Archs oral Bml. Vol.
22. pp. 167 to 174. Pergamon
Press 1977. Prmted
I” Great Br,ta,n
A HISTOLOGICAL INVESTIGATION OF THE INFLUENCE OF ADULT PORCINE GINGIVAL CONNECTIVE TISSUES IN DETERMINING EPITHELIAL SPECIFICITY T. G. HEANEY School of Dental Surgery, University of Liverpool, Pembroke Place, P.O. Box 147, Liverpool L69 3BX, England Summary-The influence of gingival lamina propria, and periosteum isolated from the gingival zone, on epithelial differentiation and organization was inliestigated. Open-fit autogenous free gingival connective tissue grafts and recombinant grafts composed of alveolar mucosal epithehum with gingival connective tissue or periosteum were placed on split-thickness dorsal skin sites together with control transplants of gingiva and alveolar mucosa, to monitor the effects of environmental stimuli. The control grafts adapted to their new environment in consistent and well-defined fashions. The behaviour of the experimental grafts, when compared with the controls, sustained the premise that gingival epithelial specificity in the pig is controlled by lamina propria, but no evidence ‘was found to indicate that periosteum has a similar role for epithelium. It was concluded that increases in the functional width of attached gingiva can be achieved most predictably in human clinical practice, by using pedicle or free gingival transplants to redistribute existing gingival lamina propria.
INTRODUCTION The importance of embryonic mesenchyme-epithelium inductive mechanisms in controlling histogenesis and morphogenesis is well established. Interactions of this type have been demonstrated in widely differing vertebrate organ rudiments including kidney and salivary gland (Grobstein, 1954) thymus (Auerbach, 1960), skin (Wessells, 1962) and tooth (Kollar and Baird, 1970; Koch, 1972). Much less is understood of the part played by the interactions of epithelium and connective tissue in maintaining differentiation and organization in the adult state. Nonetheless, it is (clear that maintenance of the specificity of some adult epithelia, including those of epidermis and dermal appendages, requires continuing stimuli from the dermis (Cohen, 1965; Billingham and Silvers, 1967; Briggaman and Wheeler, 1968). Other adult epithelia such as those of tongue, oesophagus and hamster cheek pouch apparently possess an intrinsic capacity to regulate their individual specificities and are. therefore, resistant to any connective tissue influence (Billingham and Silvers. 1967; Beer and Billingham, 1970). There is doubt about the category in which gingival tissue belongs, as Karring. Lang and Liie (1975) have recently shown that gingival lamina propria in cercopithicids may determine the differentiative pattern of the overlying epithelium, although earlier studies by Bernimoulin and Lange (1973) and by Oliver (1973) suggested that gingival epithelial specificity is an innate property. The present study was mounted to explore the possibility that the differentiation of adult gingival epithelium might be under connective tissue control, and also to show whether any such inductive control was
primarily resident in the superficial layers of lamina propria, or could be demonstrated also at deeper connective tissue levels. MATERIALS AND
METHOD
Four young domestic pigs, each weighing between 18-26 kg at the start of an experiment, and fed on a standard laboratory diet, were used. Separate strips of buccal attached gingiva and of alveolar mucosa each approximately 2 mm thick and 4 mm wide, were excised with a scalpel from each animal under general anaesthesia. Separation of these specimens into epithelial and connective tissue components was achieved by incubation in a sterile 2 per cent (w/v) solution of crude trypsin (Trypsin from Beef Pancreas, B.D.H. Chemicals Ltd., Poole, England), in Hank’s balanced salt solution (B.S.S.), and containing 0.6 mg penicillin G per ml. Incubation was carried out for 6 to 8 h at 4°C and within a pH range of 7.2 to 7.8, as described by Billingham and Silvers (1967). After incubation, the specimens were washed for 30min at 4°C in sterile Hank’s B.S.S. adjusted to the same pH range and penicillin concentration, but containing 20 per cent calf serum to inactivate residual trypsin (Rinaldini, 1958). Separation of the epithelium and connective tissue was then performed aseptically under a dissecting microscope, using fine tissue forceps while the specimens were still in the washing fluid. Separation at the level of the epithelialconnective tissue interface was assessed visually in the first instance, and confirmed subsequently by histological examination of samples taken from components of each mucosal strip. The remainders of the components were cut into 4mm squares and used to form the following test 1681
168
T. G. Heaney
and control recombinant grafts: (a) The possible effects of the trypsinization procedure were controlled by reforming the original anatomical epithelial-connective tissue partnership of some specimens of each mucosal type. (b) Test grafts for assessment of connective tissue inductive potential were made by recombining gingival connective tissue with alveolar mucosal epithelium. The epithelium was either placed in contact with the superficial connective tissue surface or the orientation of the connective tissue sheet was altered by turning it upside down, and epithelium laid on its anatomically deep surface. (c) Test grafts were prepared by pairing alveolar mucosal epithelium with periosteum isolated from the attached gingival zone. These periosteal components were prepared by excising full-thickness strips of attached gingiva and cutting them into specimens 4mm square. The deepest connective tissue layer of each was then removed to an even thickness of l-2mm and the rest discarded. The graft itself was formed by reversing this connective tissue layer and placing the epithelial sheet in contact with its periosteal surface. (d) Some free grafts of gingival connective tissue alone were used. These were of the superficial rather than the deeper layers of gingival lamina propria, and were derived from trypsinized split-thickness gingival specimens. Care was taken when forming recombinant grafts to ensure that the deep surface of epithelium was placed in contact with the requisite connective tissue surface. Once formed, orientation was maintained by storing them at 4°C in individual wells in a sterile transport chamber designed for this purpose, and filled with sterile fluid of the same composition as the washing fluid. The test and control grafts were finally transplanted as autogenous grafts on split-thickness dorsal skin beds, under a second general anaesthetic. They were placed as open-fit grafts, that is separated from one another and from the adjacent skin by wide zones of raw dermis. At the same time, further freshly cut gingival and alveolar mucosal grafts were placed on the recipient bed. These served as controls against histological changes induced by environmental variations. The grafts were dressed with a layer of sterile tulle gras overlaid by an adhesive surgical dressing. Additional protection against physical trauma was provided by covering the operative site with several layers of elastic adhesive bandage wound around the body of the animal. The numbers of the various types of graft employed were as follows: 8 trypsin controls each of gingiva and alveolar mucosa, 8 environmental controls each of gingiva and alveolar mucosa, 11 test recombinants of non-inverted gingival lamina propria with alveolar mucosal epithelium, 8 test recombinants of inverted gingival lamina propria with alveolar mucosal epithelium, 11 test recombinants of gingival periosteum with alveolar mucosal epithelium, 17 free gingival connective tissue grafts. The animals were killed 3 to 4 months after transplantation and the grafts were excised together with further intraoral specimens of attached gingiva and alveolar mucosa. After fixation in 10 per cent formolsaline, the grafts were processed, serially sectioned at 6pm and stained with haematoxylin and eosin, Wei-
gert’s method for elastic fibres counterstained with Van Gieson’s stain, Gordon and Sweets reticulin stain (1936), and the periodic acid-Schiff (P.A.S.) reaction (McManus, 1946). RESULTS
Macroscopic
appearance
The naked-eye appearance of all grafts, irrespective of their constitution, was very similar at the time of death. They were evident as circular surface elevations on the skin and were frequently covered by a rough brown scab. However, some of the grafts which contained gingival connective tissue showed a heavy flaky keratin layer instead. Although no attempt was made to measure growth rates in the grafts, most seemed to have undergone little or no change in size at the end of the experimental period, by which time each animal had doubled its body weight. Histological
appearances
Intraoral attached gingiua and alwolar mucosa. The distributions of collagen, elastic and subepithelial reticulin fibres in porcine gingiva and alveolar mucosa corresponded closely to those reported in human and macaque monkey material (Orban and Sicher, 1945; Lozdan and Squier, 1969; Karring, Ostergaard and Lee, 1971). At equal magnifications, the subepithelial reticulin fibre network of skin was of intermediate prominence, and was much better developed than that of alveolar mucosa, but not so conspicuous as that of attached gingiva. However, the distinguishing features of porcine gingival and alveolar mucosal epithelia differed from those which have been found in the equivalent human or monkey epithelia (Orban and Sicher, 1945; Trott, 1957; Karring et at., 1971; Smith C. J., 1971). In particular, the rete pegs of alveolar mucosal epithelium were much longer, more prominent, and showed a greater degree of outline irregularity than those of gingiva (Fig. 1). Attached gingival epithelium was always fully orthokeratinized and the keratin layer was strongly P.A.S.-positive. At high magnifications, the most intense reaction occurred in the zones separating individual layers of keratin although the latter also gave a strong positive response. The cells of the stratum spinosum did not react to the P.A.S. stain although the intercellular material was weakly positive. Environmental control grafts. Environmental control grafts of gingiva and alveolar mucosa showed several well-defined modifications in histological appearance when compared with their intraoral appearance. The major changes in both tissue types appeared to be confined to the epithelium. Attached gingiua. Attached gingival epithelium demonstrated a marked hyperkeratosis associated with the loss of the P.A.S.-positive reaction of the keratin, the appearance of an incomplete stratum granulosum and the occurrence of a more variable rete peg morphology (Fig. 2). The epithelium was still mostly orthokeratinized, although occasional small, limited areas of parakeratosis occurred in some specimens. There was a tendency for rete pegs to become broader or parallel-sided with rounded ends, and for the stratum granulosum to become thrown into folds
Specificity of adult porcine gingival epithelium which were mirrored by similar folds in the keratin surface (Fig. 2) although this was not found in all specimens. In sections stained by the Weigert and Van Gieson techniques, the graft connective tissue was clearly demarcated from the surrounding dermis by its palerstaining collagen bundles with their different pattern of orientation and by the absence of associated elastic fibres (Fig. 3). The d!stinction between graft and skin was further emphasin.ed by the persistence of the characteristically prominent subepithelial reticulin of the gingiva (Fig. 4) contrasting with that of the skin (Fig. 5). Alveolar mucosa. When alveolar mucosa was transferred to a cutaneous environment, the epithelium underwent a metaplastic keratinization (Fig. 6), with loss of the P.A.S.-positive surface layer. Orthokeratosis and perakeratosis occurred with equal frequency and, unlike the equivalent gingival grafts, a granular layer was rarely observed (Fig. 6). The rete peg morphology of these grafts appeared to be rather more variable than when in its normal intraoral situation. Long, narrow tapering rete peg formations alternated with short, broad epithelial downgrowths in different parts of the same graft (Fig. 6). The continuing presence of the graft connective tissue could be deduced by its pale staining collagen fibres interspersed wi::h elastic fibres. In addition, an inconspicuous subepithelial reticulin layer was always found within the connective tissue presumed to be from the graft (Fig. 7). Trypsinization control grafts. Trypsin control grafts of gingival and alveol.ar mucosal tissue resembled the environmental control grafts so closely that detailed descriptions are unnecessary. Recombinants of alveolar mucosal epithelium and gingival lamina propria. Experimental recombinant grafts of gingival connective tissue and alveolar mucosal epithelium consistently showed histological structure which closely resembled that of control gingival grafts and this was true irrespective of whether the connective tissue had been inverted or not. Hyperkeratosis and orthokeratosis, an undulating stratum granulosum and keratin surface, and long rounded or tapering rete pegs were always seen (Fig. 8). The connective tissue components of the grafts were distinguished from the dermis by the different collagen bundle arrangement, the absence of elastic fibres (Fig. 9) and a prominent subepithelial reticulin layer (Fig. 10). Recombinants of al.seolar mucosal epithelium and gingival periosteum. R.ecombinant grafts formed of alveolar mucosal epithelium and gingival periosteum once more exhibited the same range of epithelial transformations as the control gingival grafts, although the characteristic and’ clearly demarcated graft collagen bundle arrangement without elastic fibres was always found. One important additional observation was made in this group of grafts. In no case was a connective tissue layer found immediately subjacent to the epith.elium having the appearance and staining properties, of periosteum. Free gingival connective tissue grafts. Free grafts of isolated gingival lamina propria entirely free of epithelium appeared to survive well on the cutaneous recipient beds. The gingival connective tissue could be
169
distinguished from the surrounding dermis as it could in all of the other graft types. The epithelium covering these grafts, which must have been derived from skin, had acquired an appearance similar in all respects to that of the various gingival controls and recombinants (Fig. 11).
DISCUSSION The amount of mucosal tissue available in the pig for experimental purposes is relatively small because the cheeks are thick and cannot be retracted, effectively limiting available areas to those in the anterior part of the mouth. Replacing the grafts intraorally accentuates this deficiency because suitable recipient sites are also prospective donor areas. Cutaneous recipient sites were therefore chosen with appropriate controls so that these problems could be overcome. The adaptive responses of epithelium of gingiva and alveolar mucosa to an extraoral environment were sufficiently marked to require that the histological structure of experimental grafts be compared with that of the controls, rather than with the normal intraoral appearance. Conclusions drawn from such comparisons can be justified because the histological modifications occurring in the controls were always readily distinguished and consistent throughout the experiments. The similarity in behaviour of test recombinant and control grafts does not necessarily indicate that gingival connective tissue regulates epithelial specificity, because the epithelium of the control grafts of alveolar mucosa always underwent metaplastic keratinization. It is possible that control of alveolar mucosal epithelial differentiation is an intrinsic epithelial function. If this is so, the final behaviour of the recombinant grafts could be explained equally well by the successful re-establishment of a previously determined, autonomous alveolar mucosal epithelium on a gingival connective tissue substrate, and its simultaneous modulation to a keratinized form under environmental stimuli. The results achieved with the free gingival connective tissue grafts suggest the true interpretation, however, as they behaved in an identical fashion to that of the gingival controls, During healing, these grafts could only have been covered by migrating epithelial cells of skin, yet the epithelium established over the graft was always of a gingival type. The lamina propria of the gingiva must therefore possess morphogenetic and histodifferentiative control over overlying epithelium. The results of this investigation therefore are in accord with the conclusions of Karring et al. (1975). Results with recombinants containing inverted connective tissue, or periosteum together with the deepest layers of gingival connective tissue, also demonstrate that these instructional stimuli are not confined to superficial regions of the connective tissue, but extend down as far as the periosteum. It cannot be stated with certainty that periosteum itself possesses instructional competence for epithelium, because subepithelial connective tissue zones resembling periosteum were not found in any of the specimens. This is not
170
T. G. Heaney
surprising and may be of little significance as the periosteum would probably lose its characteristic structure during healing, and there is no reason to suppose that it would be reformed subsequently in an area remote from bone. Alternatively, the results might be due to the superficially placed periosteum necrosing in all grafts, and allowing epithelium from whatever source to come into contact with lamina propria. The present study cannot differentiate between these alternatives, but it seems unlikely that a tissue which is primarily concerned with osteogenesis, and which normally has no contact with tissues of ectodermal origin, could exert any influences on epithelium. Such a conclusion would be in accord with the findings of Smith R. M. (1970) which suggested that the granulation tissue derived from bone marrow might be passive to epithelium. The results of this investigation have human clinical imphcations because it is accepted that a functional width of attached gingiva is necessary for periodontal health (Nabers, 1954; Friedman and Levine, 1964; Lang and Lee, 1972). Some workers believe that the width of the gingival zone can be increased by exposing periosteum or denuding bone to induce healing by second intention (Friedman, 1962; Carranza and Carraro, 1970). The success of such procedures is unpredictable, however (Arnold and Hatchett, 1962; Bohannan, 1962; Glickman et al., 1963; Pennel et al., 1965; Carranza and Carraro, 1970), and this has been ascribed to variability in the degree of adaptation of these healing wounds to function (Ivancie, 1957; Pfeifer, 1963). The present study indicates that increasing the area of attached gingiva does not primarily depend on changing the functional environment of the wound, but of increasing the available gingival lamina propria. It is possible to redistribute and increase available lamina propria by means of pedicle and free gingival transplants. The specificities of such grafts are maintained indefinitely in new sites (Karring et al., 1971) and they can augment gingival zones with predictability (Ward, 1974; Egli et al., 1975). Acknowledgements--I wish to thank Professor E. J. H. Ford for providing the necessary animal facilities, Mr W. M. Oliver for his constant encouragement and helpful criticism, Mr J. H. Allan for his stimulating discussions, Miss D. Kenny for technical assistance, Mr J. S. Bailie and Miss J. Taylor for the illustrations, and Mrs N. Carruthers for typing the manuscript. Particular thanks are due to Mr R. S. Jones; this investigation would have been impossible without his anaesthetic skills. REFERENCES
Arnold N. R. and Hatchett C. M. 1962. A comparative investigation of two mucogingival surgical methods. .I. Periodont. 33, 129-139. Auerbach R. 1960. Morphogenetic interactions in the development of the mouse thymus gland. Deul. Biol. 2, 271-284. Beer A. E. and Billingham R. E. 1970. Implantation, transplantation and epithelial-mesenchymal relationships in the rat uterus. J. exp. Med. 132, 721-736. Bernimoulin Von J. P. and Lange D. E. 1973. Uber den Einfluss von heterotopischen Bindegewebstransplantaten auf die Epithelneubildung. Dt. zahniirztl. Ztg. 28, 202-225.
Billingham R. E. and Silvers W. K. 1967. Studies on the conservation of epidermal specificities of skin and certain mucosas of adult mammals. J. exp. Med. 125, 429-446. Bohannan H. M. 1962. Studies in the alteration of vestibular depth-11. Periosteum retention. /. Periodont. 33, 354-359. Briggaman R. A. and Wheeler C. E. 1968. Epidermaldermal interactions in adult human skin: role of dermis in epithelial maintenance. J. inaest. Dem. 51, 454-465. Carranza F. A. and Carraro J. J. 1970. Mucogingival techniques in periodontal surgery. J. Periodont. 41, 294299. Cohen J. 1965. The dermal papilla. in: Bio/og)~ of’ rhr Skin and Hair Growth (Edited by Lyne A. G. and Short B. F.), Chap. 12, pp. 183. Angus and Robertson. Sydney. Egli U., Vollmer W. H. and Rateitschak K. H. 1975. Follow-up studies of free gingival grafts, J. c/in. Pcriodont. 2, 98-104. Friedman N. 1962. Mucogingival surgery: The apically repositioned flap. J. Periodont. 33, 328-340. Friedman N. and Levine H. L. 1964. Mucogingival surgery: current status. J. Periodont. 35, 5-21. Glickman I., Smulow J., O’Brien T. and Tannen R. 1963. Healing of the periodontium following mucogingival surgery. Oral Surg. 16, 53(f538. Gordon H. and Sweets H. H. 1936. A simple method for the silver impregnation of reticulin. Am. J. Path. 12, 545-551. Grobstein C. 1954. Tissue interaction in the morphogenesis of mouse embryonic rudiments in vitro. In: Aspects of Synthesis und Order in Growth (Edited by Rudnick D.), Chap. 10, p. 233. Princeton University Press, Princeton, New Jersey. Ivancie G. P. 1957. Experimental histological investigation of gingival regeneration in vestibular surgery. J. Periodont. 28, 259-263. Karring T., Ostergaard E. and Lije H. 1971. Conservation of tissue specificity after heterotopic transplantation of gingiva and alveolar mucosa. J. periodont. Res. 6, 282-293. Karring T., Lang N. P. and Lee H. 1975. The role of gingival connective tissue in determining epithelial differentiation. J. periodont. Res. 10, l-11. Koch W. E. 1972. Tissue interaction during in vitro odontogenesis. In: Developmental Aspects of Oral Biology (Edited bv Slavkin H. C. and Bavetta L. A.). Chao. 8. p. 151. Academic Press, New York. ” ’ ’ Kollar E. J. and Baird G. R. 1970. Tissue interactions in embryonic mouse tooth germs--II. The inductive role of the dental papilla. J. Embryoi. exp. Morph. 24, 173-186. Lang N. P. and Liie H. 1972. The relationship between the width of keratinized gingiva and gingival health, J. Periodont. 43, 623-627. Lozdan J. and Squier C. A. 1969. The histology of the mucogingival junction. J. periodont. Rex 4, 83-93. McManus J. F. A. 1946. Histological demonstration of mucin after periodic acid. Nature, Lund. 158, 202. Nabers C. L. 1954. Repositioning the attached gingivae. .I. Periodont. 25, 38-39. Oliver R. F. 1973. Responses of oral epithelium to the influence of whisker dermal papillae in the adult rat. Archs oral Biol. 18, 413421. Orban B. and Sicher H. 1945. Oral mucosa. J. dent. Educ. 10, 94100. Pennel B., King K., Higgason J., Towner J., Fritz B. and Saddler J. 1965. Retention of periosteum in mucogingival surgery. J. Periodont. 36, 39-43. Pfeifer J. S. 1963. The growth of gingival tissue over denuded bone. J. Periodont. 34, l&16.
Specificity
of adult
porcine
Rinaldini L. M. J. 1958. The isolation of living cells from animal tissues. lnt. Ret>. Cytol. 7, 587-647. Smith C. J. 1971. The structure and behaviour of gingival epithelium. In: The Preczntion of Periodontal Disease (Edited by Eastoe J. E., Picton D. C. A. and Alexander A. G.), p. 67. Kimpton, London. Smith R. M. 1970. A study of the intertransplantation of alveolar mucosa. Oral Surg. 29, 328-340.
Plates
gingival
epithelium
171
Trott J. R. 1957. An histological investigation into the keratinization found in human gingiva. Br. dent. J. 103, 421427. Ward V. J. 1974. A clinical assessment of the use of the free gingival graft for correcting localized recession associated with frenal pull. J. Periodont. 45, 78-83. Wessels N. K. 1962, Tissue interactions during skin histodifferentiation. DPO~. Biol. 4, 87-107.
1 and 2 overleaf
172
T. G. Heaney
Plate Fig. 1. Normal
porcine
attached junction
1.
gingiva, (G) and alveolar mucosa, (M). The tear at the mucogingival is an artifact. Haematoxylin and eosin. x 50
Fig. 2. Gingival environmental control graft showing hyperkeratosis. an undulating and incomplete stratum granulosum and gingival rete peg morphology. Haematoxylin and eosin. x 50 Fig. 3. Transition
zone between gingival environmental in the dermis are arrowed. Weigert
Fig. 4. Gingival
environmental control graft showing characteristic of normal gingiva.
Fig. 5. Normal
dorsal
pig skin
showing
moderate Sweets.
control graft (G) and dermis. and Van Gieson. x 155
Elastic
prominent distribution of subepithelial Gordon and Sweets. x 125 amounts x 125
of subepithelial
reticuhn.
fibres
reticulin,
Gordon
and
Plate 2. Fig. 6. Junction between alveolar mucosal environmental graft epithelium has no stratum granulosum. Fig. 7. Alveolar
control graft (M) and skin. The parakeratotic Haematoxylin and eosin. x 50
mucosal environmental control graft. Section showing sparse graft subepithelial lin, characteristic of normal alveolar mucosa. Gordon and Sweets. x 125
Fig. 8. Epithelium
of a recombinant graft of non-inverted epithelium. Haematoxylin
gingival lamina propria and eosin. x 64
and alveolar
Fig. 9. Transition zone between dermis and a recombinant graft of non-inverted gingival and alveolar mucosal epithelium. Elastic fibres are absent from the graft connective and Van Gieson. x 80 Fig. 10. Recombinant graft of gingival teristic gingival distribution Fig.
11. Junction
between
free gingival
lamina propria of subepithelial connective eosin.
reticu-
mucosal
lamina propria tissue. Weigert
and alveolar mucosal epithelium with a characreticulin. Gordon and Sweets. x 125 tissue x40
graft
(G) and
skin
(S). Haematoxylin
and
Specificity of adult porcine gingival epithelium
Plate 1.
173
174
T. G. Heaney
0.2mm
Plate 2.
22. pp. 167 to 174. Pergamon
Press 1977. Prmted
I” Great Br,ta,n
A HISTOLOGICAL INVESTIGATION OF THE INFLUENCE OF ADULT PORCINE GINGIVAL CONNECTIVE TISSUES IN DETERMINING EPITHELIAL SPECIFICITY T. G. HEANEY School of Dental Surgery, University of Liverpool, Pembroke Place, P.O. Box 147, Liverpool L69 3BX, England Summary-The influence of gingival lamina propria, and periosteum isolated from the gingival zone, on epithelial differentiation and organization was inliestigated. Open-fit autogenous free gingival connective tissue grafts and recombinant grafts composed of alveolar mucosal epithehum with gingival connective tissue or periosteum were placed on split-thickness dorsal skin sites together with control transplants of gingiva and alveolar mucosa, to monitor the effects of environmental stimuli. The control grafts adapted to their new environment in consistent and well-defined fashions. The behaviour of the experimental grafts, when compared with the controls, sustained the premise that gingival epithelial specificity in the pig is controlled by lamina propria, but no evidence ‘was found to indicate that periosteum has a similar role for epithelium. It was concluded that increases in the functional width of attached gingiva can be achieved most predictably in human clinical practice, by using pedicle or free gingival transplants to redistribute existing gingival lamina propria.
INTRODUCTION The importance of embryonic mesenchyme-epithelium inductive mechanisms in controlling histogenesis and morphogenesis is well established. Interactions of this type have been demonstrated in widely differing vertebrate organ rudiments including kidney and salivary gland (Grobstein, 1954) thymus (Auerbach, 1960), skin (Wessells, 1962) and tooth (Kollar and Baird, 1970; Koch, 1972). Much less is understood of the part played by the interactions of epithelium and connective tissue in maintaining differentiation and organization in the adult state. Nonetheless, it is (clear that maintenance of the specificity of some adult epithelia, including those of epidermis and dermal appendages, requires continuing stimuli from the dermis (Cohen, 1965; Billingham and Silvers, 1967; Briggaman and Wheeler, 1968). Other adult epithelia such as those of tongue, oesophagus and hamster cheek pouch apparently possess an intrinsic capacity to regulate their individual specificities and are. therefore, resistant to any connective tissue influence (Billingham and Silvers. 1967; Beer and Billingham, 1970). There is doubt about the category in which gingival tissue belongs, as Karring. Lang and Liie (1975) have recently shown that gingival lamina propria in cercopithicids may determine the differentiative pattern of the overlying epithelium, although earlier studies by Bernimoulin and Lange (1973) and by Oliver (1973) suggested that gingival epithelial specificity is an innate property. The present study was mounted to explore the possibility that the differentiation of adult gingival epithelium might be under connective tissue control, and also to show whether any such inductive control was
primarily resident in the superficial layers of lamina propria, or could be demonstrated also at deeper connective tissue levels. MATERIALS AND
METHOD
Four young domestic pigs, each weighing between 18-26 kg at the start of an experiment, and fed on a standard laboratory diet, were used. Separate strips of buccal attached gingiva and of alveolar mucosa each approximately 2 mm thick and 4 mm wide, were excised with a scalpel from each animal under general anaesthesia. Separation of these specimens into epithelial and connective tissue components was achieved by incubation in a sterile 2 per cent (w/v) solution of crude trypsin (Trypsin from Beef Pancreas, B.D.H. Chemicals Ltd., Poole, England), in Hank’s balanced salt solution (B.S.S.), and containing 0.6 mg penicillin G per ml. Incubation was carried out for 6 to 8 h at 4°C and within a pH range of 7.2 to 7.8, as described by Billingham and Silvers (1967). After incubation, the specimens were washed for 30min at 4°C in sterile Hank’s B.S.S. adjusted to the same pH range and penicillin concentration, but containing 20 per cent calf serum to inactivate residual trypsin (Rinaldini, 1958). Separation of the epithelium and connective tissue was then performed aseptically under a dissecting microscope, using fine tissue forceps while the specimens were still in the washing fluid. Separation at the level of the epithelialconnective tissue interface was assessed visually in the first instance, and confirmed subsequently by histological examination of samples taken from components of each mucosal strip. The remainders of the components were cut into 4mm squares and used to form the following test 1681
168
T. G. Heaney
and control recombinant grafts: (a) The possible effects of the trypsinization procedure were controlled by reforming the original anatomical epithelial-connective tissue partnership of some specimens of each mucosal type. (b) Test grafts for assessment of connective tissue inductive potential were made by recombining gingival connective tissue with alveolar mucosal epithelium. The epithelium was either placed in contact with the superficial connective tissue surface or the orientation of the connective tissue sheet was altered by turning it upside down, and epithelium laid on its anatomically deep surface. (c) Test grafts were prepared by pairing alveolar mucosal epithelium with periosteum isolated from the attached gingival zone. These periosteal components were prepared by excising full-thickness strips of attached gingiva and cutting them into specimens 4mm square. The deepest connective tissue layer of each was then removed to an even thickness of l-2mm and the rest discarded. The graft itself was formed by reversing this connective tissue layer and placing the epithelial sheet in contact with its periosteal surface. (d) Some free grafts of gingival connective tissue alone were used. These were of the superficial rather than the deeper layers of gingival lamina propria, and were derived from trypsinized split-thickness gingival specimens. Care was taken when forming recombinant grafts to ensure that the deep surface of epithelium was placed in contact with the requisite connective tissue surface. Once formed, orientation was maintained by storing them at 4°C in individual wells in a sterile transport chamber designed for this purpose, and filled with sterile fluid of the same composition as the washing fluid. The test and control grafts were finally transplanted as autogenous grafts on split-thickness dorsal skin beds, under a second general anaesthetic. They were placed as open-fit grafts, that is separated from one another and from the adjacent skin by wide zones of raw dermis. At the same time, further freshly cut gingival and alveolar mucosal grafts were placed on the recipient bed. These served as controls against histological changes induced by environmental variations. The grafts were dressed with a layer of sterile tulle gras overlaid by an adhesive surgical dressing. Additional protection against physical trauma was provided by covering the operative site with several layers of elastic adhesive bandage wound around the body of the animal. The numbers of the various types of graft employed were as follows: 8 trypsin controls each of gingiva and alveolar mucosa, 8 environmental controls each of gingiva and alveolar mucosa, 11 test recombinants of non-inverted gingival lamina propria with alveolar mucosal epithelium, 8 test recombinants of inverted gingival lamina propria with alveolar mucosal epithelium, 11 test recombinants of gingival periosteum with alveolar mucosal epithelium, 17 free gingival connective tissue grafts. The animals were killed 3 to 4 months after transplantation and the grafts were excised together with further intraoral specimens of attached gingiva and alveolar mucosa. After fixation in 10 per cent formolsaline, the grafts were processed, serially sectioned at 6pm and stained with haematoxylin and eosin, Wei-
gert’s method for elastic fibres counterstained with Van Gieson’s stain, Gordon and Sweets reticulin stain (1936), and the periodic acid-Schiff (P.A.S.) reaction (McManus, 1946). RESULTS
Macroscopic
appearance
The naked-eye appearance of all grafts, irrespective of their constitution, was very similar at the time of death. They were evident as circular surface elevations on the skin and were frequently covered by a rough brown scab. However, some of the grafts which contained gingival connective tissue showed a heavy flaky keratin layer instead. Although no attempt was made to measure growth rates in the grafts, most seemed to have undergone little or no change in size at the end of the experimental period, by which time each animal had doubled its body weight. Histological
appearances
Intraoral attached gingiua and alwolar mucosa. The distributions of collagen, elastic and subepithelial reticulin fibres in porcine gingiva and alveolar mucosa corresponded closely to those reported in human and macaque monkey material (Orban and Sicher, 1945; Lozdan and Squier, 1969; Karring, Ostergaard and Lee, 1971). At equal magnifications, the subepithelial reticulin fibre network of skin was of intermediate prominence, and was much better developed than that of alveolar mucosa, but not so conspicuous as that of attached gingiva. However, the distinguishing features of porcine gingival and alveolar mucosal epithelia differed from those which have been found in the equivalent human or monkey epithelia (Orban and Sicher, 1945; Trott, 1957; Karring et at., 1971; Smith C. J., 1971). In particular, the rete pegs of alveolar mucosal epithelium were much longer, more prominent, and showed a greater degree of outline irregularity than those of gingiva (Fig. 1). Attached gingival epithelium was always fully orthokeratinized and the keratin layer was strongly P.A.S.-positive. At high magnifications, the most intense reaction occurred in the zones separating individual layers of keratin although the latter also gave a strong positive response. The cells of the stratum spinosum did not react to the P.A.S. stain although the intercellular material was weakly positive. Environmental control grafts. Environmental control grafts of gingiva and alveolar mucosa showed several well-defined modifications in histological appearance when compared with their intraoral appearance. The major changes in both tissue types appeared to be confined to the epithelium. Attached gingiua. Attached gingival epithelium demonstrated a marked hyperkeratosis associated with the loss of the P.A.S.-positive reaction of the keratin, the appearance of an incomplete stratum granulosum and the occurrence of a more variable rete peg morphology (Fig. 2). The epithelium was still mostly orthokeratinized, although occasional small, limited areas of parakeratosis occurred in some specimens. There was a tendency for rete pegs to become broader or parallel-sided with rounded ends, and for the stratum granulosum to become thrown into folds
Specificity of adult porcine gingival epithelium which were mirrored by similar folds in the keratin surface (Fig. 2) although this was not found in all specimens. In sections stained by the Weigert and Van Gieson techniques, the graft connective tissue was clearly demarcated from the surrounding dermis by its palerstaining collagen bundles with their different pattern of orientation and by the absence of associated elastic fibres (Fig. 3). The d!stinction between graft and skin was further emphasin.ed by the persistence of the characteristically prominent subepithelial reticulin of the gingiva (Fig. 4) contrasting with that of the skin (Fig. 5). Alveolar mucosa. When alveolar mucosa was transferred to a cutaneous environment, the epithelium underwent a metaplastic keratinization (Fig. 6), with loss of the P.A.S.-positive surface layer. Orthokeratosis and perakeratosis occurred with equal frequency and, unlike the equivalent gingival grafts, a granular layer was rarely observed (Fig. 6). The rete peg morphology of these grafts appeared to be rather more variable than when in its normal intraoral situation. Long, narrow tapering rete peg formations alternated with short, broad epithelial downgrowths in different parts of the same graft (Fig. 6). The continuing presence of the graft connective tissue could be deduced by its pale staining collagen fibres interspersed wi::h elastic fibres. In addition, an inconspicuous subepithelial reticulin layer was always found within the connective tissue presumed to be from the graft (Fig. 7). Trypsinization control grafts. Trypsin control grafts of gingival and alveol.ar mucosal tissue resembled the environmental control grafts so closely that detailed descriptions are unnecessary. Recombinants of alveolar mucosal epithelium and gingival lamina propria. Experimental recombinant grafts of gingival connective tissue and alveolar mucosal epithelium consistently showed histological structure which closely resembled that of control gingival grafts and this was true irrespective of whether the connective tissue had been inverted or not. Hyperkeratosis and orthokeratosis, an undulating stratum granulosum and keratin surface, and long rounded or tapering rete pegs were always seen (Fig. 8). The connective tissue components of the grafts were distinguished from the dermis by the different collagen bundle arrangement, the absence of elastic fibres (Fig. 9) and a prominent subepithelial reticulin layer (Fig. 10). Recombinants of al.seolar mucosal epithelium and gingival periosteum. R.ecombinant grafts formed of alveolar mucosal epithelium and gingival periosteum once more exhibited the same range of epithelial transformations as the control gingival grafts, although the characteristic and’ clearly demarcated graft collagen bundle arrangement without elastic fibres was always found. One important additional observation was made in this group of grafts. In no case was a connective tissue layer found immediately subjacent to the epith.elium having the appearance and staining properties, of periosteum. Free gingival connective tissue grafts. Free grafts of isolated gingival lamina propria entirely free of epithelium appeared to survive well on the cutaneous recipient beds. The gingival connective tissue could be
169
distinguished from the surrounding dermis as it could in all of the other graft types. The epithelium covering these grafts, which must have been derived from skin, had acquired an appearance similar in all respects to that of the various gingival controls and recombinants (Fig. 11).
DISCUSSION The amount of mucosal tissue available in the pig for experimental purposes is relatively small because the cheeks are thick and cannot be retracted, effectively limiting available areas to those in the anterior part of the mouth. Replacing the grafts intraorally accentuates this deficiency because suitable recipient sites are also prospective donor areas. Cutaneous recipient sites were therefore chosen with appropriate controls so that these problems could be overcome. The adaptive responses of epithelium of gingiva and alveolar mucosa to an extraoral environment were sufficiently marked to require that the histological structure of experimental grafts be compared with that of the controls, rather than with the normal intraoral appearance. Conclusions drawn from such comparisons can be justified because the histological modifications occurring in the controls were always readily distinguished and consistent throughout the experiments. The similarity in behaviour of test recombinant and control grafts does not necessarily indicate that gingival connective tissue regulates epithelial specificity, because the epithelium of the control grafts of alveolar mucosa always underwent metaplastic keratinization. It is possible that control of alveolar mucosal epithelial differentiation is an intrinsic epithelial function. If this is so, the final behaviour of the recombinant grafts could be explained equally well by the successful re-establishment of a previously determined, autonomous alveolar mucosal epithelium on a gingival connective tissue substrate, and its simultaneous modulation to a keratinized form under environmental stimuli. The results achieved with the free gingival connective tissue grafts suggest the true interpretation, however, as they behaved in an identical fashion to that of the gingival controls, During healing, these grafts could only have been covered by migrating epithelial cells of skin, yet the epithelium established over the graft was always of a gingival type. The lamina propria of the gingiva must therefore possess morphogenetic and histodifferentiative control over overlying epithelium. The results of this investigation therefore are in accord with the conclusions of Karring et al. (1975). Results with recombinants containing inverted connective tissue, or periosteum together with the deepest layers of gingival connective tissue, also demonstrate that these instructional stimuli are not confined to superficial regions of the connective tissue, but extend down as far as the periosteum. It cannot be stated with certainty that periosteum itself possesses instructional competence for epithelium, because subepithelial connective tissue zones resembling periosteum were not found in any of the specimens. This is not
170
T. G. Heaney
surprising and may be of little significance as the periosteum would probably lose its characteristic structure during healing, and there is no reason to suppose that it would be reformed subsequently in an area remote from bone. Alternatively, the results might be due to the superficially placed periosteum necrosing in all grafts, and allowing epithelium from whatever source to come into contact with lamina propria. The present study cannot differentiate between these alternatives, but it seems unlikely that a tissue which is primarily concerned with osteogenesis, and which normally has no contact with tissues of ectodermal origin, could exert any influences on epithelium. Such a conclusion would be in accord with the findings of Smith R. M. (1970) which suggested that the granulation tissue derived from bone marrow might be passive to epithelium. The results of this investigation have human clinical imphcations because it is accepted that a functional width of attached gingiva is necessary for periodontal health (Nabers, 1954; Friedman and Levine, 1964; Lang and Lee, 1972). Some workers believe that the width of the gingival zone can be increased by exposing periosteum or denuding bone to induce healing by second intention (Friedman, 1962; Carranza and Carraro, 1970). The success of such procedures is unpredictable, however (Arnold and Hatchett, 1962; Bohannan, 1962; Glickman et al., 1963; Pennel et al., 1965; Carranza and Carraro, 1970), and this has been ascribed to variability in the degree of adaptation of these healing wounds to function (Ivancie, 1957; Pfeifer, 1963). The present study indicates that increasing the area of attached gingiva does not primarily depend on changing the functional environment of the wound, but of increasing the available gingival lamina propria. It is possible to redistribute and increase available lamina propria by means of pedicle and free gingival transplants. The specificities of such grafts are maintained indefinitely in new sites (Karring et al., 1971) and they can augment gingival zones with predictability (Ward, 1974; Egli et al., 1975). Acknowledgements--I wish to thank Professor E. J. H. Ford for providing the necessary animal facilities, Mr W. M. Oliver for his constant encouragement and helpful criticism, Mr J. H. Allan for his stimulating discussions, Miss D. Kenny for technical assistance, Mr J. S. Bailie and Miss J. Taylor for the illustrations, and Mrs N. Carruthers for typing the manuscript. Particular thanks are due to Mr R. S. Jones; this investigation would have been impossible without his anaesthetic skills. REFERENCES
Arnold N. R. and Hatchett C. M. 1962. A comparative investigation of two mucogingival surgical methods. .I. Periodont. 33, 129-139. Auerbach R. 1960. Morphogenetic interactions in the development of the mouse thymus gland. Deul. Biol. 2, 271-284. Beer A. E. and Billingham R. E. 1970. Implantation, transplantation and epithelial-mesenchymal relationships in the rat uterus. J. exp. Med. 132, 721-736. Bernimoulin Von J. P. and Lange D. E. 1973. Uber den Einfluss von heterotopischen Bindegewebstransplantaten auf die Epithelneubildung. Dt. zahniirztl. Ztg. 28, 202-225.
Billingham R. E. and Silvers W. K. 1967. Studies on the conservation of epidermal specificities of skin and certain mucosas of adult mammals. J. exp. Med. 125, 429-446. Bohannan H. M. 1962. Studies in the alteration of vestibular depth-11. Periosteum retention. /. Periodont. 33, 354-359. Briggaman R. A. and Wheeler C. E. 1968. Epidermaldermal interactions in adult human skin: role of dermis in epithelial maintenance. J. inaest. Dem. 51, 454-465. Carranza F. A. and Carraro J. J. 1970. Mucogingival techniques in periodontal surgery. J. Periodont. 41, 294299. Cohen J. 1965. The dermal papilla. in: Bio/og)~ of’ rhr Skin and Hair Growth (Edited by Lyne A. G. and Short B. F.), Chap. 12, pp. 183. Angus and Robertson. Sydney. Egli U., Vollmer W. H. and Rateitschak K. H. 1975. Follow-up studies of free gingival grafts, J. c/in. Pcriodont. 2, 98-104. Friedman N. 1962. Mucogingival surgery: The apically repositioned flap. J. Periodont. 33, 328-340. Friedman N. and Levine H. L. 1964. Mucogingival surgery: current status. J. Periodont. 35, 5-21. Glickman I., Smulow J., O’Brien T. and Tannen R. 1963. Healing of the periodontium following mucogingival surgery. Oral Surg. 16, 53(f538. Gordon H. and Sweets H. H. 1936. A simple method for the silver impregnation of reticulin. Am. J. Path. 12, 545-551. Grobstein C. 1954. Tissue interaction in the morphogenesis of mouse embryonic rudiments in vitro. In: Aspects of Synthesis und Order in Growth (Edited by Rudnick D.), Chap. 10, p. 233. Princeton University Press, Princeton, New Jersey. Ivancie G. P. 1957. Experimental histological investigation of gingival regeneration in vestibular surgery. J. Periodont. 28, 259-263. Karring T., Ostergaard E. and Lije H. 1971. Conservation of tissue specificity after heterotopic transplantation of gingiva and alveolar mucosa. J. periodont. Res. 6, 282-293. Karring T., Lang N. P. and Lee H. 1975. The role of gingival connective tissue in determining epithelial differentiation. J. periodont. Res. 10, l-11. Koch W. E. 1972. Tissue interaction during in vitro odontogenesis. In: Developmental Aspects of Oral Biology (Edited bv Slavkin H. C. and Bavetta L. A.). Chao. 8. p. 151. Academic Press, New York. ” ’ ’ Kollar E. J. and Baird G. R. 1970. Tissue interactions in embryonic mouse tooth germs--II. The inductive role of the dental papilla. J. Embryoi. exp. Morph. 24, 173-186. Lang N. P. and Liie H. 1972. The relationship between the width of keratinized gingiva and gingival health, J. Periodont. 43, 623-627. Lozdan J. and Squier C. A. 1969. The histology of the mucogingival junction. J. periodont. Rex 4, 83-93. McManus J. F. A. 1946. Histological demonstration of mucin after periodic acid. Nature, Lund. 158, 202. Nabers C. L. 1954. Repositioning the attached gingivae. .I. Periodont. 25, 38-39. Oliver R. F. 1973. Responses of oral epithelium to the influence of whisker dermal papillae in the adult rat. Archs oral Biol. 18, 413421. Orban B. and Sicher H. 1945. Oral mucosa. J. dent. Educ. 10, 94100. Pennel B., King K., Higgason J., Towner J., Fritz B. and Saddler J. 1965. Retention of periosteum in mucogingival surgery. J. Periodont. 36, 39-43. Pfeifer J. S. 1963. The growth of gingival tissue over denuded bone. J. Periodont. 34, l&16.
Specificity
of adult
porcine
Rinaldini L. M. J. 1958. The isolation of living cells from animal tissues. lnt. Ret>. Cytol. 7, 587-647. Smith C. J. 1971. The structure and behaviour of gingival epithelium. In: The Preczntion of Periodontal Disease (Edited by Eastoe J. E., Picton D. C. A. and Alexander A. G.), p. 67. Kimpton, London. Smith R. M. 1970. A study of the intertransplantation of alveolar mucosa. Oral Surg. 29, 328-340.
Plates
gingival
epithelium
171
Trott J. R. 1957. An histological investigation into the keratinization found in human gingiva. Br. dent. J. 103, 421427. Ward V. J. 1974. A clinical assessment of the use of the free gingival graft for correcting localized recession associated with frenal pull. J. Periodont. 45, 78-83. Wessels N. K. 1962, Tissue interactions during skin histodifferentiation. DPO~. Biol. 4, 87-107.
1 and 2 overleaf
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Plate Fig. 1. Normal
porcine
attached junction
1.
gingiva, (G) and alveolar mucosa, (M). The tear at the mucogingival is an artifact. Haematoxylin and eosin. x 50
Fig. 2. Gingival environmental control graft showing hyperkeratosis. an undulating and incomplete stratum granulosum and gingival rete peg morphology. Haematoxylin and eosin. x 50 Fig. 3. Transition
zone between gingival environmental in the dermis are arrowed. Weigert
Fig. 4. Gingival
environmental control graft showing characteristic of normal gingiva.
Fig. 5. Normal
dorsal
pig skin
showing
moderate Sweets.
control graft (G) and dermis. and Van Gieson. x 155
Elastic
prominent distribution of subepithelial Gordon and Sweets. x 125 amounts x 125
of subepithelial
reticuhn.
fibres
reticulin,
Gordon
and
Plate 2. Fig. 6. Junction between alveolar mucosal environmental graft epithelium has no stratum granulosum. Fig. 7. Alveolar
control graft (M) and skin. The parakeratotic Haematoxylin and eosin. x 50
mucosal environmental control graft. Section showing sparse graft subepithelial lin, characteristic of normal alveolar mucosa. Gordon and Sweets. x 125
Fig. 8. Epithelium
of a recombinant graft of non-inverted epithelium. Haematoxylin
gingival lamina propria and eosin. x 64
and alveolar
Fig. 9. Transition zone between dermis and a recombinant graft of non-inverted gingival and alveolar mucosal epithelium. Elastic fibres are absent from the graft connective and Van Gieson. x 80 Fig. 10. Recombinant graft of gingival teristic gingival distribution Fig.
11. Junction
between
free gingival
lamina propria of subepithelial connective eosin.
reticu-
mucosal
lamina propria tissue. Weigert
and alveolar mucosal epithelium with a characreticulin. Gordon and Sweets. x 125 tissue x40
graft
(G) and
skin
(S). Haematoxylin
and
Specificity of adult porcine gingival epithelium
Plate 1.
173
174
T. G. Heaney
0.2mm
Plate 2.
