T H E ANATOMICAL RECORD 202:203-208 (1982) Vascular Turnover of a Continuously Growing Organ: The Rat Incisor S. PITARU, Y. MICHAELI, AND G. ZAJICEK Department of Oral Biology, School of Dentistry, Tel Auiu Uniuersity, Tel Auiu (S.l?);and the Department of Anatomy and Medicine (EM.) and the Hubert H . Humphrey Center for Experimental Medicine and Cancer Research (G.Z.1, The Hebrew Uniuersity-Hadassah Medical School, Jerusalem, Israel ABSTRACT The vascular tissue supplying the pulp of the continuously growing and developing rat incisor was quantitatively analyzed in six female rats, weighing approximately 200 gm each. One hour after a single administration of 'H-TdR, 1 pCilgm body weight, the rats were sacrificed and the lower left jaws excised and processed histologically. Every 20th section was evaluated for venular and arterial count, endothelial cell count, and percentage of labeled endothelial cells. I t was found that the proliferative capacity of the vascular tissue of the rat incisor is confined to its most apical 3.0 mm-a zone defined as the vascular progenitor compartment. Daily production of endothelial vein cells and of veins within the progenitor compartment were calculated to be 7,392 cellslday and two veinslday, respectively. Generation time of these cells was found to be 3.56 days. Endothelial cell production within the progenitor compartment provides for the formation of new vessels and for the enlargement and elongation of existing ones. The vessels originating within the progenitor compartment move in an incisal and centripetal direction, while simultaneously undergoing a continuous process of aging, until their ultimate disintegration. The proliferation of vascular tissue has been intensively studied during the past decades. Most reports on this subject deal with regenerating tissues (Clark and Clark, 1939;Cliff, 1963, 1965; Schoefl, 1963) or with the influence of solid tumors or embryonic tissues on the process of vascular proliferation and differentiation (Cavallo et al., 1973; Huseby et al., 1975; Crim and Huseby, 1976; Folkman and Cotran, 1976; Ausprunk and Folkman, 1977). Little is known, however, concerning the behavior of vascular tissue in a continuously growing adult organ, such as the rat incisor, one of the most complex organs in the mammal. The continuous attrition at the distal end of the incisor is balanced by formation of odontogenic tissues at its basal end, deep in the bony socket, and their continuous incisal migration. During their movement the basal cells differentiate and produce tooth substance. Thecells migrate at a rate of approximately 450 pmlday, equal to the rate of eruption (Michaeli and Weinreb, 1968; Zajicek, 1976). The pulp of the rat incisor is supplied by vessels that penetrate the tooth through a foramen situated at its apical end (Fig. 1).The pattern of these vessels, as represented in Figure 2, was 0003-276)(182/2022-0203$02.00 0 1982 Alan R. Liss. Inc. described in detail by Moe et al. (1979).Generally, three principle rows of vessels are observed: a centrally located row consisting of arterioles, and two rows of venules located at the lateral and medial sides. Many capillaries are located peripheral to the principle clusters of vessels, especially near the pulpodentinal junction. Different studies (Ness and Smale, 1959; Robins, 1968; Zajicek, 1976)have indicated the possible proliferative capacity of the vascular tissue. Recently, it has been suggested (Moe et al., 1979)that the vascular tissue, like other tissues forming the continuously growing incisor, is continuously renewed. However, a quantitative study of the process of continual renewal in this particular type of vascular tissue has never been undertaken. In the present investigation this process is quantitatively estimated in order to elucidate some of the properties of vascular tissue supplying a continuously growing adult organ. Received April 30. 1981: accepted J u n e 10, 1981 Dr Sandu Pitaru's present address is Medical Research Council Group in Periodontal Physiology. 4384 Medical Sciences Building. Toronto, Ontario M5S 1A8 Canada. 204 S. PITARU, Y. MICHAELI, AND G. ZAJICEK LABIAL Fig. 1. Diagrammatic representation of longitudinal section of lower rat incisor, showing the apical foramen (AF). the pattern of vein supply, and the boundaries of the progenitor compartment (PC). Bar A indicates approximate level of cross section shown in Figure 2. d, Dentine; e, enamel. MATERIAL AND METHODS Six female albino rats of the Hebrew University strain, each weighing approximately 200 gm, were intraperitoneally injected with tritiated thymidine (3H-TdR,1.9 CilmM), 1 pCilgm body weight. One hour later the animals were sacrificed, and the left mandibles were excised, fixed in Bouine-Holland solution, and decalcified in 10% EDTA. The mandibles were embedded in paraplast and cross-sectioned serially at 6 pm. The sections were dipped into Illford L4 emulsion, exposed for 3 weeks, developed in Kodak Dektol 19 for 3 minutes, fixed in Kodak Rapid Fixer for 10 minutes, and stained with hematoxylin-eosin. Starting from the basal end of the incisor, every 20th microscopic section of each left mandible was evaluated up to a distance of 3.5 mm. The evaluation of each section consisted of three procedures, performed for venules and arterioles separately: (1)mapping the position of each vessel according to its location relative to the labial side of the tooth; thus, every vessel was ranked on a labiolingual axis (Fig. 2); (2) counting the total number of arterioles and venules and the number of labeled and unlabeled cells of each vessel; and (3)measuring the diameters of each vessel with the aid of a micrometer. Using the data obtained, the following values were computed for venules and arterioles separately in every evaluated section: (1)the total endothelial cell count; (2) the percentage of labeled endothelial cells; (3)the perimeter of each vessel (computed by assuming each vessel had a perfect elliptical form);(4) the average endothelial cell count per vessel; and (5) the mean internuclear distance, which equals the average diameter of an endothelial cell (computed by dividing the average perimeter of arterioles or venules by the average endothelial cell count per vessel). The progenitor compartment of endothelial cells was defined according to Zajicek (1976). The total number of endothelial cells (venular or arterial) in the compartment was computed using the formula: n, N = f n(x) dx (1) n, where N represents the total number of endothelid cells in the progenitor compartment and n(x) the number of endothelial cells per section at a certain distance from the basal end of the rat incisor. Daily endothelial cell production was estimated by determining the average diameter (D) of an endothelid cell in a transverse section of the progenitor compartment; on the assumption that the same value is valid in a longitudinal direction, each endothelial cell progresses daily a distance of S cells, such that: S= 450 pm (Daily eruption rate) D p n (average diameter of an endothelial cell). Since the total number of endothelial cells at the distal limit of the progenitor compartment VASCULAR TURNOVER O F RAT INCISOR PULP LABIAL 205 in the progenitor compartment by the length (in millimeters) of the compartment, and multiplying the result by the daily rate of eruption of the rat incisor. RESULTS The results obtained for arterioles and venules were almost identical. Since the majority of studies that have investigated the development, growth, and turnover of vascular tissue have dealt with the arterial part, it was decided to describe in the present study the venular part. The observed and calculated results obtained for the venules are graphically depicted in Figures 3-7 in which the abscissa represents the length of the tooth, and in Figures 8 and 9 in a 0 0 0 0 0 7 4 15 10 @ 5 0 Fig. 3. Graphic illustration of distribution of the percentage of labeled endothelial vein cells along sagittal axis of the tooth. The abscissa represents the distance from the basal end of the tooth. Each point represents the mean, and the bar one standard error of the mean. LINGUAL Fig. 2. Diagrammatic representation of cross section of lower rat incisor, showing general pattern of venules (v)and arterioles (a) within the pulp cavity. Vessels are ranked according to their position on the labiolingual axis of the tooth. d, Dentine; e, enamel. equals n,(x),daily cell production P,,, is calculated as: Pidl= S n,(x) (3) Taking into account all endothelial cells comprising the progenitor compartment cycle and the growth fraction G F = 1, the average turnover time t,,,equals the generation time of cells in the compartment (Michaeliet al., 1978);thus, The daily production of veins was computed by dividing the total number of veins produced Fig. 4. Graphic illustration of progressive increase in total endothelial vein cell count per section along sagittal axis of the tooth up to 3 mm from its basal end, the distal limit of the progenitor compartment. Dotted area beneath curve represents the total number of endothelial vein cells found in the progenitor compartment. Each point represents the mean, and the bar one standard error of the mean. S. PITARU, Y. MICHAELI, AND G. ZAJICEK 206 1 5 1 2 3 5 mm Fig. 7. Graph illustrating mean number of endothelial cells per vein along the sagittal axis of the tooth. Each point represents the mean, and the bar one standard error of the mean. Fig. 5. Graph depicting mean internuclear distance of endothelial vein cells along the sagittal axis of the incisor. Average distance in the middle of the progenitor compartment is approximately 13.4 pm. 1 2 3 mm Fig. 6. Graphic illustration of mean number of veins per section along sagittal axis of incisor. The increase in the number of veins per section levels off at the distal limit of the progenitor compartment. Each point represents the mean, and the bar one standard error of the mean. Daily production of vein cells The curve in Figure 4 reveals that the total vein cell count per section increased in almost linear progression throughout the progenitor compartment, from 35 cells per section at its proximal limit to 220 cells per section at its distal boundary. Further distal to this point the total vein cell count leveled off. The internuclear distance of the vein cells also increased progressively (Fig. 5), from 11.4 pm at the proximal end of the progenitor compartment to approximately 15.2 pm at the distal border. The average diameter of a progenitor endothelial cell was approximately 13.4 pm. According to equation (2), each endothelial cell progresses a distance S of 33.6 cells. Using formula (3), the daily vein cell production P,,, was found to be 7,392 cellslday. Total cell count of the progenitor compartment The total cell count of the progenitor compartment N is represented graphically by the dotted area in Figure 4. Using equation (l),N was which the abscissa represents the ranking num- found to equal 26,305 cells. ber of venules on the labiolingual axis of the rat Generation time evaluation incisor. The great majority of labeled endothelial vein Based upon the value determined for N and cells was noted within the most apical 3.0 mm of for P,,,and by using equation (4),the generation the basal end of the rat incisor (Fig. 3). The per- time was estimated to be 3.56 days. Thus, the centage of labeled cells increased gradually in growth process requires that each endothelial successive sections from the basal end and cell in the progenitor compartment reproduce reached a peak at a distance of approximately every 3.56 days. 1.3 mm. It thereafter decreased, reaching the Daily production of veins lowest value a t a distance of 3.0 mm from the basal end. From this point on the number of The number of veins per section increased labeled cells was negligible. This part of the throughout the length of the progenitor compulp, within which endothelial cell proliferation partment and levelled off at its distal end (Fig. takes place, is defined as the progenitor com- 6). A total of 13.3 veins are produced throughpartment of the pulp vascular tissue. out the 3.0-mm length of the compartment, a 207 VASCULAR TURNOVER OF RAT INCISOR PULP mean of 4.4 veinslmm (13.313). Since the eruption rate of the incisor is 450 pmlday, vein p r o duction is 4.4 X 0.45, or two veinslday. The mean number of endothelial cells per vein (Fig. 7) increased progressively throughout the vascular progenitor compartment and leveled off at its distal end. The mean venular perimeter increased linearly throughout the compartment, reflecting the increase in the number of endothelial cells and in their diameter. The mean cell count of all venules with identical ranking numbers was determined for all the evaluated sections (Fig. 8).The results indicate a relation between the position of a vein on the labiolingual axis of the tooth and its cell count; that is, the more centrally located the vein, the greater the number of cells that comprise it. The mean percentage of labeled vein cells of all venules with identical ranking numbers was computed in a similar manner. The results were divided into three equal groups representing the labial, central, and lingual zones of the tooth. The results of each group, represented in Figure 9, were subjected to an analysis of variance and were found to differ statistically at F = 0.025. It is evident that the more centrally located veins have a lower ratio of labeled cells. I 1 2 3 4 5 6 7 8 9 Fig. 8. Graph illustrating change in mean cell count per section of all veins with identical ranking numbers, according to their position along the labiolingual axis of the tooth (see Fig. 2). The abscissa depicts the labiolingual axis, the numbers represent the ranking numbers of the vessels. The vein ranked No. 1 is the most labial. Each point represents the mean. and the bar one standard error of the mean. DISCUSSION The present study indicates that the vascular tissue supplying the rat incisor pulp is endowed with proliferative properties which are confined within the most apical 3.0 mm of the pulp-a zone defined as the vascular progenitor compartment. The new endothelial cells originating in this compartment are utilized in different ways. They contribute to the formation of new vessels and to the enlargement of existing ones, as deduced from the progressive increase calculated in the number of veins and in the number of endothelial cells per vein throughout the progenitor compartment (Figs. 6 , 7 ) . Moreover, the continuous eruption of the rat incisor displaces the tissues produced at its basal end incisally, a process requiring continuous elongation of the supplying vessels and, obviously, the formation of additional new endothelial cells. Careful consideration of the data reveals that the procedure described herein is a course of related events, all part of an overall process of continuous renewal of the vascular tissue supplying the rat incisor. The continuous elongation of the vessels at their base enables migration of the older parts in an incisal direction. Therefore, the more incisal the location of a 1 2 1 3 GROUP Fig. 9. Histogram showing mean percentage distribution of labeled endothelial vein cells arranged in three groups: 1 = three most labial vessels; 2 = three central vessels; 3 = three most lingual vessels. Bar represents one standard error of the mean. given segment of a vessel, the older it is. Since the number of cells per vein (Fig. 7) also increases in a basoincisal direction, this value may be used as an indirect index for estimating the age of a vein at a given distance from the apex. The curve in Figure 8 describes the relation between the number of cells per vein and the 208 S. PITARU, Y. MICHAELI, AND G. ZAJICEK location of the vein on the labiolingual axis of the tooth. I t is evident that the higher the cell count per vein-and consequently the older the vessel-the more centrally the vein is located. It can therefore be concluded that new vessels are formed within the vascular progenitor compartment at theperiphery of thepulp, functioningas suppliers for newly formed segments of the Pulp. Further evidence for this is provided by the data in Figure 9, which indicate a higher percentage of labeled cells in the peripheral group of veins than in the centrally located veins, a fact in accordance with the low proliferative capacity of mature endothelial cells (Folkman and Cotran, 1976). As mentioned above, the continuous eruption of the tooth displaces the young tissue-vessel complex in a distal direction. Since the pulp is cone-shaped, tapering at its incisal edge, concomitant with the vessel's incisal migration is its centripetal displacement. Two simultaneous and related events are therefore characteristic of the vessels supplying the rat incisor pulp: bidirectional displacement accompanied by continuous aging. Since the tooth is in a steady-state condition, the same amount of vessels formed must be destroyed. The most centrally located vessels are the oldest, so they are expected to disintegrate together with the tissues they supply, now located in the most distal part of the tooth. In a histologic study on the arterial part of the vascular tissue supplying the pulp of the upper rat incisor, Moe et al. (1979)also suggested the continuous cycling of the arterial bed, thus providing further evidence for this concept. The distribution of mitotic figures observed in arterial endothelialcells was generally similar to the distribution of venular and arterial labeled endothelial cells found in our study. However, in Moe's study the mitotic figures were observed in arterial endothelial cells also in the middle and incisal parts of the pulp. Since the number of mitotic figures observed is not reported, a comparison between the two findings is not possible. The histopathologic findings of regression and degeneration observed by Moe et al. (1979) in the centrally located arterioles supplying the most incisal part of the tooth confirm our kinetic findings regarding the relation between the location of a vessel and its age. The present study reveals that vascular tissue supplying the rat incisor has a progenitor compartment, like all other tissues forming the rat incisor (Ness and Smale, 1959; Smith and Warchawsky, 1975; Zajicek et al., 1979). This may serve as a good model for further investigation concerning vascular tissue proliferation, differentiation, maturation, and disintegration. ACKNOWLEDGMENTS The authors wish to express their gratitude to Prof. M.M. Weinreb for his helpful remarks during the preparation of this manuscript. The authors thank Misses Sylvie Mayer, Jardena Mazor, and Gloria Ganzach for their help in preparing this manuscript. LITERATURE CITED Ausprunk. D.H., and J. Folkman (1977) Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvasc. Res. 14: 53-65. Cavallo, T.. R. Sade, J. Folkman, and R.S. Cotran (1973)Tumor angiogenesis: Rapid induction of endothelial mitoses demonstrated by autoradiography. J. Cell Biol., 54: 408-420. Clark, E.R., and E.L. Clark (1939) Microscopic observation on growth of blood capillaries in the living mammal. Am. J. Anat., 64: 251-299. Cliff, W.J. (1963)Ohservationsonhealingtissue:Acombined light and electron microscopic investigation. Philos. Trans. R. SOC.Lond. (Biol.),246: 305-325. Cliff, W.J. (1965) Kinetics of wound healing in rabbit ear chamber, a time lapse cinemacroscopic study. Q. J. Exp. Phys., 50: 79-89. Crim. J.W., and R.A. Huseby (1976) Initial events in the vascularization of day-old mouse testes implanted into the inguinal gland fat p a d A light microscopic and autoradiographic study. Microvasc. Res., 12: 141-156. Folkman. J., and R. Cotran (1976)Relation of vascular proliferation to tumor growth. Int. Rev. Exp. Pathol. 16: 208-245. Huseby, R.A., C. Currie, V.A. Langerborg, and S. Garb (1975) Angiogenesis about and within grafts of normal testicular tissue: A comparison with transplanted neoplastic tissue. Microvasc. Res., 10: 396-413. Michaeli, Y.. and M.M. Weinreb (1968)Role of attrition in the physiology of the rat incisor. 111. Prevention of attrition and occlusal contact in the non-articulating incisor. J. Dent. Res.. 47: 633-638. Michaeli. Y., G . Zajicek, and J. Regev (1978)The adaptation of a two compartment cell renewal system to external demands. Cell Tissue Res., 194: 163-170. Moe, H., N. Thorball, and H.W. Nielson (1979) Structural alterations in proliferating remodeling and regressing tooth pulp arterioles. Cell Tissue Res., 203: 339-354. Ness. A.R.. and D.E. Smale (1959) The distribution of mitoses and cells in the tissues bounded by the socket wall of the rabbit mandibular incisor. Proc. R. SOC.Br., 151: 106-128. Robins, M.W. (1968)Growth and Eruptionof theRat Incisor. Royal Dental Hospital of London, School of Dental Surgery (Ph.D. thesis). Schoefl. G.J. (1963)Studies on inflammation. 111. Growing capillaries: Their structure and permeability. Virchows Arch. [Pathol. Anat. Phys.]. 337: 97-141. Smith, C.E., and H. Warshawsky (1975) Histological and three-dimensional organization of the odontogenic organ in the lower incisor of 100 gram rats. Am. J. Anat., 142: 403-430. Zajicek. G. (1976)The rodent incisor tooth proliferon. Cell. Tissue Kinet., 9: 207-214. Zajicek, G., Y. Michaeli. and J. Regev (1979) On progenitor cell migration velocity Cell Tissue Kinet.. 12: 453-460.