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Responses of oral epithelium to the influence of whisker dermal papillae in the adult rat
Arch oral Bid.
Vol.18,pp.413421, 1973. Pergamon Press. Printed inGreatBritain.
RESPONSES OF ORAL EPITHELIUM OF WHISKER DERMAL PAPILLAE
TO THE INFLUENCE IN THE ADULT RAT
R. F. OLIVER*
A.R.C. Institute of Animal Physiology,
Babraham,
Cambridge, England
Sunnnary-Subcutaneous implants into the pinna of gingival and labial mucosae, or their epithelia alone, essentially maintained the oral pattern of keratinization and did not develop epidermal appendages. In contrast, oral epithelium implanted into this site in recombination with whisker dermal papillae developed various epidermal modulations. These included generalised hyperplasia and the development of spikes of keratinized cells, hair follicles, sebaceous gland cells and stellate reticulum-like configurations reminiscent of the tooth germ.
INTRODUCTION THE DERMALpapilla
is established very early in the ontogenetic development of hair follicles as a permanent population of specialised fibroblasts (WESSELLSand ROESSNER, 1965; BREATHNACHand SMITH, 1968). There is good evidence that the papilla anlage, which eventually becomes enclosed by the epidermal matrix of the hair bulb, induces follicle development (JACOBSON,1966; KOLLAR, 1968; 1970). It is also likely that the dermal papilla induces hair growth in the subsequent hair growth cycles since whisker growth occurs when dermal papillae are implanted into the bases of the superficial regions of adult rat whisker follicles (OLIVER, 1966, 1967). Moreover, adult whisker papillae induce the development of new hair follicles in adult rat epidermis of nonwhisker origin including, apparently, keratinizing oral epithelium (OLIVER, 1970). This last finding is of special interest because, although dermal-epidermal recombination experiments have shown that surface skin epidermis responds readily to a variety of inductive stimuli from the underlying dermis, adult oral epithelium has been considered to be a possible example of an epithelium which is innately determined in its behavioural characteristics (BILLINGHAM and SILVERS, 1965, 1967, 1968). A more detailed investigation of adult oral epithelium was therefore undertaken to establish the ease with which it could be made to respond to foreign dermis. MATERIALS
AND
METHODS
Operations All operations were performed on 2- to 4-month-old animals from inbred strains of hooded and Wistar rats. Donors were killed with ether and thin sheets of oral mucosa were removed free-hand with a scalpel. Gingival mucosa was obtained from the anterior aspect of the lower incisor root region and immediately adjacent labial mucosa (Fig. 1) from the central region of the inner lower lip ex* Present address: Department
of Biological Sciences, University of Dundee, Dundee, Scotland. 413
414
R. F. OLIVER
tending to the base of the lip sulcus. The epithelia from these sites were virtually identical histologically, both having a poorly developed granular layer and being of the “complete” orthokeratinizing type according to the classification of ALVARESand MEYER (1971). The mucosa was further trimmed, where necessary, to remove adherent musculature and incubated in 0.5 per cent trypsin (Burroughs Wellcome 1: 303) at 4°C for approximately 60 min. In the meantime, the whisker follicles on the left upper lip were exposed and their bulbar regions removed and placed into Hanks’s solution. Dermal papillae were dissected from the bulbs as previously described (OLIVER, 1967) and put into fresh Hanks’s solution cooled in an icebath. From each rat, 2-8 dermal papillae were isolated which were considered to be free of contaminating whisker epidermal matrix cells as judged by microscopic examination. At completion of incubation, the mucosa was transferred to 50 per cent calf serum in Hanks’s solution and the epithelium separated from the dermis using watchmaker’s forceps. The epithelium was then washed in fresh Hanks’s solution and cut into rectangular sheets cu. l-3 mm square. Littermate recipients were anaesthetized by the intraperitoneal injection of a x 10 diluted solution of Nembutal (Abbott), 0.65 ml/100 g body weight. For each of the grafting procedures, a small flap of skin was reflected on the dorsum of one of the ears and dermal papillae were grouped on the exposed dermal or muscle bed. The epithelial sheet was carefully lowered, basal surface down, on the papillae and the skin flap gently repositioned (OLIVER, 1970). Similarly, trypsinized but unseparated sheets of oral mucosa and sheets of separated epithelium were implanted under ear skin without papillae. Design of experiments
A total of 89 dermal papillae were implanted into 17 rats: 43 papillae in association with gingival epithelium (as 9 separate recombinants, each with 4-6 papillae) and 46 papillae in association with labial epithelium (as 11 separate recombinants, each with 2-6 papillae). In addition, the following were implanted: 5 gingival mucosa, 5 gingival epithelium, a single labial mucosa and 3 labial epithelium implants. One animal was killed 12 days after operation, and the remainder between 35 and 68 days, and the implant areas removed. Histology
Implant areas were fixed in formol-saline, serially sectioned at 8 pm and stained in a combination of Weigert’s haematoxylin, alcian blue and Curtis’s Ponceau S. Alcian blue is specific for acid mucopolysaccharides. It stains dermal papillae blue-green in anagen hair follicles (i.e. those follicles producing hair) and, as will be seen, acted as a useful marker for implanted papillae, being particularly prominent in papillary tissue which was in active association with epidermis or epithelium.
RESULTS
All the following sections of results refer to implantation littermate recipients.
into ear dermis of
Gingival and labial mucosa and their epithelia alone
All of the above will be considered together as they did not differ significantly. Of the 9 out of 14 grafts which were not sloughed from the ear, 2 became incorporated into the ear surface (Fig. 2) while the remainder gave rise to 9 separate cysts. The cysts were large, thin-walled and keratinizing into a central lumen (Fig. 3). The squamous keratin was either thick and yellow or more dense, less obviously laminate, and orange. The dermal+pidermal junction was smooth and flat throughout and the pattern of keratinization never resembled that of ear epidermis. There were no indications of histogenesis of epidermal appendages or sebaceous glands. Therefore, regardless of whether epithelium was implanted with its own dermis or alone, when it necessarily acquired an ear dermis substratum, it retained its oral type of “complete” orthokeratinization with a poorly developed granular layer.
RESPONSES
OF ORAL
EPITHELIUM
TO WHISKER
DERMAL
PAPILLAE
415
Oral epithelium-dermal papilla recombinants
A total of 21 epithelium-papilla associations were found, each incorporating a variable number of papillae, but not all of the dermal papillae had remained in contact with the epithelium. Seventeen papillae were found isolated in ear dermis and a further 6 were in contact with ear epidermis at the edge of the implant area. An indeterminate number, probably not more than 10, were also lost as a result of epidermal undergrowth and subsequent sloughing of part of the implant area during the post-operative healing phase. Isolated dermal papillae
All of the 17 papillae isolated in ear dermis were entirely free of adherent whisker epidermal matrix cells, as were all of a greater number of similarly isolated papillae examined in a previous study (OLIVER, 1970). The papillae remained as discrete entities, with apparently little loss of cell number, although they were often compacted with a decrease in cellular cytoplasmic volume. Most had acquired a capillary supply and were stained in varying degrees by the alcian blue. Obviously their integrity was not dependent on contact with epidermal cells. Epithelium-dermal papilla associations
Papillae, which had either retained their discreteness or, more commonly, had fused into a mass of papillary tissue, were readily distinguishable from the surrounding ear mesoderm because they stained with alcian blue. When associated with epithelium, the papilla cells invariably presented the “foamy” cytoplasmic appearance characteristic of such cells in dermal papillae of anagen hair follicles. Seventeen of the 21 papilla-epithelium associations were essentially intradermal cysts, a further two were cystic but opened on the ear surface, and two were more completely incorporated into the ear surface. In those regions where the epithelium was not immediately associated with papillary tissue, or where it had formed separate cysts, the oral pattern of keratinization was preserved exactly as described above for oral implants alone. However, where papillary tissue was contiguous with epithelium, a more complex and variable histological picture prevailed. In all instances, adjacent papillary tissue locally promoted epithelial hyperplasia with mitotic figures present both suprabasally and in increased numbers in a columnar basal layer (Figs. 4, 6-l 1). The epithelium was also commonly thrown into large projections or folds with an inner core of diffuse, well vascularised, papillary tissue (Fig. 4). The inner aspect of these folds was often ridged and thick squamous keratin was being produced. Sebaceous cells, singly or in groups, were found in 6 of the implants where they either opened into the lumen of cysts or were isolated within the thickness of the epithelium (Fig. 5). They were frequently associated with the localised and restricted development of a granular layer. Of especial interest was the presence in 7 separate implants of areas of epithelium in which the cells had, in varying degrees, assumed a stellate reticulum-like configuration in a manner reminiscent of the enamel organ in developing teeth (Figs. 6-8).
416
R.F.
OLIVER
Intercellular contact was achieved through attentuated cytoplasmic processes with, apparently, an intervening intercellular accumulation of fluid. There was no evidence that this intraepithelial condition represented a form of pathological oedema and there was no accompanying inflammatory response. The dermal+epithelial interface in these regions was particularly distinct and smooth and the epithelial basal layer was markedly columnar. There was, however, no differentiation of recognizable ameloblasts nor was there any sign of papillary cells either differentiating into odontoblasts or showing a significantly increased cellular density adjacent to the basement membrane. Indentations of papillary tissue in small epithelial projections associated with the localized development of large, fibrous epithelial cells were a frequent occurrence in most of the recombinants (Fig. 9). These cells were very similar to the cortex cells of the hair shaft as seen at the level of the keratogeneous zone of hair follicles, except that they were not as elongated and narrow. Furthermore, they were the immediate cellular precursors of horny spikes of keratin, as opposed to squames, which histologically resembled mature hair cortex cells. No other features characteristic of hair follicles were present in these particular configurations. However, a single small anagen follicle was found confluent with gingival epithelium as were two similar follicles associated with labial epithelium. None of these follicles had sebaceous glands; the small papillae were apparently derived from adjacent whisker papillary tissue. Three other follicular configurations were present in labial epithelium. One of these consisted of an epithelial matrix organized around a large papilla, with peripherally distributed trichohyalin granules. The latter are characteristically synthesised by the inner root sheath cells of hair follicles. A rather better developed but extremely short follicular structure was also seen, with distinct outer and inner root sheaths, which was producing large, loosely associated cortex-like cells containing wispy keratin fibres (Fig. 10). Finally, there was a small aberrant hair follicle with normal follicular features developed only on one side (Fig. 1l), which was producing a spike of hairlike keratin via large fibrous cells identical to those already described above. Ear epidermis-dermal papilla associations
Two of the 6 dermal papillae in contact with surface ear epidermis had, at 12 days, locally organized the development of epidermal matrices containing basal and suprabasal mitoses. A matrix configuration was also observed in association with a dermal papilla at 63 days. Trichohyalin granules were present peripherally but, apart from some sebaceous gland cells, there were no other follicular features. The remaining three papillae had induced the formation of hair follicles. These follicles were shorter and stouter and had larger bulbs than local ear hair-follicles and were producing thick, whisker-like hairs. Their papillae, which were not vascularized, consisted of hundreds of cells; fewer than when implanted and yet many more than ear hairpapillae which contain 30-50 cells. None of these follicles had developed normal sebaceous glands with ducts opening into the necks of the follicles and their inner root sheaths were present adhering to the hair shafts beyond the ear surface. In
RESPONSES
OF ORAL
EPITHELIUM
TO WHISKER
DERMAL
PAPILLAE
417
naturally occurring hair follicles, the inner root sheath fragments or is lysed just below the opening of the sebaceous duct. DISCUSSION It has been shown that adult rat gingival and labial epithelia essentially retained their typically oral pattern of keratinization when implanted into ear dermis. These observations are in accord with the findings of BILLINGHAM and SILVERS(1968) who demonstrated that mucosal epithelia from guinea-pig and hamster tongue and oesophagus maintained their site of origin characteristics when recombined with ear and sole of foot dermis. Such results, in themselves, are suggestive that these epithelia are innately determined in their behavioural characteristics. In contrast, surface skin epidermis “modulates” when recombined with ectopic dermis so that, under the specific influence of the dermis, it comes to assume the characteristics typical of the epidermis which normally overlies such dermis (BILLINGHAM and SILVERS,1965, 1967, 1968). A similar but more complex modulation is demonstrated by the induction of hair follicle development in adult epidermis by whisker dermal papillae (OLIVER, 1970). This was incidentally confirmed in the present study where follicle development was observed following the chance contact of ear epidermis by dermal papillae. The various developmental responses of oral epithelium when recombined with whisker dermal papillae clearly demonstrated that, provided the dermal influence is strong enough, oral epithelium can also exhibit modulatory behaviour. In its generalised response to the contiguous presence of papillary tissue, oral epithelium, by becoming hyperplastic and in the development of large projections, resembled ear and scrotal sac epidermis (OLIVER, 1970). It retained, however, the oral pattern of keratinization. Superimposed on these features, and in strict relation to small protrusions of papillary tissue, oral epithelium uniquely developed horny spikes of keratin which resemble the cortical keratin of hair shafts. Perhaps as a progressive response there were also attempts, with varying degrees of success, at the formation of recognizable but aberrant hair follicles which were either small or otherwise incompletely developed and thus lacking in certain follicular features. It may be significant that, with one exception, they developed from labial epithelium which obviously is normally immediately confluent with hair-bearing skin. Sebaceous gland cells had also developed in the epithelium of some of the recombinants but never in relation to papillary tissue or to an induced hair follicle. They differed in this respect from their more normal developmental position, when present, in follicles experimentally induced in ear and scrotal sac epidermis (OLIVER,1970). It is interesting to note that naturally occurring sebaceous glands, similarly unassociated with hair follicles, have been occasionally encountered in rat gingival epithelium (BERNICKand BAVETTA,1962; FRANDSEN,1962; RULLI and MARTINELLI,1971). Perhaps the most intriguing response of the epithelium, which corroborated two similar observations described in a previous paper (OLIVW, 1970), was the development of stellate reticulum-like configurations. The histological picture of a columnar basal layer giving rise to an epithelial stellate reticulum, separated in some instances by A.O.B. 18/3-H
418
R. F. OLIVER
an intermediate cell layer arranged parallel to a prominent basement membrane, closely resembled enamel organ of developing teeth. This conformation may have represented attempts by adult oral epithelium to participate in tooth-forming activities, with obvious and interesting developmental implications, or merely a fortuitous and misleading histological analogy. Little more can be said at this stage other than nothing resembling such a distinctive conformation was observed in recombinants of whisker papillae with whisker follicle, ear, or scrotal sac epidermis. It should be recalled that in this context embryonic mouse lip-furrow epithelium and foot plate ectoderm can become true enamel organ when recombined with dental papilla mesenchyme from tooth germs (KOLLAR and BAIRD, 1970a,b). How whisker dermal papillae revealed these various developmental potentials in adult oral epithelium can only be guessed at. Some explanation may be found within the suggestion that tissue determination is achieved by permanent blocking of part of the genome and the refinements of differentiation by the reversible suppression of the remaining genome (SLAVKIN, 1970; WESSELLS, 1970). Thus the potential of adult oral epithelium to become follicular epidermis, sebaceous gland cells or even, perhaps, to attempt to become tooth formative epithelium is normally suppressed yet in the present studies was expressed by some permissive activity on the part of whisker dermal papilla fibroblasts in an ear dermis milieu.
Acknowledgements-I would like to express my thanks to Mr. L. G. JARVIS and Mr. A. L. GALLUP who were responsible for the histology and photography. R&urn&-Des implantations sous-cutanees dans le point saillant de la muqueuse gingivale et labiale ou seulement dans leur epithelium, maintinrent essentiellement l’aspect oral de keratinisation et ne developpa pas d’appendices Cpidermiques. Par contraste, l’epithelium oral ‘implant6 dans cette region, en combinaison nouvelle avec des papilles dermiques a poils, developpa diverses modulations Cpidermiques. Ces-ci incluaient la hyperplasie gendralis6e et le developpement d’epis de cellules keratinisees, des follicules a poils, des cellules de glandes sCbacQs et des configurations stellaires d’aspect reticulaire, reminiscentes de germes des dents. Zusammenfassung-Subkutane Verpflanzung in die Ohrmuschel der Gingiva- und labialen SchleimhPute oder nur in ihre Epithels behielt im wesentlichen das Mundmuster der Keratinisierung bei und entwickelte keine Epidernralfortsatze. Im Gegensatz dazu entwickelte Mundepithel, das in diese Stelle in Wiedervereinigung mit dermalen Bartpapillen verpflanzt wurde, verschiedene Epidermallnderungen. Dazu gehiirten allgemeine Hyperplasie und die Entwicklung von Spitzen keratinisierter Zellen, Haarfollikel, Talgdriisenzellen und sternartige Netzstrukturen, die an Zahnkeim erinnerten. REFERENCES ALVARES,0. F. and MEYER,J. 1971. Variable features and regional differences in oral epithelium. In: Current Concepts of the Histology of Oral Mucosa (edited by SQUIER,C. A. and MEYER,J.) p. 97. Thomas, Springtield. BERNICK,S. and BAVETTA,L. A. 1962. The development of gingival sebaceous-like glands and cysts in rats of the Holzman Strain. Oral Surg. 15, 351-354. BILLINGHAM,R. E. and SILVERS,W. K. 196% Some unsolved problems in the biology of skin, In: Biology of the Skin and Hair Growth (edited by LYNE, A. G. and SHORT,B. F.), p. 1. Angus & Robertson, Sydney.
RESPONSES OF ORALEPITHELIUM TO WHISKERDERMALPAPILLAE
419
BILLINGHAM,R. E. and S~~veas, W. K. 1967. Studies on the conservation of epidermal specifities of skin and certain mucosas in adult mammals. J. exp. Med. 125, 429-446. BILLINOHAM,R. E. and SILVERS,W. K. 1968. Dermo-epidermal interactions and epithelial specificity. In : Epithelial-Mesenchymal Interactions (edited by FLEISCHMAIER,R. and BILLINGHAM,R. E.), p. 252. Williams & Wilkins, Baltimore. BREATHNACH, A. S. and SMITH,J. 1968. Fine structure of the early hair germ and dermal papilla in the human foetus. J. Anat. 102, 51 l-526. FRANDSEN,A. M. 1962. Sebaceous glands in the gingiva of the rat. Archs oral Biol. 7, 247-248. JACOBSON,C. M. 1966. A comparative study of the mechanisms by which x-irradiation and genetic mutation cause loss of vibrissae in embryo mice. J. Embryol. exp. Morph. 16, 369-379. KOLLAR,E. J. 1968. The inhibition of vibrissa development in vitro by beta-2-thienylalanine. J. invest. Derm. 50, 319-322. KOLLAR, E. J. 1970. The induction of hair follicles by embryonic dermal papillae. J. invest. Derm. 55, 374-378. KOLLAR, E. J. and BAIRD, G. R. 1970a. Tissue interactions in embryonic mouse tooth germs. I. Reorganization of the dental epithelium during tooth-germ reconstruction. J. Embryol. exp. Morph. 24, 159-171. KOLLAR,E. J. and BAIRD, G. R. 1970b. Tissue interactions in embryonic mouse tooth germs. II. The inductive role of the dental papilla. J. Embryol. exp. Morph. 24, 173-186. OLIVER,R. F. 1966. Whisker growth after removal of the dermal papilla and lengths of follicle in the hooded rat. J. Embryol. exp. Morph. 15,331-347. OLIVER,R. F. 1967. The experimental induction of whisker growth in the hooded rat by implantation of dermal papillae. J. Embryol. exp. Morph. l&43-51. OLIVER,R. F. 1970. The induction of hair follicle formation in the adult hooded rat by vibrissa dermal papillae. J. Embryol. exp. Morph. 23, 219-236. RULLI, M. A. and MARTINELLI,C. 1971. Free sebaceous glands in the gingiva of some rats. Archs. oral Bi. 16,831-832. SLAVKIN, H. C. 1970. Epithelial-mesenchymal interactions related to periodental disease. J. Periodont. 41, 373-381. WESSELLS,N. K. 1970. Some thoughts on embryonic inductions in relation to determination. J. inuest. Derm. 55,221-225. WESSELLS,N. K. and ROESSNER,K. D. 1965. Nonproliferation in dermal condensations of mouse vibrissae and pelage hairs. Devl. Biol. 12,419-433.
R. F. OLIVER
420
PLATE1 All sections stained with Weigerts
haematoxylin,
alcian blue and Curtis Ponceau S.
FIG. 1. Labia1 mucosa.
x 125
FIGS. 2-6. Sections of implants in ear at various times after implantation. FIG. 2. Gingival mucosa (left) incorporated into ear surface. Note abrupt junction with ear epidermis and the poorly developed granular layer and densely laminated comified layer of the gingival epithelium in comparison with ear epidermis. 44 days. x100 FIG. 3. Large, thin-walled
labial epithelium cyst in ear. 64 days. x 65
FIGS. 4-6. Sections of oral epithelium-whisker
dermal papilla associations
in ear.
FIG. 4. Extremely large projections
of thickened gingival epithelium with inner cores of dermal papilla tissue extending beyond the ear surface. 68 days. x 45
FIG. 5. Sebaceous gland cells in gingival epithelium some distance from papillary tissue. 44 days. x 265 FIG. 6. Papillary tissue associated with hyperplastic labial epithelium which has a columnar basal layer and is at a presumed early stage in the development of a stellate reticulumlike appearance. 51 days. x 165
RtSPONSES
OF ORAL
EPITHELIUbt
TO WHISKER
DERMAI.
PAPILLAF
PLATt
1
A.0 B. f.p. 410
RESPONSES
PLATE
2
OF ORAL
EPITHELIUM
TO WHISKER
DERMAL
PAPILLAE
RESPONSES OF ORAL EPITHELIUM TO WHISKER
DEKMAL PAPIL.LAE
421
PLATE 2 All
sections stained with Weigerts haematoxylin,
alcian blue and Curtis Ponceau S.
FIGS. 7-11. Sections of oral epithelium-whisker dermal papillae associations various times after implantation. FIG. 7. Hyperplastic
in ear at
gingival epithelium with an area of early stellate reticulum-like formation. 62 days. x 110
FIG. 8. Region of hyperplastic labial epithelium, adjacent to papillary tissue, which has a pronounced columnar basal layer, a layer of “intermediate” cells and above this a welldeveloped stellate reticulum-like configuration. 35 days. x 250 FIG. 9. Small projection of labia1 epithelium associated with a small papillary indentation and showing the lower region of a keratinized spike. 62 days. x 165 FIG. 10. Bulbar hair follicle structure induced by a single whisker dermal papilla in labial epithelium. 62 days. x 165 FIG. 11. Small, incompletely
developed hair follicle induced in labial epithelium papillary cells. 51 days. x 165
by
Vol.18,pp.413421, 1973. Pergamon Press. Printed inGreatBritain.
RESPONSES OF ORAL EPITHELIUM OF WHISKER DERMAL PAPILLAE
TO THE INFLUENCE IN THE ADULT RAT
R. F. OLIVER*
A.R.C. Institute of Animal Physiology,
Babraham,
Cambridge, England
Sunnnary-Subcutaneous implants into the pinna of gingival and labial mucosae, or their epithelia alone, essentially maintained the oral pattern of keratinization and did not develop epidermal appendages. In contrast, oral epithelium implanted into this site in recombination with whisker dermal papillae developed various epidermal modulations. These included generalised hyperplasia and the development of spikes of keratinized cells, hair follicles, sebaceous gland cells and stellate reticulum-like configurations reminiscent of the tooth germ.
INTRODUCTION THE DERMALpapilla
is established very early in the ontogenetic development of hair follicles as a permanent population of specialised fibroblasts (WESSELLSand ROESSNER, 1965; BREATHNACHand SMITH, 1968). There is good evidence that the papilla anlage, which eventually becomes enclosed by the epidermal matrix of the hair bulb, induces follicle development (JACOBSON,1966; KOLLAR, 1968; 1970). It is also likely that the dermal papilla induces hair growth in the subsequent hair growth cycles since whisker growth occurs when dermal papillae are implanted into the bases of the superficial regions of adult rat whisker follicles (OLIVER, 1966, 1967). Moreover, adult whisker papillae induce the development of new hair follicles in adult rat epidermis of nonwhisker origin including, apparently, keratinizing oral epithelium (OLIVER, 1970). This last finding is of special interest because, although dermal-epidermal recombination experiments have shown that surface skin epidermis responds readily to a variety of inductive stimuli from the underlying dermis, adult oral epithelium has been considered to be a possible example of an epithelium which is innately determined in its behavioural characteristics (BILLINGHAM and SILVERS, 1965, 1967, 1968). A more detailed investigation of adult oral epithelium was therefore undertaken to establish the ease with which it could be made to respond to foreign dermis. MATERIALS
AND
METHODS
Operations All operations were performed on 2- to 4-month-old animals from inbred strains of hooded and Wistar rats. Donors were killed with ether and thin sheets of oral mucosa were removed free-hand with a scalpel. Gingival mucosa was obtained from the anterior aspect of the lower incisor root region and immediately adjacent labial mucosa (Fig. 1) from the central region of the inner lower lip ex* Present address: Department
of Biological Sciences, University of Dundee, Dundee, Scotland. 413
414
R. F. OLIVER
tending to the base of the lip sulcus. The epithelia from these sites were virtually identical histologically, both having a poorly developed granular layer and being of the “complete” orthokeratinizing type according to the classification of ALVARESand MEYER (1971). The mucosa was further trimmed, where necessary, to remove adherent musculature and incubated in 0.5 per cent trypsin (Burroughs Wellcome 1: 303) at 4°C for approximately 60 min. In the meantime, the whisker follicles on the left upper lip were exposed and their bulbar regions removed and placed into Hanks’s solution. Dermal papillae were dissected from the bulbs as previously described (OLIVER, 1967) and put into fresh Hanks’s solution cooled in an icebath. From each rat, 2-8 dermal papillae were isolated which were considered to be free of contaminating whisker epidermal matrix cells as judged by microscopic examination. At completion of incubation, the mucosa was transferred to 50 per cent calf serum in Hanks’s solution and the epithelium separated from the dermis using watchmaker’s forceps. The epithelium was then washed in fresh Hanks’s solution and cut into rectangular sheets cu. l-3 mm square. Littermate recipients were anaesthetized by the intraperitoneal injection of a x 10 diluted solution of Nembutal (Abbott), 0.65 ml/100 g body weight. For each of the grafting procedures, a small flap of skin was reflected on the dorsum of one of the ears and dermal papillae were grouped on the exposed dermal or muscle bed. The epithelial sheet was carefully lowered, basal surface down, on the papillae and the skin flap gently repositioned (OLIVER, 1970). Similarly, trypsinized but unseparated sheets of oral mucosa and sheets of separated epithelium were implanted under ear skin without papillae. Design of experiments
A total of 89 dermal papillae were implanted into 17 rats: 43 papillae in association with gingival epithelium (as 9 separate recombinants, each with 4-6 papillae) and 46 papillae in association with labial epithelium (as 11 separate recombinants, each with 2-6 papillae). In addition, the following were implanted: 5 gingival mucosa, 5 gingival epithelium, a single labial mucosa and 3 labial epithelium implants. One animal was killed 12 days after operation, and the remainder between 35 and 68 days, and the implant areas removed. Histology
Implant areas were fixed in formol-saline, serially sectioned at 8 pm and stained in a combination of Weigert’s haematoxylin, alcian blue and Curtis’s Ponceau S. Alcian blue is specific for acid mucopolysaccharides. It stains dermal papillae blue-green in anagen hair follicles (i.e. those follicles producing hair) and, as will be seen, acted as a useful marker for implanted papillae, being particularly prominent in papillary tissue which was in active association with epidermis or epithelium.
RESULTS
All the following sections of results refer to implantation littermate recipients.
into ear dermis of
Gingival and labial mucosa and their epithelia alone
All of the above will be considered together as they did not differ significantly. Of the 9 out of 14 grafts which were not sloughed from the ear, 2 became incorporated into the ear surface (Fig. 2) while the remainder gave rise to 9 separate cysts. The cysts were large, thin-walled and keratinizing into a central lumen (Fig. 3). The squamous keratin was either thick and yellow or more dense, less obviously laminate, and orange. The dermal+pidermal junction was smooth and flat throughout and the pattern of keratinization never resembled that of ear epidermis. There were no indications of histogenesis of epidermal appendages or sebaceous glands. Therefore, regardless of whether epithelium was implanted with its own dermis or alone, when it necessarily acquired an ear dermis substratum, it retained its oral type of “complete” orthokeratinization with a poorly developed granular layer.
RESPONSES
OF ORAL
EPITHELIUM
TO WHISKER
DERMAL
PAPILLAE
415
Oral epithelium-dermal papilla recombinants
A total of 21 epithelium-papilla associations were found, each incorporating a variable number of papillae, but not all of the dermal papillae had remained in contact with the epithelium. Seventeen papillae were found isolated in ear dermis and a further 6 were in contact with ear epidermis at the edge of the implant area. An indeterminate number, probably not more than 10, were also lost as a result of epidermal undergrowth and subsequent sloughing of part of the implant area during the post-operative healing phase. Isolated dermal papillae
All of the 17 papillae isolated in ear dermis were entirely free of adherent whisker epidermal matrix cells, as were all of a greater number of similarly isolated papillae examined in a previous study (OLIVER, 1970). The papillae remained as discrete entities, with apparently little loss of cell number, although they were often compacted with a decrease in cellular cytoplasmic volume. Most had acquired a capillary supply and were stained in varying degrees by the alcian blue. Obviously their integrity was not dependent on contact with epidermal cells. Epithelium-dermal papilla associations
Papillae, which had either retained their discreteness or, more commonly, had fused into a mass of papillary tissue, were readily distinguishable from the surrounding ear mesoderm because they stained with alcian blue. When associated with epithelium, the papilla cells invariably presented the “foamy” cytoplasmic appearance characteristic of such cells in dermal papillae of anagen hair follicles. Seventeen of the 21 papilla-epithelium associations were essentially intradermal cysts, a further two were cystic but opened on the ear surface, and two were more completely incorporated into the ear surface. In those regions where the epithelium was not immediately associated with papillary tissue, or where it had formed separate cysts, the oral pattern of keratinization was preserved exactly as described above for oral implants alone. However, where papillary tissue was contiguous with epithelium, a more complex and variable histological picture prevailed. In all instances, adjacent papillary tissue locally promoted epithelial hyperplasia with mitotic figures present both suprabasally and in increased numbers in a columnar basal layer (Figs. 4, 6-l 1). The epithelium was also commonly thrown into large projections or folds with an inner core of diffuse, well vascularised, papillary tissue (Fig. 4). The inner aspect of these folds was often ridged and thick squamous keratin was being produced. Sebaceous cells, singly or in groups, were found in 6 of the implants where they either opened into the lumen of cysts or were isolated within the thickness of the epithelium (Fig. 5). They were frequently associated with the localised and restricted development of a granular layer. Of especial interest was the presence in 7 separate implants of areas of epithelium in which the cells had, in varying degrees, assumed a stellate reticulum-like configuration in a manner reminiscent of the enamel organ in developing teeth (Figs. 6-8).
416
R.F.
OLIVER
Intercellular contact was achieved through attentuated cytoplasmic processes with, apparently, an intervening intercellular accumulation of fluid. There was no evidence that this intraepithelial condition represented a form of pathological oedema and there was no accompanying inflammatory response. The dermal+epithelial interface in these regions was particularly distinct and smooth and the epithelial basal layer was markedly columnar. There was, however, no differentiation of recognizable ameloblasts nor was there any sign of papillary cells either differentiating into odontoblasts or showing a significantly increased cellular density adjacent to the basement membrane. Indentations of papillary tissue in small epithelial projections associated with the localized development of large, fibrous epithelial cells were a frequent occurrence in most of the recombinants (Fig. 9). These cells were very similar to the cortex cells of the hair shaft as seen at the level of the keratogeneous zone of hair follicles, except that they were not as elongated and narrow. Furthermore, they were the immediate cellular precursors of horny spikes of keratin, as opposed to squames, which histologically resembled mature hair cortex cells. No other features characteristic of hair follicles were present in these particular configurations. However, a single small anagen follicle was found confluent with gingival epithelium as were two similar follicles associated with labial epithelium. None of these follicles had sebaceous glands; the small papillae were apparently derived from adjacent whisker papillary tissue. Three other follicular configurations were present in labial epithelium. One of these consisted of an epithelial matrix organized around a large papilla, with peripherally distributed trichohyalin granules. The latter are characteristically synthesised by the inner root sheath cells of hair follicles. A rather better developed but extremely short follicular structure was also seen, with distinct outer and inner root sheaths, which was producing large, loosely associated cortex-like cells containing wispy keratin fibres (Fig. 10). Finally, there was a small aberrant hair follicle with normal follicular features developed only on one side (Fig. 1l), which was producing a spike of hairlike keratin via large fibrous cells identical to those already described above. Ear epidermis-dermal papilla associations
Two of the 6 dermal papillae in contact with surface ear epidermis had, at 12 days, locally organized the development of epidermal matrices containing basal and suprabasal mitoses. A matrix configuration was also observed in association with a dermal papilla at 63 days. Trichohyalin granules were present peripherally but, apart from some sebaceous gland cells, there were no other follicular features. The remaining three papillae had induced the formation of hair follicles. These follicles were shorter and stouter and had larger bulbs than local ear hair-follicles and were producing thick, whisker-like hairs. Their papillae, which were not vascularized, consisted of hundreds of cells; fewer than when implanted and yet many more than ear hairpapillae which contain 30-50 cells. None of these follicles had developed normal sebaceous glands with ducts opening into the necks of the follicles and their inner root sheaths were present adhering to the hair shafts beyond the ear surface. In
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naturally occurring hair follicles, the inner root sheath fragments or is lysed just below the opening of the sebaceous duct. DISCUSSION It has been shown that adult rat gingival and labial epithelia essentially retained their typically oral pattern of keratinization when implanted into ear dermis. These observations are in accord with the findings of BILLINGHAM and SILVERS(1968) who demonstrated that mucosal epithelia from guinea-pig and hamster tongue and oesophagus maintained their site of origin characteristics when recombined with ear and sole of foot dermis. Such results, in themselves, are suggestive that these epithelia are innately determined in their behavioural characteristics. In contrast, surface skin epidermis “modulates” when recombined with ectopic dermis so that, under the specific influence of the dermis, it comes to assume the characteristics typical of the epidermis which normally overlies such dermis (BILLINGHAM and SILVERS,1965, 1967, 1968). A similar but more complex modulation is demonstrated by the induction of hair follicle development in adult epidermis by whisker dermal papillae (OLIVER, 1970). This was incidentally confirmed in the present study where follicle development was observed following the chance contact of ear epidermis by dermal papillae. The various developmental responses of oral epithelium when recombined with whisker dermal papillae clearly demonstrated that, provided the dermal influence is strong enough, oral epithelium can also exhibit modulatory behaviour. In its generalised response to the contiguous presence of papillary tissue, oral epithelium, by becoming hyperplastic and in the development of large projections, resembled ear and scrotal sac epidermis (OLIVER, 1970). It retained, however, the oral pattern of keratinization. Superimposed on these features, and in strict relation to small protrusions of papillary tissue, oral epithelium uniquely developed horny spikes of keratin which resemble the cortical keratin of hair shafts. Perhaps as a progressive response there were also attempts, with varying degrees of success, at the formation of recognizable but aberrant hair follicles which were either small or otherwise incompletely developed and thus lacking in certain follicular features. It may be significant that, with one exception, they developed from labial epithelium which obviously is normally immediately confluent with hair-bearing skin. Sebaceous gland cells had also developed in the epithelium of some of the recombinants but never in relation to papillary tissue or to an induced hair follicle. They differed in this respect from their more normal developmental position, when present, in follicles experimentally induced in ear and scrotal sac epidermis (OLIVER,1970). It is interesting to note that naturally occurring sebaceous glands, similarly unassociated with hair follicles, have been occasionally encountered in rat gingival epithelium (BERNICKand BAVETTA,1962; FRANDSEN,1962; RULLI and MARTINELLI,1971). Perhaps the most intriguing response of the epithelium, which corroborated two similar observations described in a previous paper (OLIVW, 1970), was the development of stellate reticulum-like configurations. The histological picture of a columnar basal layer giving rise to an epithelial stellate reticulum, separated in some instances by A.O.B. 18/3-H
418
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an intermediate cell layer arranged parallel to a prominent basement membrane, closely resembled enamel organ of developing teeth. This conformation may have represented attempts by adult oral epithelium to participate in tooth-forming activities, with obvious and interesting developmental implications, or merely a fortuitous and misleading histological analogy. Little more can be said at this stage other than nothing resembling such a distinctive conformation was observed in recombinants of whisker papillae with whisker follicle, ear, or scrotal sac epidermis. It should be recalled that in this context embryonic mouse lip-furrow epithelium and foot plate ectoderm can become true enamel organ when recombined with dental papilla mesenchyme from tooth germs (KOLLAR and BAIRD, 1970a,b). How whisker dermal papillae revealed these various developmental potentials in adult oral epithelium can only be guessed at. Some explanation may be found within the suggestion that tissue determination is achieved by permanent blocking of part of the genome and the refinements of differentiation by the reversible suppression of the remaining genome (SLAVKIN, 1970; WESSELLS, 1970). Thus the potential of adult oral epithelium to become follicular epidermis, sebaceous gland cells or even, perhaps, to attempt to become tooth formative epithelium is normally suppressed yet in the present studies was expressed by some permissive activity on the part of whisker dermal papilla fibroblasts in an ear dermis milieu.
Acknowledgements-I would like to express my thanks to Mr. L. G. JARVIS and Mr. A. L. GALLUP who were responsible for the histology and photography. R&urn&-Des implantations sous-cutanees dans le point saillant de la muqueuse gingivale et labiale ou seulement dans leur epithelium, maintinrent essentiellement l’aspect oral de keratinisation et ne developpa pas d’appendices Cpidermiques. Par contraste, l’epithelium oral ‘implant6 dans cette region, en combinaison nouvelle avec des papilles dermiques a poils, developpa diverses modulations Cpidermiques. Ces-ci incluaient la hyperplasie gendralis6e et le developpement d’epis de cellules keratinisees, des follicules a poils, des cellules de glandes sCbacQs et des configurations stellaires d’aspect reticulaire, reminiscentes de germes des dents. Zusammenfassung-Subkutane Verpflanzung in die Ohrmuschel der Gingiva- und labialen SchleimhPute oder nur in ihre Epithels behielt im wesentlichen das Mundmuster der Keratinisierung bei und entwickelte keine Epidernralfortsatze. Im Gegensatz dazu entwickelte Mundepithel, das in diese Stelle in Wiedervereinigung mit dermalen Bartpapillen verpflanzt wurde, verschiedene Epidermallnderungen. Dazu gehiirten allgemeine Hyperplasie und die Entwicklung von Spitzen keratinisierter Zellen, Haarfollikel, Talgdriisenzellen und sternartige Netzstrukturen, die an Zahnkeim erinnerten. REFERENCES ALVARES,0. F. and MEYER,J. 1971. Variable features and regional differences in oral epithelium. In: Current Concepts of the Histology of Oral Mucosa (edited by SQUIER,C. A. and MEYER,J.) p. 97. Thomas, Springtield. BERNICK,S. and BAVETTA,L. A. 1962. The development of gingival sebaceous-like glands and cysts in rats of the Holzman Strain. Oral Surg. 15, 351-354. BILLINGHAM,R. E. and SILVERS,W. K. 196% Some unsolved problems in the biology of skin, In: Biology of the Skin and Hair Growth (edited by LYNE, A. G. and SHORT,B. F.), p. 1. Angus & Robertson, Sydney.
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BILLINGHAM,R. E. and S~~veas, W. K. 1967. Studies on the conservation of epidermal specifities of skin and certain mucosas in adult mammals. J. exp. Med. 125, 429-446. BILLINOHAM,R. E. and SILVERS,W. K. 1968. Dermo-epidermal interactions and epithelial specificity. In : Epithelial-Mesenchymal Interactions (edited by FLEISCHMAIER,R. and BILLINGHAM,R. E.), p. 252. Williams & Wilkins, Baltimore. BREATHNACH, A. S. and SMITH,J. 1968. Fine structure of the early hair germ and dermal papilla in the human foetus. J. Anat. 102, 51 l-526. FRANDSEN,A. M. 1962. Sebaceous glands in the gingiva of the rat. Archs oral Biol. 7, 247-248. JACOBSON,C. M. 1966. A comparative study of the mechanisms by which x-irradiation and genetic mutation cause loss of vibrissae in embryo mice. J. Embryol. exp. Morph. 16, 369-379. KOLLAR,E. J. 1968. The inhibition of vibrissa development in vitro by beta-2-thienylalanine. J. invest. Derm. 50, 319-322. KOLLAR, E. J. 1970. The induction of hair follicles by embryonic dermal papillae. J. invest. Derm. 55, 374-378. KOLLAR, E. J. and BAIRD, G. R. 1970a. Tissue interactions in embryonic mouse tooth germs. I. Reorganization of the dental epithelium during tooth-germ reconstruction. J. Embryol. exp. Morph. 24, 159-171. KOLLAR,E. J. and BAIRD, G. R. 1970b. Tissue interactions in embryonic mouse tooth germs. II. The inductive role of the dental papilla. J. Embryol. exp. Morph. 24, 173-186. OLIVER,R. F. 1966. Whisker growth after removal of the dermal papilla and lengths of follicle in the hooded rat. J. Embryol. exp. Morph. 15,331-347. OLIVER,R. F. 1967. The experimental induction of whisker growth in the hooded rat by implantation of dermal papillae. J. Embryol. exp. Morph. l&43-51. OLIVER,R. F. 1970. The induction of hair follicle formation in the adult hooded rat by vibrissa dermal papillae. J. Embryol. exp. Morph. 23, 219-236. RULLI, M. A. and MARTINELLI,C. 1971. Free sebaceous glands in the gingiva of some rats. Archs. oral Bi. 16,831-832. SLAVKIN, H. C. 1970. Epithelial-mesenchymal interactions related to periodental disease. J. Periodont. 41, 373-381. WESSELLS,N. K. 1970. Some thoughts on embryonic inductions in relation to determination. J. inuest. Derm. 55,221-225. WESSELLS,N. K. and ROESSNER,K. D. 1965. Nonproliferation in dermal condensations of mouse vibrissae and pelage hairs. Devl. Biol. 12,419-433.
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PLATE1 All sections stained with Weigerts
haematoxylin,
alcian blue and Curtis Ponceau S.
FIG. 1. Labia1 mucosa.
x 125
FIGS. 2-6. Sections of implants in ear at various times after implantation. FIG. 2. Gingival mucosa (left) incorporated into ear surface. Note abrupt junction with ear epidermis and the poorly developed granular layer and densely laminated comified layer of the gingival epithelium in comparison with ear epidermis. 44 days. x100 FIG. 3. Large, thin-walled
labial epithelium cyst in ear. 64 days. x 65
FIGS. 4-6. Sections of oral epithelium-whisker
dermal papilla associations
in ear.
FIG. 4. Extremely large projections
of thickened gingival epithelium with inner cores of dermal papilla tissue extending beyond the ear surface. 68 days. x 45
FIG. 5. Sebaceous gland cells in gingival epithelium some distance from papillary tissue. 44 days. x 265 FIG. 6. Papillary tissue associated with hyperplastic labial epithelium which has a columnar basal layer and is at a presumed early stage in the development of a stellate reticulumlike appearance. 51 days. x 165
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1
A.0 B. f.p. 410
RESPONSES
PLATE
2
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DERMAL
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PLATE 2 All
sections stained with Weigerts haematoxylin,
alcian blue and Curtis Ponceau S.
FIGS. 7-11. Sections of oral epithelium-whisker dermal papillae associations various times after implantation. FIG. 7. Hyperplastic
in ear at
gingival epithelium with an area of early stellate reticulum-like formation. 62 days. x 110
FIG. 8. Region of hyperplastic labial epithelium, adjacent to papillary tissue, which has a pronounced columnar basal layer, a layer of “intermediate” cells and above this a welldeveloped stellate reticulum-like configuration. 35 days. x 250 FIG. 9. Small projection of labia1 epithelium associated with a small papillary indentation and showing the lower region of a keratinized spike. 62 days. x 165 FIG. 10. Bulbar hair follicle structure induced by a single whisker dermal papilla in labial epithelium. 62 days. x 165 FIG. 11. Small, incompletely
developed hair follicle induced in labial epithelium papillary cells. 51 days. x 165
by