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Gingival tissue reactions to orthodontic closure of extraction sites
Gingival
tissue reactions to
orthodontic closure of extraction sites Histologic
and histochemical
studies
Assar RBnnerman, D.D.S., Odont.D.*, Birgit Thilander, D.D.S., Odont.D.,** and Guy Heyden, D.D.S., Odont.D.*** Gtiteborg
and Jiinkijping,
Sweden
The formation of u hyperplastic gin&d tissue with invugination wus obsrn~ed in the extraction ureas in seven patients aged 1.2 to 17 years who, qfter c~xtruc’ticm of the maxillary first premolar-s. were undergoing orthodontic treatment with ~717 edgewise appliance. This tissue was excised when about 2 mtn. oj’the space remained to be closed. Histologic und histochemical analyses of the biopsy areas demonstrated hyperplasia ar;d increased metabolism in the invaginuted epithelium NJ ~~41 a.$ increased production of glucose aminoglycans in the surrounding connecti~~c ti,>.\ue. Loss of collagen was noted in the same regions. There WUJ evidence of’ hone remodeling rather than only bone resorption in the biopsy area. It was concluded that stimulation from the orthodontic forces was responsible for the hyperplcrstic tissue reaction und that the increased umounts oj’glucose aminoglyans muy be responsible ,for possible relapses.
Key words: Gingival tissue, orthodontic closure, extraction, histology, histochemistry
A
fter orthodontic closure of an extraction site with a fixed appliance, the gingival tissue as a rule becomes hyperplastic when the space diminishes. The teeth that are moved together thereby push the gingiva in front of them, and a fold or invagination of epithelium and connective tissue is formed. Erikson and associates,j Thompson and colleagues,” and Parker,x among other authors, have stated that compression of the transseptal fibers in man and in the monkey can cause relapse after closure of the extraction site. Picton and Moss9 have also stated that the transseptal fiber system may play an important part in approximal drift. According to Stubley , I0 human transseptal fibers are very tough and resistant. Edwards,4 on the other hand, found that these fibers had a normal morphologic appearance, which may indicate a rapid reorganization and adaptation to new positions of orthodontically approximated teeth. He also found that the keratinization of *Head of the Department of Orthodontics, Institute for Postgraduate Dental Education, S-552 55 Jankiiping. **Professor and Head of the Department of Orthodontics, Faculty of Odontology, University of G&eborg, S-400 33 Giiteborg. ***Professor and Head of the Department of Oral Pathology, Faculty of Odontology, University of Giiteborg, S-400 33 GCteborg.
620
0002~9416/80/@50620+06$00.6010
0
1980 The C. V. Mosby
Co.
Volume 71 Number 6
Orthodontic closure of extraction sites 621
I
la
BONE
Fig. 1. a, Drawing of the biopsy area in the present investigation. b, Survey over the microscopic picture of the biopsy area with indications of the tissue regions to be presented in Figs. 2 to 5. the epithelium increased and that the rete pegs appeared more complex in the cleft area. Morphologically, the connective tissue was highly distorted and signs of inflammation were seen. Therefore, Edwards4 recommended surgical removal of the excess gingival tissue that appears in papillary form buccally and lingually between the teeth that have moved together. This excess tissue has been associated with orthodontic relapse in extraction areas. The aim of the present investigation was to study further, by histologic and histochemical methods, changes in the epithelium and in the connective tissue in gingival areas where orthodontic space closure with a fixed appliance is being performed. Materials and methods The formation of hyperplastic gingival tissue with invagination was observed in the extraction areas in seven patients aged 12 to 17 years who, after extraction of the maxillary first premolars, were undergoing orthodontic treatment with an edgewise appliance (Fig. la). This tissue was excised when about 2 mm. of the space remained to be closed. After local anesthesia, avoiding injection in the proximity of the area to be biopsied, incisions were made buccally and lingually (Fig. la) and the intermediate tissue was extirpated in one piece. To obtain control material, gingival tissue (papillae) was excised from corresponding areas in four patients, 12 to 13 years of age, in connection with extraction before orthodontic treatment. The biopsy specimens were immersed in ice-cold Histocon (Histo-Lab, GGteborg, Sweden) immediately after excision and transported in this solution to the laboratory. The tissue specimens were then mounted on cryostat chucks and frozen in isopentane chilled to about - 140’ C. by means of liquid nitrogen. The frozen tissue was stored in a freezer at -70” C. Sections (8 microns) were cut in a cryostat at - 20” C. The histomorphologic investigations comprised staining of fixed (3 percent glutaraldehyde) cold microtome sections with hematoxylin and eosin and by the hematoxylin-van Gieson method. The chemical component histochemical studies involved staining of neutral polysac-
622 Riinnrrman, Thikmder. rendHtwitw
Fig. 2. Histologic appearance of the invagination area(i) with deep proliferations of the oral epithelium (E). C denotes connective tissue, weakly stained as a result of loss of collagen. (van Gieson stain. Magnification, x 125.) Fig. 3. NADH,-diaphorase recording of the area shown in Fig. 2, showing high levels of oxidative enzyme activity, especially in the hyperplastic basal cell layers (arrowsj. (Magnification, x 125.) charides with the McManus PAS-technique and sulfated polysaccharides (GAG’s) with alcian blue 8 GS at pH 0.5 and 2.5. The cold microtome sections were fixed, as described above, before the staining procedures. The enzyme histochemical analyses were performed on unfixed cold microtome sections. Recordings were made of NADH2- and NADPH,-diaphorase activities,” leucine aminopeptidase activity,7 acid phosphatase activity (azo-dye method),’ and alkaline phosphatase activity (azo-dye method).2 The usual controls for any nonenzymatic staining reactions were performed. Results Clinically, the experimental region was pale to deep red, sometimes with a bluish tint. The light-microscope examinations were focused on any differences between clinically changed tissue and control tissue, The following changes were observed: Histomorphologically , the oral mucosa subjected to the orthodontic forces showed an invagination with strong epithelial hyperplasia (Figs. lb and 2). Deep proliferations of the epithelium occasionally revealed signs of early formation of keratin pearls. The connective tissue in this area showed increased “basophilia” compared with peripheral parts of
Volume
II
Number 6
Orthodontic closure of extraction sites 623
Fig. 4. Alkaline phosphatase activity in the connective tissue below the area of epithelial invagination (Fig. 2). Deep proliferations of oral epithelium (e) lacking enzyme activity are locally surrounding bone spicules (b). It must be noted that only cells with positive reactions of alkaline phosphatase are being stained in the histochemical recording. Negative cells, still present in the tissue section, are therefore not visible in the picture. Because of the sectioning of undecalcified tissue, solitary bone fragments have been lost (sectioning artifacts). (Magnification, x 125.) Fig. 5. Leucine aminopeptidase activity in the area shown in Fig. 4 showing marked enzyme reactions chiefly in juxtaepithelial regions (filled arrows). Connective tissue cells in direct contact with marginal bone do not reveal any marked hydrolytic activity (open arrow). For comments on unstained cells, see Fig. 4. (Magnification, x 125.)
the tissue specimens. Staining by the hematoxylin-van Gieson method demonstrated loss of collagen in the “basophilic” region. The experimental tissue did not show any marked inflammatory reactions. However, the connective tissue was rich in blood vessels and vessel proliferations. The chemical component histochemical investigations indicated increased amounts of, e,specially, hyaluronic acid and occasionally also sulfated mucopolysaccharides in the areas showing loss of collagen (see above). Because of the weak contrasts in black and white reproductions, this increase cannot be satisfactorily demonstrated in the present article. In some specimens increased stainability was also observed with the PAS technique in histomorphologically changed connective tissue regions. Positive PAS reactions suggesting the presence of glycogen in superficial epithelial layers were also observed. The enzyme histochemical analyses demonstrated that the oxidative metabolism
(diaphorase activities) in the epithelium in the invagination area was not inhibited in the experimental situation (Fig. 3). In fact, the reverse reactions (that is, increased oxidative enzyme activities) could sometimes be recorded. In tissue specimens including bone tissue, metabolically active osteoblastemas could be demonstrated. These cells showed strong alkaline phosphatase activity and comparatively low acid phosphatase activity, indicating active bone formation rather than resorption (Figs. 4 and 5). In some specimens the deep epithelial proliferations in the invagination area reached the marginal bone tissue and even surrounded some of the bone projections. There was no evidence of active osteolysis in the area. The comparatively strong activity of leucine aminopeptidase, usually surrounding epithelial proliferations, did not seem to affect the bone tissue, probably because of the local epithelial protection. An increased number of juxtaepithelial blood vessels with high alkaline phosphatase activity were also observed in the experimental regions. Discussion As mentioned in the introduction, one cause of relapse after orthodontic space closure has been related to the compression of the transseptal fibers and their general toughness and resistance. 5, x. lo, ii It appears from the results of the present investigation that one reason for relapse may be an increased appearance of glucose aminoglycans in the intercellular substance of the connective tissue. Such substances may cause u very elastic gelatinous tissue, facilitating relapse ufter the orthodontic closure of the extruction site. As already mentioned, alkaline phosphatase activity was comparably strong at bone surfaces. This indicated that bone remodeling exceeded bone resorption. Erikson and associates”stated that the stress from the compressed gingiva might cause resorption of the underlying bone. Furthermore, Zachrisson and Alnaes,” ‘ in a radiographic study, found slight bone loss after the final closure of the extraction sites, particularly in the pressure zones of the retracted canines. Our investigation, however, has not supported the view of any significant bone resorption. Existing divergencies in results may be due to differences in the type of appliances, treatment time, and forces used as well as in analytical methods employed. It should be emphasized that the histologic and morphologic findings in our investigation were supported by the complementary enzyme histochemical analyses. A radiographic study using the same experimental model6 also supports the present histologic and histochemical results. In conclusion, the lack of inflammatory cells, the ingrowth of blood vessels, and the collagen fibers being replaced by increased amounts of glucose aminoglycans, as found in our study, indicated that the mechanical forces employed caused noninflammatory, sublethal damage, stimulating a hyperplastic tendency of tissue components, except collagen formation, in the area. REFERENCES 1. Barka, T., and Anderson, P. J.: Histochemistry: Theory, practice, and bibliography, New York, 1965, Hoeber Medical Division, Harper & Row, p. 245. 2. Burstone, M. S.: The relationship between fixation and techniques for the histochemical localization of hydrolytic enzymes, J. Histochem. Cytochem. 6:322-328, 1958. 3. Chayen, J., Bitensky, L., and Butcher, R. G.: Practical histochemistry, London, 1973, John Wiley & Sons. 4. Edwards, J. G.: The prevention of relapse in extraction cases, AM. J. ORTHOD. 60:128-141, 1971. 5. Erikson, B. E., Kaplan, H., and Aisenberg, M. S.: Orthodontics and transseptal fibers, AM. J. ORTHOD. 31:1-20,
1945.
Volume 71 Number 6
Orthodontic closure of extraction sites
625
6 Hollender, L., Ronnerman, A., and Ibilander, B.: Marginal bone support, clinical crown length and root resorption in orthodontically treated patients, Eur. J. Orthod. (In press.) 7 Nachlas, M., Monis, B., Rosenblatt, D., and Seligman, A. M.: Improvement of the histochemical locahzation of leucine aminopeptidase with a new substrate, L-leucyl-4methoxy-2-napthylamide, J. Biophys. Biochem. Cytol. 7:261-264, 1960. 8. Parker, G. R.: Transseptal fibers and relapse following bodily retraction of teeth: A histologic study, AM. J. ORTHOD. 61:331-344, 1972. 9. Picton, D. C. A., and Moss, J. P.: The part played by the trans-septal fibre system in experimental approximal drift of the cheek teeth of monkeys (Mucaca irus), Arch. Oral Biol. l&669-680, 1973. 10. Stubley, R.: The influence of transseptal fibers on incisor position and diastema formation, AM. J. ORTHOD. 70:645-662, 1976. 11. Thompson, H. E., Myers, H. I., Waterman, J. M., and Flanagan, V. D.: Preliminary macroscopic observations concerning the potentiality of supra-alveolar collagenous fibers in orthodontics, AM. J. ORTHOD. 44:485-497,
1958.
12. Zachrisson, B. U., and Alnaes, L.: Periodontal condition in orthodontically treated and untreated individuals: Alveolar bone loss; radiographic findings, Angle Orthod. 44~48-55, 1974.
tissue reactions to
orthodontic closure of extraction sites Histologic
and histochemical
studies
Assar RBnnerman, D.D.S., Odont.D.*, Birgit Thilander, D.D.S., Odont.D.,** and Guy Heyden, D.D.S., Odont.D.*** Gtiteborg
and Jiinkijping,
Sweden
The formation of u hyperplastic gin&d tissue with invugination wus obsrn~ed in the extraction ureas in seven patients aged 1.2 to 17 years who, qfter c~xtruc’ticm of the maxillary first premolar-s. were undergoing orthodontic treatment with ~717 edgewise appliance. This tissue was excised when about 2 mtn. oj’the space remained to be closed. Histologic und histochemical analyses of the biopsy areas demonstrated hyperplasia ar;d increased metabolism in the invaginuted epithelium NJ ~~41 a.$ increased production of glucose aminoglycans in the surrounding connecti~~c ti,>.\ue. Loss of collagen was noted in the same regions. There WUJ evidence of’ hone remodeling rather than only bone resorption in the biopsy area. It was concluded that stimulation from the orthodontic forces was responsible for the hyperplcrstic tissue reaction und that the increased umounts oj’glucose aminoglyans muy be responsible ,for possible relapses.
Key words: Gingival tissue, orthodontic closure, extraction, histology, histochemistry
A
fter orthodontic closure of an extraction site with a fixed appliance, the gingival tissue as a rule becomes hyperplastic when the space diminishes. The teeth that are moved together thereby push the gingiva in front of them, and a fold or invagination of epithelium and connective tissue is formed. Erikson and associates,j Thompson and colleagues,” and Parker,x among other authors, have stated that compression of the transseptal fibers in man and in the monkey can cause relapse after closure of the extraction site. Picton and Moss9 have also stated that the transseptal fiber system may play an important part in approximal drift. According to Stubley , I0 human transseptal fibers are very tough and resistant. Edwards,4 on the other hand, found that these fibers had a normal morphologic appearance, which may indicate a rapid reorganization and adaptation to new positions of orthodontically approximated teeth. He also found that the keratinization of *Head of the Department of Orthodontics, Institute for Postgraduate Dental Education, S-552 55 Jankiiping. **Professor and Head of the Department of Orthodontics, Faculty of Odontology, University of G&eborg, S-400 33 Giiteborg. ***Professor and Head of the Department of Oral Pathology, Faculty of Odontology, University of Giiteborg, S-400 33 GCteborg.
620
0002~9416/80/@50620+06$00.6010
0
1980 The C. V. Mosby
Co.
Volume 71 Number 6
Orthodontic closure of extraction sites 621
I
la
BONE
Fig. 1. a, Drawing of the biopsy area in the present investigation. b, Survey over the microscopic picture of the biopsy area with indications of the tissue regions to be presented in Figs. 2 to 5. the epithelium increased and that the rete pegs appeared more complex in the cleft area. Morphologically, the connective tissue was highly distorted and signs of inflammation were seen. Therefore, Edwards4 recommended surgical removal of the excess gingival tissue that appears in papillary form buccally and lingually between the teeth that have moved together. This excess tissue has been associated with orthodontic relapse in extraction areas. The aim of the present investigation was to study further, by histologic and histochemical methods, changes in the epithelium and in the connective tissue in gingival areas where orthodontic space closure with a fixed appliance is being performed. Materials and methods The formation of hyperplastic gingival tissue with invagination was observed in the extraction areas in seven patients aged 12 to 17 years who, after extraction of the maxillary first premolars, were undergoing orthodontic treatment with an edgewise appliance (Fig. la). This tissue was excised when about 2 mm. of the space remained to be closed. After local anesthesia, avoiding injection in the proximity of the area to be biopsied, incisions were made buccally and lingually (Fig. la) and the intermediate tissue was extirpated in one piece. To obtain control material, gingival tissue (papillae) was excised from corresponding areas in four patients, 12 to 13 years of age, in connection with extraction before orthodontic treatment. The biopsy specimens were immersed in ice-cold Histocon (Histo-Lab, GGteborg, Sweden) immediately after excision and transported in this solution to the laboratory. The tissue specimens were then mounted on cryostat chucks and frozen in isopentane chilled to about - 140’ C. by means of liquid nitrogen. The frozen tissue was stored in a freezer at -70” C. Sections (8 microns) were cut in a cryostat at - 20” C. The histomorphologic investigations comprised staining of fixed (3 percent glutaraldehyde) cold microtome sections with hematoxylin and eosin and by the hematoxylin-van Gieson method. The chemical component histochemical studies involved staining of neutral polysac-
622 Riinnrrman, Thikmder. rendHtwitw
Fig. 2. Histologic appearance of the invagination area(i) with deep proliferations of the oral epithelium (E). C denotes connective tissue, weakly stained as a result of loss of collagen. (van Gieson stain. Magnification, x 125.) Fig. 3. NADH,-diaphorase recording of the area shown in Fig. 2, showing high levels of oxidative enzyme activity, especially in the hyperplastic basal cell layers (arrowsj. (Magnification, x 125.) charides with the McManus PAS-technique and sulfated polysaccharides (GAG’s) with alcian blue 8 GS at pH 0.5 and 2.5. The cold microtome sections were fixed, as described above, before the staining procedures. The enzyme histochemical analyses were performed on unfixed cold microtome sections. Recordings were made of NADH2- and NADPH,-diaphorase activities,” leucine aminopeptidase activity,7 acid phosphatase activity (azo-dye method),’ and alkaline phosphatase activity (azo-dye method).2 The usual controls for any nonenzymatic staining reactions were performed. Results Clinically, the experimental region was pale to deep red, sometimes with a bluish tint. The light-microscope examinations were focused on any differences between clinically changed tissue and control tissue, The following changes were observed: Histomorphologically , the oral mucosa subjected to the orthodontic forces showed an invagination with strong epithelial hyperplasia (Figs. lb and 2). Deep proliferations of the epithelium occasionally revealed signs of early formation of keratin pearls. The connective tissue in this area showed increased “basophilia” compared with peripheral parts of
Volume
II
Number 6
Orthodontic closure of extraction sites 623
Fig. 4. Alkaline phosphatase activity in the connective tissue below the area of epithelial invagination (Fig. 2). Deep proliferations of oral epithelium (e) lacking enzyme activity are locally surrounding bone spicules (b). It must be noted that only cells with positive reactions of alkaline phosphatase are being stained in the histochemical recording. Negative cells, still present in the tissue section, are therefore not visible in the picture. Because of the sectioning of undecalcified tissue, solitary bone fragments have been lost (sectioning artifacts). (Magnification, x 125.) Fig. 5. Leucine aminopeptidase activity in the area shown in Fig. 4 showing marked enzyme reactions chiefly in juxtaepithelial regions (filled arrows). Connective tissue cells in direct contact with marginal bone do not reveal any marked hydrolytic activity (open arrow). For comments on unstained cells, see Fig. 4. (Magnification, x 125.)
the tissue specimens. Staining by the hematoxylin-van Gieson method demonstrated loss of collagen in the “basophilic” region. The experimental tissue did not show any marked inflammatory reactions. However, the connective tissue was rich in blood vessels and vessel proliferations. The chemical component histochemical investigations indicated increased amounts of, e,specially, hyaluronic acid and occasionally also sulfated mucopolysaccharides in the areas showing loss of collagen (see above). Because of the weak contrasts in black and white reproductions, this increase cannot be satisfactorily demonstrated in the present article. In some specimens increased stainability was also observed with the PAS technique in histomorphologically changed connective tissue regions. Positive PAS reactions suggesting the presence of glycogen in superficial epithelial layers were also observed. The enzyme histochemical analyses demonstrated that the oxidative metabolism
(diaphorase activities) in the epithelium in the invagination area was not inhibited in the experimental situation (Fig. 3). In fact, the reverse reactions (that is, increased oxidative enzyme activities) could sometimes be recorded. In tissue specimens including bone tissue, metabolically active osteoblastemas could be demonstrated. These cells showed strong alkaline phosphatase activity and comparatively low acid phosphatase activity, indicating active bone formation rather than resorption (Figs. 4 and 5). In some specimens the deep epithelial proliferations in the invagination area reached the marginal bone tissue and even surrounded some of the bone projections. There was no evidence of active osteolysis in the area. The comparatively strong activity of leucine aminopeptidase, usually surrounding epithelial proliferations, did not seem to affect the bone tissue, probably because of the local epithelial protection. An increased number of juxtaepithelial blood vessels with high alkaline phosphatase activity were also observed in the experimental regions. Discussion As mentioned in the introduction, one cause of relapse after orthodontic space closure has been related to the compression of the transseptal fibers and their general toughness and resistance. 5, x. lo, ii It appears from the results of the present investigation that one reason for relapse may be an increased appearance of glucose aminoglycans in the intercellular substance of the connective tissue. Such substances may cause u very elastic gelatinous tissue, facilitating relapse ufter the orthodontic closure of the extruction site. As already mentioned, alkaline phosphatase activity was comparably strong at bone surfaces. This indicated that bone remodeling exceeded bone resorption. Erikson and associates”stated that the stress from the compressed gingiva might cause resorption of the underlying bone. Furthermore, Zachrisson and Alnaes,” ‘ in a radiographic study, found slight bone loss after the final closure of the extraction sites, particularly in the pressure zones of the retracted canines. Our investigation, however, has not supported the view of any significant bone resorption. Existing divergencies in results may be due to differences in the type of appliances, treatment time, and forces used as well as in analytical methods employed. It should be emphasized that the histologic and morphologic findings in our investigation were supported by the complementary enzyme histochemical analyses. A radiographic study using the same experimental model6 also supports the present histologic and histochemical results. In conclusion, the lack of inflammatory cells, the ingrowth of blood vessels, and the collagen fibers being replaced by increased amounts of glucose aminoglycans, as found in our study, indicated that the mechanical forces employed caused noninflammatory, sublethal damage, stimulating a hyperplastic tendency of tissue components, except collagen formation, in the area. REFERENCES 1. Barka, T., and Anderson, P. J.: Histochemistry: Theory, practice, and bibliography, New York, 1965, Hoeber Medical Division, Harper & Row, p. 245. 2. Burstone, M. S.: The relationship between fixation and techniques for the histochemical localization of hydrolytic enzymes, J. Histochem. Cytochem. 6:322-328, 1958. 3. Chayen, J., Bitensky, L., and Butcher, R. G.: Practical histochemistry, London, 1973, John Wiley & Sons. 4. Edwards, J. G.: The prevention of relapse in extraction cases, AM. J. ORTHOD. 60:128-141, 1971. 5. Erikson, B. E., Kaplan, H., and Aisenberg, M. S.: Orthodontics and transseptal fibers, AM. J. ORTHOD. 31:1-20,
1945.
Volume 71 Number 6
Orthodontic closure of extraction sites
625
6 Hollender, L., Ronnerman, A., and Ibilander, B.: Marginal bone support, clinical crown length and root resorption in orthodontically treated patients, Eur. J. Orthod. (In press.) 7 Nachlas, M., Monis, B., Rosenblatt, D., and Seligman, A. M.: Improvement of the histochemical locahzation of leucine aminopeptidase with a new substrate, L-leucyl-4methoxy-2-napthylamide, J. Biophys. Biochem. Cytol. 7:261-264, 1960. 8. Parker, G. R.: Transseptal fibers and relapse following bodily retraction of teeth: A histologic study, AM. J. ORTHOD. 61:331-344, 1972. 9. Picton, D. C. A., and Moss, J. P.: The part played by the trans-septal fibre system in experimental approximal drift of the cheek teeth of monkeys (Mucaca irus), Arch. Oral Biol. l&669-680, 1973. 10. Stubley, R.: The influence of transseptal fibers on incisor position and diastema formation, AM. J. ORTHOD. 70:645-662, 1976. 11. Thompson, H. E., Myers, H. I., Waterman, J. M., and Flanagan, V. D.: Preliminary macroscopic observations concerning the potentiality of supra-alveolar collagenous fibers in orthodontics, AM. J. ORTHOD. 44:485-497,
1958.
12. Zachrisson, B. U., and Alnaes, L.: Periodontal condition in orthodontically treated and untreated individuals: Alveolar bone loss; radiographic findings, Angle Orthod. 44~48-55, 1974.