Tooth Transplantation in the Mouse I. THE USE OF PROCION DYES AND TRITIATED PROLINE IN A STUDY OF SYNGENEIC TOOTH GERM TRANSPLANTATION '

JAN KLEIN. WALTER R. SECOSKY2 AND DAGMAR KLEIN Departments of Oral Biology, Oral Surgery arid Human , The University of Michigari, Ann Arbor, Michigan 481 04

ABSTRACT Tooth germs of first molars from four- to six-day old mice were transplanted heterotopically into the connective tissue under the dorsal skin of adult syngeneic recipients. The fate of the transplants was studied by histologic staining, vital staining with procion dyes, and autoradiography. Together these methods provided the following picture of the development of the transplant. Ex- traction of the tooth germ severs its vascular connections and disturbs the nutri- tional environment of the tooth so that many cells undergo necrosis and degen- eration. The resulting inflammatory response clears the transplant of necrotic tissue, leaving a fibrous pulp of low cellularity and an extensively disrupted odontoblastic layer; the enamel is reduced to only a few epithelial cells. In the second week following transplantation, however, the surviving cells start to proliferate and the tissues of the transplant begin to reorganize. Reorganiza- tion starts at the apical foramen and spreads to the rest of the tooth. The central pulp is penetrated by new vessels, its cellular content is reestablished, and its morphology returns to normal. Odontoblasts start producing dentin which at first is highly irregular and contains many cells (osteodentin). Later the dentin formation becomes more and more regular and the dentin assumes its normal tubular structure. New enamel is not formed at any time after transplantation. The cells of the enamel organ undergo squamous metaplasia and form epithelial cysts. Abortive root formation is observed but typical roots never develop. Rudi- ments of periodontal ligament develop rarely. The transplants are enclosed in a capsule of connective tissue. It is concluded that the heterotopically transplanted tooth germs maintain their capability to recover, develop, and differentiate in syngeneic recipients. Al- though almost all of the developmental processes continue in the transplant after a period of adjustment, some of them are abortive.

The literature on tooth transplantation technically. Between these two extremes is enormous. A bibliography compiled in lie various concepts, for example, host- 1962 (Ivhnyi, '62) lists over 300 publica- tissue acceptance of both syngeneic and tions, and since then some 300 more have allogeneic grafts (Haley and Costich, '69), appeared (Natiella et al., '70). Widely acceptance of syngeneic grafts and rejec- contrasting views on the antigenicity of tion of allografts (IvBnyi and Vacek, '64), teeth have been expressed. It has been re- rejection of some allografts and acceptance ported that tooth xenografts survive (Flem- of others according to the strength of the ming, '55; Cserepfalvi and Price, '68), histocompatibility barrier (IvAnyi, '68; which implies that the tooth tissues are f Supported by grant DE-02731 from the National non-antigenic. Other investigators, how- Institute for Dental Research, PHS. 2 Portions of this investigation are being submitted ever, have concluded that even syngeneic as partial fulfillment of the requirements for the Mas- grafts do not survive (Lefkowitz, '61) and ter of Science deeree in oral surgery, Horace H. Rack- ham School of Graduate Studies, Ann Arbor, Mich- that transplantation of teeth is not possible igan.

AM. J. ANAT., 131: 371-386. 3 71 372 JAN KLEIN, WALTER R. SECOSKY AND DAGMAR KLEIN

Weinreb et al., ’68), and induction of the transplant was prepared by making an transplantation by allogeneic incision in the skin just behind the ears hard tissue (Shulman, ’64; Mincer and and opening a subcutaneous pouch by Jennings, ’70 ) and soft tissue of the tooth blunt dissection with iridectomy scissors. Zaleski et al.. ’67). The tooth germ was inserted as far into The confusion in the field of transplanta- the pouch as possible and the incision tion stems from two main closed. The operation was performed under factors: first, the use of laboratory animals intraperitoneal Nembutal anesthesia (0.5 with nondescript genetic backgrounds, mg pentobarbital sodium per 10 gm body and second, the use of different criteria weight). The recipients were two- to three- for evaluating acceptance or rejection of month old mice. To avoid possible sex- the graft. In most studies the experimental linked histoincompatibility, only males subjects were “monkeys,” randomly bred were used. dogs. rabbits, guinea pigs, or rats. Only a Histologic eramination. At termination few investigators have used inbred strains, of the experiment, the transplanted teeth, and to our knowledge no one has used together with the surrounding tissue, were congenic resistant lines. As for evaluation dissected out and fixed in 4% paraform- methods, many of these have involved only aldehyde dissolved in 0.2 M phosphate buf- gross observation of the tooth graft; others fer, pH 7.4. After fixation for 24 to 36 have included histologic techniques, but hours, the specimens were decalcified with the histologic criteria for survival or de- chelating agents in dilute HC13 and em- struction of the tooth graft vary from one bedded in paraffin. Serial sections of 10 ,u report to another. thickness were cut and stained with The experiments described below were hematoxylin and eosin (H and E). undertaken to reexamine the role of genetic Grossly the transplant usually appeared and nongenetic factors in tooth trans- as a small bulge at the site of the opera- plantation. The subjects were mice with tion, at least in the first postoperative days. known genetic background, and the meth- Later, when the transplant was being sur- ods involved standard histologic techniques rounded by connective tissue, the bulge augmented by the use of procion dyes and usually disappeared but the tooth was still tritiated proline as markers for the study palpable. About 1% of the transplants of dentinogenesis in transplanted teeth. could not be found; they were probably lost The first set of experiments focused on the either by migration or by technical failure. morphologic characteristics of syngeneic In some instances the grafts were lost be- he tero topic transplants. cause of infection. Vital staining with procion dyes. The MATERIALS AND METHODS following dyes were tested for in vivo Mice. Mice used in this work were of toxicity and €or efficacy observed in mark- the highly inbred strain C57BL/lOScSn ing hard tissues of normal teeth: red (abbreviated to B10) and its congenic re- M-8BS, scarlet M-GS, orange M-GS, olive sistant lines BlO.BY, BlO.A, BlO.LPa, and green M-3CS, grey M-3GS, purple H-3RS, E10.129(21M). Breeding pairs were sup- and blue H-5GS.4 The dyes were dissolved plied by Dr. George D. Snell, The Jackson in distilled water and injected intraperi- Laboratory, Bar Harbor, Maine, and were toneally. Toxicity was evaluated by inject- maintained in our colony by brother-x-sister ing groups of five mice with the dye solu- matings. Skin grafts exchanged between tions in dosages ranging from 2 to 5 mg/lO individuals of the same line survived gm body weight (b.w.) and recording the permanently. deaths each day for one week (table 1). Transplantation of tooth germs. Maxil- Marked difference in tolerance to different lary or mandibular first molar germs of dyes was observed: procion orange M-GS four- to six-day old donors were carefully and scarlet M-GS were tolerated in doses dissected out with 25-gauge syringe needles up to 5 mg/lO gm b.w.; whereas procion under a dissecting microscope and trans- purple H-3RS had some lethal effect even planted into the dorsal connective tissue 1 The Rupp and Bowman Co , Hlghland Park. Mich- igan of‘ the recipient. The site for reception of 4 Colab Laboratories, Inc , Glenwood, Illino~s. TOOTH TRANSPLANTATION 373

TABLE 1 In viuo toxicity of different procion dyes

~ No. of mice dead at days Procion after injection dye Dose No. of mice mg/lO gm b.w. injected 12345 Red M-8BS 5 5 221-- 4 5 12000 3 5 1211- 2 5 00000 Scarlet M-GS 5 5 00000 4 5 00000 3 5 00000 2 5 00000 Purple H-3RS 5 5 32--- 4 5 5---- 3 5 131-- 2 5 02000 Orange M-GS 5 5 00000 4 5 10000 3 5 00000 2 5 00000 Olive green M3GS 5 5 30100 4 4 10000 3 5 00000 2 5 10000 Grey M-3GS 5 5 20000 4 5 11000 3 5 00000 2 5 00000 f Blue H-5GS 5 5 11111 4 5 10000 3 4 00000 2 4 00000 Mice were injected intraperitoneally with water solutions of the dyes. Dose range: 2 to 5 mg per 1Ogm of body weight. in the low dosage of 2 mg/lO gm b.w. The mercury lamp, a BG12 exciter filter, and efficacy of the procion dyes was evaluated Zeiss barrier filter inserts (47 and 50). by their capacity to reveal distinct incre- The seven tested dyes fell into three groups mental zones of growth in dentin of dc- according to their marking suitability. In calcified sections of normal teeth cut the first group, procion red M-8BS and at 7 to 10 p. Groups of three to five mice scarlet M-GS were considered the most one to three weeks old were injected suitable of those tested. Zones of dentin with the individual dyes at the highest non- stained with these two dyes were visible toxic dosage as determined in the previous under the standard microscope but only experiment. One week after the injection, as pale lines with poor contrast against the mice were sacrificed and their teeth ex- the background. For this reason standard tracted, fixed, decalcified, and sectioned. microscopy was abandoned. With ultra- Half of the sections were stained with H violet light, the two dyes yielded bright and E and the other half remained un- fluorescence of excellent visibility (fig. 9). stained. The unstained sections were ex- Although the fluorescence had the same amined by means of a standard microscope orange tint, the procion red M-8BS was with direct or oblique illumination, and by slightly more intense and for this reason a fluorescence microscope (GFL) equipped it was preferred over scarlet M-GS for sub- with an Osram HBO 200W super-pressure sequent experiments. In the second group, 374 JAN KLEIN, WALTER R. SECOSKY AND DAGMAR KLEIN zones stained with procion grey M-3GS '57; Hay, '61) and, therefore, will not be and orange M-GS were visible only under discussed here. the fluorescence microscope. The intensity Sections also were made from extracted of the fluorescence was relatively low in five-day old tooth germs to determine mor- both cases, and the dyes were judged not phologic features before transplantation. suitable for transplantation experiments. A typical extracted tooth germ (fig. 2) con- In the third group, zones stained with sisted of enamel matrix, dentin, and un- procion blue H-5GS and purple H-3RS disturbed pulp with a continuous layer of were not visible under the conditions odontoblasts. Of the enamel organ, usually described. only the inner dental epithelium (amelo- Mice carrying syngeneic tooth trans- blasts), stratum intermedium, and small plants were injected with procion red areas of stellate reticulum were preserved. M-8BS at different intervals after trans- The continuity of these tissues was dis- plantation, killed one week after the in- turbed in some grafts. Only a small por- jection, and tooth sections examined with tion of the dental sac was present. Some the fluorescence microscope. grafts also included small pieces of Azctoradiography. Tritiated proline was alveolar bone. used as a marker of dentinogenesis in both Development of syngeneic transplants normal and transplanted teeth. In the as revealed by histological stains. Tooth studies of normal dentinogenesis, groups germs from four- to six-day old mice were of five-day old mice were injected sub- transplanted under the dorsal skin of cutaneously with 3H-proline,5 50 ,Ci per adult syngeneic recipients, i.e., BlO-+BlO, mouse. The mice were killed 7 and 14 BlO.BY-+BlO.BY, BlO.A+BlO.A, B1O.LPa days later, their first molars extracted and +BlO.LPa and B10.129 (21M)+B10.129 sectioned. The sections were covered with (21M). Since no difference in the behavior photographic emulsion NTB; exposed for of transplants in the different strains was ten days, and then developed, fixed, and observed, the results were pooled. Alto- stained with H and E, In the transplant gether, 95 successful transplantations were studies, another group of five-day old mice performed. The transplanted teeth were were injected with 3H-proline, killed 24 excised from the recipients starting at hours later, and the first molars extracted day 3 after transplantation, then every odd and transplanted into adult mice of the day up to 29 days, and then every ten days same strain. Thirty days after transplanta- up to 100 days. At least three or four grafts tion, the recipients were killed and the at each interval were available for sec- transplants processed histologically and tioning. As a means of gauging develop- autoradiographically. ments in the transplants, the histologic changes were noted on a week-to-week OBSERVATIONS basis. Normal development of the first molars. First postoperative week. As a conse- As a control for the transplants, sections quence of the surgical trauma, drastic were made of normal first molars (man- changes occurred in the transplanted tooth. dibular and maxillary) at different ages In the first two days the vascular system from 0 to 24 days after birth. In another of the pulp underwent degeneration ac- experiment, procion red M-8BS was used companied by hemorrhage and thromboses. as a marker of dentin formation. In this Concurrently with the loss of vascularity, experiment 10 mice were injected with the the typical triangular or spindle contour dye once a week for four weeks starting at of the cells of the central pulp changed to five days after birth. The mice were killed round or oval. The odontoblastic layer be- one week after the last injection and the came disorganizecl and lost its continuity. sections of their molars examined with the The odontoblasts were cuboidal, their nu- fluorescence microscope (fig. 9). The ob- clei pycnotic and the cytoplasm vacuolar. served major events in the postnatal de- Dentinogenesis and amelogenesis ceased, velopment of the first molar (fig. 1) were and the predentin formed before trans- basically in agreement with what has been 5 New England Nuclear, Boston, Massachusetts. reported on this subject (Gaunt, '56; Cohn, 6 Eastman Kodak Co., Rochester, New York. TOOTH TRANSPLANTATION 375

Fig. 1 Time table of events in the postnatal development of the mouse first molar (mandibular). (The dotted bars indicate activity, the empty bars indicate absence OP activity. ) plantation did not calcify. The enamel Second week. The pulp was again cel- organ (if present) and Hertwig’s root lular (fig. 4). At first, the pulp tissue was sheath also showed signs of degeneration composed of a pleomorphic mixture of ir- and necrosis. The degenerative changes in regularly dispersed round, oval, triangular, the tooth transplant were most prominent and spindle-shaped cells, and a few histio- at about four to five days after the opera- cytes. Toward the end of the second week, tion. At the same time, the transplant be- however, the composition shifted toward came a target of an inflammatory response. normal, with a predominance of triangular At first, round cells infiltrated the connec- and spindle-shaped cells. Deposition of tive tissue surrounding the transplant and dentin matrix was concentrated in the then invaded the pulp through the apical apex, almost occluding the lower part of foramen. The necrotic cells of the central the pulp chamber. At the beginning of the pulp were destroyed, leaving behind a net- second week the pattern of matrix deposi- work of pale ground substance and connec- tion and calcification was uneven; the tive fibers (fig. 3). The few remaining matrix showed no tubules but contained cells were round, granular, and poorly many irregularly dispersed or clustered stained. The integrity of the central pulp cells in small lacunae. It will be referred was lost. In some tooth germs the odonto- to as osteodentin. Toward the end of the blastic layer was limited to small strands second week, the matrix exhibited more or scattered atypical cells. In others, long regular deposition and the incorporation segments of the layer were preserved. At of fewer cells. Tubules appeared at this the end of the week, however, signs of time, gradually increasing in number and new organization appeared. The round- becoming oriented parallel to each other. infiltration in the pulp subsided and re- The odontoblastic layer in the apex also vascularization developed; cells in the apex underwent progressive changes. During the started to synthesize and deposit extra- week the layer became more organized; the cellular material. cells changed from round and irregular to 376 JAN KLEIN, WALTER R. SECOSKY AND DAGMAR KLEIN cuboidal and later columnar, and arranged dentin beyond the dentino-enamel junction themselves side by side into a continuous was occasionally observed. In some in- stratum. The nuclei of the columnar cells stances, definite layers of acellular and moved toward the eccentric position typi- cellular cementum were deposited (fig. 6). cal for normal odontoblasts. The odonto- The connective tissue around the apex was blasts of the crown, however, still produced quite often organized into a structure little or no dentin, and in large segments which resembled the hammock ligament, of the pulp chamber the odontoblasts were and in some cases a rudimentary perio- still missing. The tooth appeared deformed dontal ligament also developed (fig. 6). by the pressure of the overlying skin. Some transplants were surrounded by al- Third zueeh. By the third week the pulp veolar bone which had developed from was similar to that of a normal tooth. The pieces inadvertently included in the trans- odontoblastic layer was continuous in some planted tooth germ. The osseous cavities transplants and interrupted in others. were filled with bone marrow. In rare in- Dentin was deposited not only in the apex stances, bone formation had occurred in but also in other areas of the pulp chamber the pulp chamber. where the odontoblasts had reorganized. The dentigerous cysts enlarged and Masses of dentin at the apex in some cases sometimes encapsulated the crown of the reduced the apical foramen to a thin canal. tooth, arid where secondary infection had In some transplants, granulation tissue occurred, the cyst cavity was filled with surrounded the crown (fig. 5) and the infl ainmatory cells. whole tooth was enclosed in a connective Procion dyes as markers for study of tissue capsule. The most conspicuous dentinogenesis in tooth transplants. The event at this stage was the appearance of procion dye stained hard tissues (dentin. areas of stratified squamous epithelium enamel matrix, bone) selectively, but the (fig. 7), which spread rapidly until the cellular components of the transplant were whole crown was covered in some in- also visible because of their autofluores- stances. This epithelium probably origi- cence. Moreover, the tissues were dis- nated from the enamel organ. Most of the tinguished by variations in color, the cells of the inner dental epithelium and odontoblasts appearing bright green, cen- the stratum intermedium had undergone tral pulp cells brown, and the external necrosis in the first postoperative week connective-tissue capsule yellow. This dis- and were removed by the inflammatory tinction greatly iacilitated evaluation of response; cells that survived became hyper- the grafts. The entire enamel matrix, when plastic. In some transplants this epithelial present, was stained uniformly without layer underwent degeneration, and vesicles any sign of banding. Its bright orange developed that had all the characteristics fiuorescence of the early stages later faded. of dentigerous cysts (fig. 7). Young cysts During the later post-graft periods the were composed mostly of epithelial cells; enamel matrix became more porous. The in older cysts, fibrous connective tissue absence of banding in the enamel matrix surrounded the inner epithelial layer. The was taken as evidence that no new enamel inner layer was continuously shed off into was formed after transplantation. The pre- the growing cavity of the cysts, which in transplant enamel matrix was not lost by addition to these squamous cells also con- decalcification, indicating that it did not tained polymorphonuclear leucocytes in mature. some instances. The staining pattern of the osteodentin From the fourth week to the end of the was irregular and diffuse (fig. lo). The p~xperiment. Dentinogenesis continued, lacunae showed a green lining surrounded filliiig the chamber so that the pulp was by orange fluorescence; apparently the reduced to small islands (fig. 8). Typical cells entrapped in the osteodentin were roots never formed, although abortive root producing extracellular material. In the formation was noted in some transplants. second week after transplantation an Rudimentary root sheath and epithelial orange line was observed parallel to the diaphragm were often present in the early pulp surface of the dentin in the apex stages, and the massive formation of (fig. 10, L), and separated from the odonto- TOOTH TRANSPLANTATION 377 blasts by a green layer of dentin (fig. 10, Dj. cally transplanted tooth germs indicates At first, the line was present only in the that the inflammatory response is non- apex, but later it extended toward the specific and distinct from the allograft coronal part of the graft (fig. 11, L) until reaction. it formed a continuous ring. Three weeks We found few reports in the literature after transplantation the line was “em- dealing with syngeneic tooth transplanta- bedded in the dentin at the apex, while tion, apparently because few investigators in the crown it lined the inner surface of have used inbred strains of laboratory ani- the pulp chamber. Five weeks after trans- mals. An exception is the large series re- plantation the line was “embedded’ ported by IvBnyi (IvBnyi and Vacek, ’64: throughout the entire layer of dentin. At IvBnyi, ’65), who transplanted tooth germs the end of the experiment the line was in newborn and adult rats of an inbred again present on the inner surface of the strain AVN and observed the grafts histo- pulp chamber. The presence of the line logically up to 50 days after transplanta- was taken as evidence of dentinogenesis at tion. She found that all the grafts were a given time and position in the transplant. accepted and developed roots with ce- During the fourth postoperative week, a mentum and a periodontal ligament. She second line appeared in some transplants also observed early degenerative changes on the host side of the apex. This line ex- in the upper part of the pulp and in the tended only to the dentino-enamel junc- enamel organ. She did not mention the fate tion, and was evidently associated with of the enamel and dentin and did not de- cementogenesis in the rudimentary roots. scribe the development of the different tis- Tritiated proline as a marker for study sues of the tooth transplant. With this of dentinogenesis. In both the normal reservation, our results are in good agree- and transplanted teeth, most of the isotope ment with hers. was concentrated in the dentin (fig. 12); Another set of syngeneic tooth trans- enamel matrix, at least after decalcifica- plants was performed by Lefkowitz (’61), tion, and cellular components of the tooth in an inbred strain of Sherman albino rats. showed little or no radioactivity. Some The number of transplants was relatively radioactivity was noted in the tissues sur- small, and the period of study rather brief. rounding the transplant, probably as a. Lefkowitz observed that despite the alleged result of misplacement of the tracer. In isogenicity of the hosts and donors, the the dentin, the isotope formed a granular transplanted tooth germs were destroyed band which was separated from the pulp by a process which he termed a typical wherever a considerable amount of new allograft reaction. Since the recipients dentin was formed after the injection and were all males, sex-linked histoincompati- transplantation (fig. 13), and extended bility should not have been respmsible onto the pulpal surface wherever little for the rejection. The most likely explana- dentin formed. tion of these results is that the rats were not inbred and the grafts were actually DISCUSSION allogeneic. Fate of syngeneic tooth transplants. Nonacceptance of syngeneic tooth trans- The results of our studies indicate that plants was also reported by Grewe and syngeneic, heterotopically transplanted Felts (’68) and by Mincer and Jennings tooth germs do resume development after (’70). In both cases the grafts were mouse an interval of adjustment. During this lag incisors, transplanted in the former case period the host tissues respond to the into mandibular incisor sockets of new- graft by an inflammatory reaction resem- born animals, and in the latter case sub- bling the cellular response to an allograft. cutaneously in adult mice. In both cases The inflammatory process can easily he the transplants failed to grow and ulti- confused with the rejection phenomenon, mately their soft tissues were completely and apparently this happened in some of destroyed. Whether this “rejection” was the studies reported by other investigators. related to the nature of the grafts or to The fact that there is complete recovery technical problems is not clear. A limited from the degenerative changes in syngenei- number of successful syngeneic grafts was 378 JAN KLEIN, WALTER R. SECOSKY AND DAGMAR KLEIN reported by Yoshioka and Gonzales ('59), concentration of the dye in the serum drops Fleming ('52; '53; '55), Coburn and to a low level. When staining ceases, an Henriques ('64) and by Haley and unstained material is again deposited and Costich ('69). thus a stained band is formed in the in- Unlike syngeneic transplantation, auto- cremental zone of growth. Since the band transplantation of teeth is well documented becomes an integral part of the tissue, it in the literature (Ivfinyiovfi, '63; Natiella persists even after decalcification. The et al., '70). Autotransplantation is not width of the band depends primarily upon strictly comparable to the type of syngeneic the rate of growth, providing that the rate transplantation reported here because it o€ clearance of the dye from the serum of is usually performed between adult ani- different animals is the same. Thus, both mals. A detailed description of tooth auto- the width of the stained band and the transplants has been published recently by width of the unstained adjacent material. Weinreb et al. ('67). These authors trans- are good indicators of the growth of the planted maxillary molars from ten-day old tissue in the interval between the injection rats into the dorsal subcutaneous connec- of the dye and killing of the animal. Of tive tissue and followed the grafts up to the seven dyes tested in this study only two six months after transplantation. Their were good markers of dentin-formation in criteria for continued growth of the trans- normal teeth, i.e., procion red M-8BS and plants included the presence of the calcio- procion scarlet RI-GS. Procion red H-8BS traumatic line, postoperative dentin forrna- was used successfully by Sherman ('69) tion, and the presence of odontoblasts. as a marker of' cementogenesis in replanta- Their results are similar to ours in that tion studies. Procion red M-8BS and scar- all transplants continued to grow after a let M-GS were used by Goland and Grant period of adjustment during which tem- ('68) with good results for staining teeth porary degenerative changes accompanied and bones in situ. Good staining of dentin by an inflammatory reaction were observed. was also reported for procion blue H-5GS Even after six months the transplants were (Prescott et al., '68) and olive green M-3GS still in perfect condition. (Seiton and Engle, '70). Why these dyes In summary, the studies reported here gave such poor results in our tests is not and the few reported in the literature lead clear. to the conclusion that syngeneic tooth germ The second marker, 3H-proline, is incor- transplants are generally accepted. Devel- porated into the collagen of the dentin and opmental disturbances of the transplant eukeratin of the enamel. According to (incomplete root formation, absence of Anderson ('67:) the total tritium activity amelogenesis) can be explained as an ef- in the serum of an animal injected with fect of the transplantation into a hetero- "-proline reaches its peak from four to topical (non-physiologic ) site. six hours after intraperitoneal administra- Markers of dentinogenesis. Two mark- tion of the isotope, and then declines grad- ers of dentin-formation were used in ually until a plateau is reached at about the present study, procion dyes and H'- 20 days. The isotope is picked up first by proline. Procion dyes are monochloro-s- cells, beginning 15 minutes after injection, triazinyl (procion H) or dichloro-s-tri- and then, after about four hours, is trans- azinyl (procion M) derivatives of cyanuric ferred into extracellular structures. There chloride (2,4,6-trichloro-s-triazine).The is essentially no change in the amount of one unsubstituted chlorine atom (two isotope in the dentin at 60 days after in- atoms in procion M) can react with func- jection, whereas the cellular content is de- tional groups of proteins and form stable creased sharply at that time. The auto- covalent bonds with the matrix of bones radiographs of tooth transplants labeled and teeth (Goland and Grand, '68). When with 3€i-prolinc show concentration of the injected into an animal (Seiton and tracer in the dentin where it forms a gran- Engel, '70), the dye is deposited within ular band running parallel to the surface three to six hours at sites of growth in of the tooth. In the experiment described bones and teeth. The uptake of the dye in this paper, the availability of the tracer continues for two to three days, until the was terminated by the grafting, and the TOOTH TRANSPLANTATION 379 effects in the dentin did not last longer Goland, P. P., and N. G. Grand 1968 Chloro-s- than 24 hours. Consequently, the tracer triazines as markers and fixatives for the study band was relatively narrow and clearly of growth in teeth and bones. Am. J. Phys. Anthrop., 29: 201-218. separated the portions of dentin formed be- Grewe, J. M., and W. J. L. Felts 1968 Auto- fore and after the operation. The enamel radiographic investigation of tritiated thymidine matrix, as well as the cellular tissues of incorporation into replanted and transplanted the transplant, showed only low and rather mouse mandibular incisors. J. Dent. Res., 47: diffuse radioactivity. 108-114. Thus, both procion dyes and tritiated Haley, E. W., and E. R. Costich 1969 Im- munological studies on allografts of teeth in proline proved to be excellent markers of inbred hamsters. Transplantation, 8: 91-97. dentinogenesis in transplanted teeth. We Hay, M. F. 1961 The development in. vivo and judged procion dyes as slightly better in vitro of the lower incisor and molars of the markers for the following reasons. They are mouse. Arch. Oral Biol., 3: 86-109. less expensive, their use is much simpler IvPnyi, D. 1962 Bibliography of tooth trans- and less time-consuming, and they are less plantation. Transplant. Bull., 30: 76-83. 1965 Immunologic studies on tooth demanding technically. Also, the band germ transplantation. Transplantation, 3: formed by procion dyes in dentin is sharper 572-576. than the band formed by the radioactive 1968 The role of the major histocom- tracer. patibility system in tooth germ transplantation of the rat. Transplantation, 6: 594-597. ACKNOWLEDGMENTS IvPnyi, D., and Z. Vacek 1964 Homologous and isologous transplantation of tooth germs in We thank Mr. Suresh Savarirayan for rats. Folia Biol. (Praha), 10: 285-292. excellent technical assistance, Dr. James IvPnyiovP, D. 1963 Biological aspects of the K. Avery for providing facilities for the transplantation of teeth. (in Czech). Cs. histological work and for his valuable ad- Stomatol., 63: 51-67. Lefkowitz, W. 1961 Homotransplantation OP vice, and Dr. James R. Hayward for his tooth buds. Arch. Oral Biol., 3: 129-136. critical review of the manuscript. The use Mincer, H. H., and B. R. Jennings 1970 Mor- of vital stains was suggested to us by Drs. phologic and immunologic studies of subcutane- Nathaniel H. Rowe and Melvyn J. Baer. ous incisors allograft in mice. J. Dent. Res., 49: 381-388. LITERATURE CITED Natiella, J. R., J. E. Armitage and G. W. Greene 1970 The replantation and transplantation of Anderson, A. A. 1967 The protein matrixes of teeth. Oral Surg., 29: 397419. the teeth and periodontium in hamsters: A tri- Prescott, G. H., D. F. Mitchell and H. Fahmy tiated proline study. J. Dent. Res., 46: 67-78. 1968 Procion dyes as matrix markers in grow- Coburn, R. J., and B. L. Henriques 1964 Im- ing bone and teeth. Am. J. Physical Anthrop., munological considerations in experimental 29: 219-224. tooth transplantation. Dent. Res., 43: 895 J. Seiton, C., and M. B. Engel 1969 Reactive (Abstract). E. Cohn, S. A. 1957 Development of the molar dyes as vital indicators of bone growth. Am. J. Anat., 126: 373-392. teeth in the albino mouse. Am. J. Anat., 101: 295-319. Sherman, P. 1968 Intentional replantation of Cserepfalvi, M. P., and P. J. Price 1969 Trans- teeth in dogs and monkeys. J. Dent. Res., 47: plantation of a preserved tooth to a monkey: 1066-1071. Preliminary report of a case. J. Dent. Res., 47: Shulman, L. B. 1964 The transplantation 641-643. antigenicity of tooth homografts. Oral Surg., Fleming, H. S. 1952 Early influence of methyl- 17: 389-394. cholanthrene on transplanted tooth germs: A Weinreb, M. M., 1’. Sharav and M. Ickowicz 1967 histopathogenic study. J. Dent. Res., 31: Behaviour and fate of transplanted tooth buds. 398-41 1. I. Influence of bone from different sites on tooth 1953 Effect of certain concentrations of bud autografts. Transplantation, 5: 379-389. fluorides on enamel and dentine as formed in Yoshioka, W., and F. Gonzales 1959 Survival tooth germs. J. Dent. Res., 32: 469-485. of mouse tooth germs after freezing in liquid 1955 Observations concerning the den- nitrogen as demonstrated by differentiation of tal pulp in tooth germ transplants. Oral Surg., isologous transplants. J. Dent. Res., 38: 983-993. 8: 198-205. Zaleski, M., A. Oberszyn, T. Rymaszewska-Kos- Gaunt, W. A. 1956 The development of enamel sakowska, J. Jedrzejczk and A. Sankowski and dentine on the molars of the mouse, with 1967 Immunizing properties of allogeneic den an account of the enamel-free areas. Acta Anat., tal pulp grafts in rabbits. Transplantation, 28: 111-134. 5: 589-596. PLATE 1

EXPLANATION GF FIGURES

2 Mandibular first molar dissected from a five-day old mouse. P, pulp: D, dentin; EM, enamel matrix; EO, remnants of the enamel organ: ERS, epithelial root sheath. H and E. x 40. 3 First molar transplant five days postoperatively. Notice the reduced cellularity of the pulp (P), degeneration of the enamel organ (EO) and disruption of the odontoblastic layer (0).H and E. ;< 40. 4 Apical area of a first molar transplant 12 days postoperatively. Notice the cellularity of the pulp (P), organization of odontoblasts (0) and mass of osteodentin (0s). H and E. X 100. 5 First molar transplant 17 days postoperatively. Notice the formation of a connective tissue capsule (C) around the graft, and granulation tissue (G) around the crown. H and E. X 40.

380 TOOTH TRANSPLANTATION PLATE 1 Jan Klein, Walter R. Secosky and Dagmar Klein

381 PLATE 2

EXPLANATION OF FIGURES

6 Root area of a first molar transplant 21 days postoperatively. PL. periodontal ligament; CE, cementum; D, dentin. H and E. X 200. 7 Coronal area of a first molar transplant 25 days postoperatively. SE, squamous epithelium; CY, epithelial cyst containing degenerated squamous cells. H and E. x 100. 8 First molar transplant 90 days postoperatively Notice the reduction of the pulp chamber (P). A calciotraumatic line (CL) separates pre- and postoperatively formed dentin. H and E. >: 40. 9 First maxillary molar with three incremental growth zones (1-3) in the dentin resulting from three weekly intraperitoneal injections of procion red M-8BS. The mouse was sacrified seven days after the last injection. Photomicrograph of unstained decalcified section was taken with the fluorescence microscope. P, pulp. X 100.

382 TOOTH TRANSPLANTATION PLATE 2 Jan Klein, Walter R. Secosky and Dagmar Klein

383 PLATE 3

EXPLANATION OF FIGURES

10 Apical area of a firs: molar transplant 14 days postoperatively. The recipient was injected with procion red M-8BS seven days prior to sacrifice. Photomicrograph of ;n unstained section was taken with the fluorescence microscope. Notice the irregular staining pattern of the osteodentin (0j and the straight line (L j in the dentin due to the procion dye. x 150. 11 Coronal area of a first molar transplant 19 days postoperatively. The recipient was injected with procicn red M-8BS seven days prior to sacrifice. Photomicrograph of an unstained section was taken with the fluorescence microscope. D, dentin; EM, enamel matrix; P, pulp; L, line due to the procion dye. x 100. 12 Autoradiograph of the coronal area of a first molar transplant, 30 days after transplantation. Notice the heavy labeling of the dentin (D) and the diffuse pattern of labeling of the enamel matrix (EM). The pulp (P) has almost no label at all. The donor was injected with 3H-proline 24 hours prior to sacrifice. H and E. x 100. 13 Higher magnification of area in figure 12. Notice the displacement of the label throughout the newly formed dentin. P, pulpal surface of the dentin. H and E. ;< 200.

384 TOOTH TRANSPLANTATION PLATE 3 Jan Klein, Walter R. Secosky and Dagmar Klein

385