to DNCB in the animals was assessed by skin tests (Nobreus et al. 1974c) on days 0, 25, 50 and 75 of the experiment. During the pre-experimental period the condition of the gingiva was assessed weekly as described by Attstrom, Graf de Beer & Schroeder (1975). At the start of the experiment (day 0), each animal had a normal gingiva according to the clinical criteria of Attstrom et al. (1975). The various operative procedures in the animals before day 0 were carried out under intravenous anesthesia with 0.5 ml/kg bodyweight Penthotal sodium*. During the period day 0 to day 75 the anesthesia was injected intraperitoneally at a dosage of 1 ml/kg bodyweight to keep the animals immobilized for a period of about 4 hours. Experimental period (Day 0 to Day 75) On day 0 a paste designed to adhere to mucous membranes (Orahase®, Squibb and Sons, England) containing 0.05 % DNCB was applied to the buccal marginal gingiva from the second premolars to the first molars on the right side of the jaws (P^j, P-g, PSg, P*4, Ml). Orabase without DNCB was applied to the contralateral side. Precautions were taken to avoid contralateral DNCB contamination. Prior to application the teeth were cleaned as described above. The applications were performed three times weekly for a duration of at least three hours, during which time the animals were kept sleeping. The experimental period lasted 75 days and included 33 applications. The clinical condition of the gingiva was assessed from color photos obtained at weekly intervals from day 0 to day 75. Probing into the junctional epithelium was * Abbot, Belgium.
C E L L U L A R
I M M U M E
R E A C T I O N S
I N
G I N G I V A
microscope. The morphometric determinations of various structural tissue components were made at three levels (I, 111 and IV) as described by Schroeder & Munzel-Pedrazzoli (1973) and Lindhe & Rylander (1975). Each biopsy was divided into four blocks and two sections from one block picked at random were selected for analyses. 16 experimental and 16 control sections were analyzed from each dog. At level I (Magn. 200 X, lattice with 42 points) a morphometric anaiysis of the cross-section of the free part of the gingival tissue (GT) was undertaken. The volumetric density was determined for oral epithelium (OE), junctional epithelium (JE), non-infiltrated connective tissue (NCT) as well as for the infiltrated connective tissue (ICT) below the oral epithelium (IOTQE) and below the junctional epithelium (ICTjj.^) (Fig. ]).
Fig. 1. Schematic illuslration of the dei..,.. a^-a region. The oral epithelium (OE) and the junctional epithelium (JE) were divided into three portions of equal vertical height (area A, B and C). ICToi;, ICTip.: infiifrated connective tissue below the oral and the junctional epithelium lesp. NCT: non-infiltrated connective tissue. CEJ: cemenio-enamel junction. AB: alveolar bone.
avoided in order to prevent the penetration of DNCB through a hroken epithelial lining. During the cleaning procedures trauma to the gingival margin was kept to a minimum. On day 75, gingival biopsies were harvested from premolars and molars in the upper and lower jaws (^P', ^P^g, ^gPSg, 't^V\, jMj). The biopsies consisted of the free and part of the attached gingival tissue and a buccal portion of the tooth and alveolar bone. The biopsy procedure and the histological processing of the tissues were done according to Schroeder et al. (1973b). ^'orphometric analyses of gingival tissues T le hucco-Iingual cross sections (2—3 ^im ^1 ick) were analyzed with a Wild® sampling
K\ \e-*>ul \\\ (Magn. iOOO X, Valtice v*'ith 400 points) the volumetric densities were determined for neutrophilic granulocytes (NG) and monocytes/macrophages (MO) residing within the junctional epithelium and oral epithelium. The analyses were performed in three different regions of equal vertical height, A, B and C as shown in Fig. 1. At level IV (Magn. 1000 X, lattice with 100 points) the structural composition was determined for the infiltrated connective tissue portion below the oral epithelium (ICTQE) and the junctional epithelium (ICTjj^), as well as the non-infiltrated connective tissue. The volumetric density of the following parameters were determined: collagen fibers (CO), fihroblasts (Fi), neutrophiiic granuiocytes (NG), monocytes, lymphocytes and macrophages (MO), plasma cells (PC), vessels (V) and residual tissue (R: lymphatic vessels, nerves, unidentified cells/structures and ground substance). In summary: The overall composition of the gingival tissue (GT) was determined at
m
Fig. 2. Cross-sections of the fre ICToi;, iCTir: the infiltrated con NCT: non-infiltrated connective
RYLANDER
ET
AL.
representing Day 75. A: DNCB side. B; control side, area below ttie oral epittielium and the juhctional epilhelii
Level I, the size and volumetric density of OE and JE as well as the volumetric density of leukocytes in OE and JE were determined at Level UL The size and composition of ICToE, ICTjK and NCT were assessed at Level IV. The experimental design allowed for a comparison between contralateral biopsy sections. Hence, the units to which DNCB had been applied could be compared with the contralateral units which received only the vehicle for DNCB. Volumetric density and percentages are given as average (X + SD) values per dog. Statistical anal)'ses were performed using Student's t-test for intraindividual differences.
Skin biopsies Skin biopsies obtain L-d from DNCB-test sites following the sensitizing procedure and on days 0, 25, 50 and 75 den^onstrated large numbers of mononuclear inflammatory cc^ls in the connective tissue adjacent to the: epithelium. Also the epithelium showed signs of intraepithelial edema. In biopsies taken from control sites no reactions were observed. The skin reactions were identical to those described previously (Nobreus et al. 1974c) and indicated that all dogs were sensitized to DNCB at the start of the experiment and that the sensitivity was maiitained during the experimental period.
C E L L U L - A R
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ig. 3. Higher magnification of the c epithelium from the DNCB section (A and C) and from the control section (B and D).
Clinical observations A; the start of the experiment (day 0) the gi'igivae of all dogs were in a healthy state. Aready after I week of DNCB applicatif-ns the first minor signs of gingivites (i.e. re 'ness and swelling) was seen and on day 75 the buccal gingivai margins as well as
a part of the alveolar mucosa in the DNCBtreated regions were markedly inflamed. On the control sides no signs of inflammation could be noted. General morphology Experimental regions: The sections from
Fig. 4. Cross-section of the fre© gmgival u microbial plaque was located in ths gingiv^ lo the microbial plaque (arrow Fig. B).
representing Day 75 of the control side in s biopsy where a thin auicus. Note the accumulation of leukocytes in the sulcus apical
DNCB-treated gingival regions exhibited signs of inflammation in the connective tissue subjacent to the oral and junctiona! epithelium. Infiltrates of leukocytes were consistently present at these locations. The ICTju extended apically to and in some sections even below the cemento-enamel junction (Fig. 2 A). The oral epithelium in the marginal third of the gingiva frequently lacked a fully developed keratin layer, instead a nonkeratinezed or parakeratotic layer could be observed. An undulating interface occurred between the junctional epithelium and the connective tissue (Fig. 2 A, 3 A). The marginal oral epithelium as well as the junctional epithelium harboured inflammatory cell infiltrates consisting predominantly of
mononuclear leukocytes (Figs. 2 A, 3 A and 3 C). ln areas of heavy leukocyte infiltration in the oral epithelium, acanthosis and intercellular edema were observed (Fig. 3 C). In the midportion of the connective tissue of the free gingiva a dense network of collagen fiber bundles was present. In the regions of ieukocyte infiltration in the connective tissue few collagen fiber bundles could be observed. Also, in these areas numerous vessels were present (Fig. 3 A). In around 40 per cent of the sections a thin microbial plaque was observed. The plaque extended approximately 0.5 mm subgingivally. Control regions: In 60 per cent cf the control sections the buccal gingival connei tve
C E L L U L A R
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R E A C T I O N S
IN
G I N G I V A
5
Table 1 Structural composition of gingivai tissue (GT) in DNCB sections, all control sections and control sections with and without a celluSar intiUrate subjacent the junctional epithelium. The data are expressed as mmVlOO mm^ GT. X arithmetical mean S.D. standard deviation Conf ml s,ecti ons al
DNCE3 iectioi ns
PEiramelter X
S. D.
X
Cont rol sections w ith ICTii.:
S .D.
S.D.
X
Contmi s
Experimental cellular immune reactions in the gingiva of beagledogs H. RYLANDER, R. ATTSTROM, J. LINDHE AND N . NOBREUS
Departments of Periodontology, Universities of Goteborg and Lund, Sweden and Royal Dental College, Aarhus, Denmark Four Beagle dogs were sensitized to l-dinitro-2.4 chlorobenzene (DTSCB) during a preexperimental period of three months. The animals were fed a hard diet and subjected to plaque removal three times weekly throughout the study. During the experimental period of 75 days DNCB was applied to the gingiva three times weekly on one side of the jaws and the vehicle (Orabase) was applied on the other. Gingivai biopsies were taken on day 75 of the experiment. Semithin sections were subjected to light microscopic analysis using morphometric point counting procedures. The morphometric data showed that the gingiva on the experimental (DNCB) side contained leukocyte infiltrates dominated by mononuclear inflammatory cells, in the marginal oral epithelium, junctions] epithelium as well as in the connective tissue immediately below these epithelia. The oral epithelium was the site of severe alterations such as intercellular edema and lack of a stratum corneum. In the diseased portion of the cormective tissue the establishment of a lymphocyte-mactophage dominated infiltrate was secured simultaneously with a drastic reduction of the collagen content. The results of the study indicate that the reaction elicited in the tissues was related to delayed hypersensitivity and that the establishment of an experimental cellular immune reaction caused damage to the gingival structures. (Accepted for publication April 20, 1977)
Introduction
In the early plaque induced gingival lesion, lymphocytes predominate in the inflammatory cell infiltrate which is iocated in the connective tissue subjacent to the junctional epithelium (Zachrisson 1968, Listgarten & Ellegaard 1973, Payne et al. 1975, Schroeder et al. 1973a). It has heen suggested that lymphocytes within the gingival infiltrate are involved in delayed hypersenitivity reactions and that such T-cell-delendent reactions may play an important 'ole in connective tissue destruction in gin;;ivitis and periodontitis (Schroeder et al.
1973a, Schroeder & Page 1972. Horton et al. 1974, Page & Schroeder 1976). Indeed, in vitro studies hy Ivanyi & Lehneh (1970, 1971), Ivanyi et al. (1972), Horton, Oppenheim & Mergenhagen (1973, 1974), Lehner et al. (1974), Lang (1975) and others have shown that cell-mediated imnaunity may play a role in the initiation and further development of periodontitis in humans. In dogs with plaque-dependent gingivitis, short-term immunosuppression by antithymocyte serum gave some alterations of the cellular composition of the connective tissue infiltrate, but did not influence the clinical intensity of gingivitis as measured
514
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by amounts of crevicular leukocytes and gingival fluid (Nobreus. Attstrom & Egelberg 1974a, b). These results were interpreted to indicate that cellular immunity plays a minor role in the pathogenesis of vascular and cellular changes in gingivitis. Recent experiments, however, have revealed that the number of crevicular leukocytes and the amount of gingival exudate not only indicate the presence (and size) of an infiltrate in the gingival connective tissue but also reflect the presence of chemotactic and permeability-inducing factors in the gingival sulcus (Hellden & Lindhe 1973, Hellden & Kahnberg 1973, Kahnberg et al. 1976). Short-term experiments in dogs have shown that experimental contact hypersensitivity reactions elicited in normal gingiva of sensitized animals gave rise to symptoms of gingivitis (Nobreus & Attstrom 1974). Immuno-suppression by anti-thymocyte serum decreased the intensity of these experimental gingival lesions both clinically and histopathoiogically. Contact hypersensitivity reactions in the gingiva during long time may cause a tissue damage comparable to lesions in plaqueinduced gingivites and periodontitis. The aim of the present study was to characterize histopathoiogically the gingival lesion following prolonged gingival challenge with DNCB in sensitized Beagle dogs with healthy gingiva. Material and Methods Animals: Four one-year-old Beagle dogs were used. Pre-experimental period (Day -90 to Day 0), On day -90 the teeth were thoroughly scaled and polished and the animals were from this day and onward given a hard diet {Egelberg 1965) which did not allow gross plaque formation. In addition the teeth were clean-
ET
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ed three times weekly with rubber cups and pumice. The dogs were sensitized against l-chloro-2.4-dinitrochIoro benzene (DNCB) as described previously (Nobreus et al. 1974c). Contact hypersensitivity to DNCB in the animals was assessed by skin tests (Nobreus et al. 1974c) on days 0, 25, 50 and 75 of the experiment. During the pre-experimental period the condition of the gingiva was assessed weekly as described by Attstrom, Graf de Beer & Schroeder (1975). At the start of the experiment (day 0), each animal had a normal gingiva according to the clinical criteria of Attstrom et al. (1975). The various operative procedures in the animals before day 0 were carried out under intravenous anesthesia with 0.5 ml/kg bodyweight Penthotal sodium*. During the period day 0 to day 75 the anesthesia was injected intraperitoneally at a dosage of 1 ml/kg bodyweight to keep the animals immobilized for a period of about 4 hours. Experimental period (Day 0 to Day 75) On day 0 a paste designed to adhere to mucous membranes (Orahase®, Squibb and Sons, England) containing 0.05 % DNCB was applied to the buccal marginal gingiva from the second premolars to the first molars on the right side of the jaws (P^j, P-g, PSg, P*4, Ml). Orabase without DNCB was applied to the contralateral side. Precautions were taken to avoid contralateral DNCB contamination. Prior to application the teeth were cleaned as described above. The applications were performed three times weekly for a duration of at least three hours, during which time the animals were kept sleeping. The experimental period lasted 75 days and included 33 applications. The clinical condition of the gingiva was assessed from color photos obtained at weekly intervals from day 0 to day 75. Probing into the junctional epithelium was * Abbot, Belgium.
C E L L U L A R
I M M U M E
R E A C T I O N S
I N
G I N G I V A
microscope. The morphometric determinations of various structural tissue components were made at three levels (I, 111 and IV) as described by Schroeder & Munzel-Pedrazzoli (1973) and Lindhe & Rylander (1975). Each biopsy was divided into four blocks and two sections from one block picked at random were selected for analyses. 16 experimental and 16 control sections were analyzed from each dog. At level I (Magn. 200 X, lattice with 42 points) a morphometric anaiysis of the cross-section of the free part of the gingival tissue (GT) was undertaken. The volumetric density was determined for oral epithelium (OE), junctional epithelium (JE), non-infiltrated connective tissue (NCT) as well as for the infiltrated connective tissue (ICT) below the oral epithelium (IOTQE) and below the junctional epithelium (ICTjj.^) (Fig. ]).
Fig. 1. Schematic illuslration of the dei..,.. a^-a region. The oral epithelium (OE) and the junctional epithelium (JE) were divided into three portions of equal vertical height (area A, B and C). ICToi;, ICTip.: infiifrated connective tissue below the oral and the junctional epithelium lesp. NCT: non-infiltrated connective tissue. CEJ: cemenio-enamel junction. AB: alveolar bone.
avoided in order to prevent the penetration of DNCB through a hroken epithelial lining. During the cleaning procedures trauma to the gingival margin was kept to a minimum. On day 75, gingival biopsies were harvested from premolars and molars in the upper and lower jaws (^P', ^P^g, ^gPSg, 't^V\, jMj). The biopsies consisted of the free and part of the attached gingival tissue and a buccal portion of the tooth and alveolar bone. The biopsy procedure and the histological processing of the tissues were done according to Schroeder et al. (1973b). ^'orphometric analyses of gingival tissues T le hucco-Iingual cross sections (2—3 ^im ^1 ick) were analyzed with a Wild® sampling
K\ \e-*>ul \\\ (Magn. iOOO X, Valtice v*'ith 400 points) the volumetric densities were determined for neutrophilic granulocytes (NG) and monocytes/macrophages (MO) residing within the junctional epithelium and oral epithelium. The analyses were performed in three different regions of equal vertical height, A, B and C as shown in Fig. 1. At level IV (Magn. 1000 X, lattice with 100 points) the structural composition was determined for the infiltrated connective tissue portion below the oral epithelium (ICTQE) and the junctional epithelium (ICTjj^), as well as the non-infiltrated connective tissue. The volumetric density of the following parameters were determined: collagen fibers (CO), fihroblasts (Fi), neutrophiiic granuiocytes (NG), monocytes, lymphocytes and macrophages (MO), plasma cells (PC), vessels (V) and residual tissue (R: lymphatic vessels, nerves, unidentified cells/structures and ground substance). In summary: The overall composition of the gingival tissue (GT) was determined at
m
Fig. 2. Cross-sections of the fre ICToi;, iCTir: the infiltrated con NCT: non-infiltrated connective
RYLANDER
ET
AL.
representing Day 75. A: DNCB side. B; control side, area below ttie oral epittielium and the juhctional epilhelii
Level I, the size and volumetric density of OE and JE as well as the volumetric density of leukocytes in OE and JE were determined at Level UL The size and composition of ICToE, ICTjK and NCT were assessed at Level IV. The experimental design allowed for a comparison between contralateral biopsy sections. Hence, the units to which DNCB had been applied could be compared with the contralateral units which received only the vehicle for DNCB. Volumetric density and percentages are given as average (X + SD) values per dog. Statistical anal)'ses were performed using Student's t-test for intraindividual differences.
Skin biopsies Skin biopsies obtain L-d from DNCB-test sites following the sensitizing procedure and on days 0, 25, 50 and 75 den^onstrated large numbers of mononuclear inflammatory cc^ls in the connective tissue adjacent to the: epithelium. Also the epithelium showed signs of intraepithelial edema. In biopsies taken from control sites no reactions were observed. The skin reactions were identical to those described previously (Nobreus et al. 1974c) and indicated that all dogs were sensitized to DNCB at the start of the experiment and that the sensitivity was maiitained during the experimental period.
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ig. 3. Higher magnification of the c epithelium from the DNCB section (A and C) and from the control section (B and D).
Clinical observations A; the start of the experiment (day 0) the gi'igivae of all dogs were in a healthy state. Aready after I week of DNCB applicatif-ns the first minor signs of gingivites (i.e. re 'ness and swelling) was seen and on day 75 the buccal gingivai margins as well as
a part of the alveolar mucosa in the DNCBtreated regions were markedly inflamed. On the control sides no signs of inflammation could be noted. General morphology Experimental regions: The sections from
Fig. 4. Cross-section of the fre© gmgival u microbial plaque was located in ths gingiv^ lo the microbial plaque (arrow Fig. B).
representing Day 75 of the control side in s biopsy where a thin auicus. Note the accumulation of leukocytes in the sulcus apical
DNCB-treated gingival regions exhibited signs of inflammation in the connective tissue subjacent to the oral and junctiona! epithelium. Infiltrates of leukocytes were consistently present at these locations. The ICTju extended apically to and in some sections even below the cemento-enamel junction (Fig. 2 A). The oral epithelium in the marginal third of the gingiva frequently lacked a fully developed keratin layer, instead a nonkeratinezed or parakeratotic layer could be observed. An undulating interface occurred between the junctional epithelium and the connective tissue (Fig. 2 A, 3 A). The marginal oral epithelium as well as the junctional epithelium harboured inflammatory cell infiltrates consisting predominantly of
mononuclear leukocytes (Figs. 2 A, 3 A and 3 C). ln areas of heavy leukocyte infiltration in the oral epithelium, acanthosis and intercellular edema were observed (Fig. 3 C). In the midportion of the connective tissue of the free gingiva a dense network of collagen fiber bundles was present. In the regions of ieukocyte infiltration in the connective tissue few collagen fiber bundles could be observed. Also, in these areas numerous vessels were present (Fig. 3 A). In around 40 per cent of the sections a thin microbial plaque was observed. The plaque extended approximately 0.5 mm subgingivally. Control regions: In 60 per cent cf the control sections the buccal gingival connei tve
C E L L U L A R
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R E A C T I O N S
IN
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5
Table 1 Structural composition of gingivai tissue (GT) in DNCB sections, all control sections and control sections with and without a celluSar intiUrate subjacent the junctional epithelium. The data are expressed as mmVlOO mm^ GT. X arithmetical mean S.D. standard deviation Conf ml s,ecti ons al
DNCE3 iectioi ns
PEiramelter X
S. D.
X
Cont rol sections w ith ICTii.:
S .D.
S.D.
X
Contmi s
