Effect of surgical sympathectomy on bone remodeling at rat incisor and molar root sockets.код для вставкиСкачать
THE ANATOMICAL RECORD 219:32-38 (1987) Effect of Surgical Sympathectomy on Bone Remodeling at Rat Incisor and Molar Root Sockets H.S. SANDHU, M.S. HERSKOVITS, AND I.J. SINGH Division of Oral Biology, Faculty o f Dentistry, The University of Western Ontario, London, Canada N6A 5 C l (H.S.S.); Department ofdnatomy, New York College of Osteopathic Medicine, Old Westbury, Ny 11568 (M.S. H.); Department of Anatomy, New York University Dental Center, New York,NY 10010 (I.J.S.) ABSTRACT Sympathectomy was carried out in 4-week-old Sprague-Dawley rats by unilateral surgical removal of the superior cervical ganglion. Sham-treated rats served as controls. All rats were injected with tetracycline hydrochloride a t surgery a s well as 36 h r prior to sacrifice. Rats were killed at 7, 14, or 21 days following sympathectomy. Mandibular periosteal and endosteal surfaces were analyzed by fluorochrome morphometry. Osteoclasts were identified by acid phosphatase staining, and incisor and molar root sockets were analyzed morphometrically. Following sympathectomy, periosteal and endosteal apposition as well as the rate of mineralization were significantly lower. At the same time, a significant increase in the number of osteoclasts per socket as well as in active and inactive bone resorption surfaces was also seen. All parameters, however, returned to normal values 2-3 weeks after sympathectomy. The data provide the first direct quantitative evidence that sympathetic neurons modulate bone resorption and bone remodeling in vivo. The presence of adrenergic nerves in bone is well documented (Duncan and Shim, 1977), and several recent studies have reported the effects of sympathetic denervation on skeletal tissues. Osteoblastic activity was decreased following sympathetic denervation (Singh et al., 1981,1982; Herskovits and Singh, 1984).The changes in osteogenesis following sympathectomy could possibly be attributed to vascular changes andlor to the loss of direct neurotrophic influences of adrenergic neurons on the osteoblasts. However, there is little information on the effects of denervation on the activity of osteoclasts during bone remodeling. The vascular abnormalities resulting from the reflex sympathetic dystrophy syndrome are known to cause irregular bone remodeling (Basle et al., 1983). Fell (1949) also noted a n increase in resorption spaces in long bones of cats following lumbar sympathectomy. More recently, vasoactive peptides that stimulate osteoclastic activity in vitro (Hohmann et al., 1983) have been shown to be sympathetic in origin (Hohmann et al., 1986). Similar vasoactive peptides have also been identified in human osteosarcoma cells (Hohmann and Tashjian, 1984).These studies suggest that sympathetic denervation may also influence the resorption component of bone remodeling. In the present investigation, the rat mandible model described by Liu and Baylink (1984) was used to study the effects of surgical sympathectomy on bone remodeling at the incisor socket and the molar root socket. MATERIALS AND METHODS Four-week-old male Sprague-Dawley rats were used. The left superior cervical ganglion was surgically removed under sodium pentobarbital anesthesia (5 mgl 100 gm b.wt.) in 15 rats. Another 15 rats served as sham0 1987 ALAN R. LISS. INC treated controls in which all surgicai procedures were accomplished, i.e. the superior cervical ganglion and the sympathetic trunk were visualized but not cut. All rats were injected with tetracycline hydrochloride (15 mg/kg b.wt.) immediately following surgery and again 36 hr prior to sacrifice. The presence of ptosis was considered indicative of a successful sympathectomy. Five experimental and five control rats were killed at 7,14, and 21 days following surgery. At autopsy, left mandibles were freed of soft tissue and fixed in 10%phosphate-buffered formalin (pH 7.4). The mandibles were divided into a medial block and a lateral block between the second and third molars (Fig. 1). Fluorescence Microscope Morphometry The medial blocks were embedded in Spurr medium, and 60-pm-thick sections were cut on a n Isornet@slowcutting machine and lapped down to sections 25 f 5 pm thick on a Moruto lapping machine. These sections were used to analyze the periosteal a s well as the endosteal deposition of bone. Linear periosteal deposition, linear endosteal deposition, and width of the second periosteal and endosteal tetracycline labels were measured on a semiautomatic lBM computer-based image analyzer. The rates of periosteal and endosteal apposition (pmlday) were derived by dividing the average distance between the two tetracycline labels by the number of days between injections (Sandhu and Jande, 1982; Frost, 1983). Baylink et al. (1970) have shown that tetracycline diffuses into bone that has attained up to 20% of its maximum mineral concentration. The time required to reach Received July 10, 1986; accepted March 6, 1987. 33 BONE REMODELING FOLLOWING SYMPATHECTOMY MEOIAL BLOCK 7 ' Y'Y -Embedded in Spurr -60pm section lapped down to 2 5 p m -Fluorochrome Morphometry PERIOSTEAL APPOSITION LATERAL 6?LOCK -Decalcified in 5% Formic Acid -Embedded according to Westen et 01. (1981) -4pm sections lncuboted for Acid Phos. histochemistry (Evans et al., 1979) MOLAR SOCKET INCISOR SOCKET ENDOSTEAL APPOSITION Rate of Initial Mineralization -Active Resorbing Surface -Inactive Resorbing Surface -Number of Osteoclasts/mm -Number of Nuclei/Osteoclast -Area of Osteoclast Fig. 1 . Schematic of a rat mandible showing landmarks and measurements. 20% of maximum mineralization is determined by converting, by means of the apposition rate, the width of the final tetracycline label to units of time. The maximum concentration divided by the time yields the initial mineralization rate (% maximum/hr). Histochemical Morphometry The lateral blocks of mandibles were decalcified in 5% formic acid containing 5% formalin. The bones were embedded in a mixture of 2-hydroxyethyl methacrylate (54.5%); methyl methacrylate (27.25%); 2-hydroxyethyl acrylate (9.10%); propanol (9.10%); and BPO (1%). The whole procedure was done at 0-4°C. This method is very well suited for enzyme histochemistry of bone (Westen et al., 1981). Sections 4 pm thick were stained for acid phosphatase activity according to a method described by Evans et al. (1979). Napthol AS-TFt phosphate was used as substrate and hexazotized pararosaniline as a coupler in 0.1 M acetate buffer (pH 5.5) at 37"C, with or without 10 mM tartrate, and counterstained with methyl green. Osteoclasts were identified by the presence of tartrateresistant acid phosphatase. Since multinucleated cells can appear mononuclear depending upon the plane of the section (Thompson et al., 1975), number of nuclei was not used as a criterion for osteoclastic identification. All sections were cut perpendicularly to the long axis of the incisor (Fig. 4a). The number of osteoclasts and osteoclast nuclei were counted separately in the incisor socket and the third molar root socket. The inner perimeters of incisor socket and molar root were measured as total length of these respective sockets (excluding the marrow spaces). Morphometry was performed with a semiautomatic image analysis system (Southern Micro Instruments, Atlanta, Georgia): The images of bone sections were projected to a video screen and measurements were made on a digitizing pad. The length of the incisor socket did not differ significantly from animal to animal; therefore, data were expressed as the number of osteoclasts per socket, whereas the osteoclast-lined bone surfaces and acid phosphatase-positive bone surfaces were expressed in absolute millimeters. The length of the molar root showed considerable variability. Therefore, the distribution of osteoclasts was expressed as the num- ber of cells per 1 mm of bone surface. The length of osteoclast-lined bone surfaces was calculated as a percentage of the total bone length of the socket. A 2 0 ~ objective was used and the final magnification at the video screen was 81x . The bone surfaces lined by osteoclasts were measured as active resorbing surfaces, whereas acid phosphatase-positive bone surfaces devoid of osteoclasts were termed inactive bone surfaces. The area of osteoclasts was estimated from cell perimeter. Data Analyses Several sections per bone specimen were examined and Student t tests were applied to means & standard errors of the mean. RESULTS All sympathectomized animals but none of the sham controls showed ptosis on the operated side. The average body weight was not significantly different between control and sympathectomized rats a t any time during the experiment. Measures of Bone Formation Figures 2 and 3 show the typical appearance of the double-tetracycline labels in periosteal and endosteal bone, respectively. Linear periosteal apposition, endosteal apposition, and the rate of initial mineralization of periosteum and endosteum were significantly lower in sympathectomized rats than in the controls 1week after denervation (Table 1). However, 21 days after sympathectomy, periosteal and endosteal apposition as well a s the rate of mineralization were not significantly different between the sympathectomized rats and the controls. Measures of Bone Resorption The typical appearance of the molar and incisor root sockets is shown in Figures 4a and 4b, respectively. Acid phosphatase-positive osteoclasts are seen in Figure 4c. Following sympathectomy, the length of the bone surface of the incisor socket is not significantly altered a t either 1 or 2 weeks following surgery (Table 2). The osteoclastic parameters at days 7 and 14 after sympathectomy are presented in Tables 2 and 3 for the incisor 34 H.S. SANDHU, M.S. HERSKOVITS, AND I.J. SINGH Fig. 4. a: A composite of the buccolingual section of rat mandible showing the molar root socket (partly indicated by arrows) and the incisor socket (indicated by triangles). The sections were stained for acid phosphatase. x7. b: A part of the incisor socket showing enamel Fig. 3.Endosteal surface of a mandible from a rat killed 1week after (E);periodontal ligament (PDL); osteoclasts (arrows); and bone (B). The surgery. Tetracycline was administered at surgery and at 36 hr before section was stained for acid phosphatase and counterstained with sacrifice. UV light, ~ 5 3 . methyl green. ~ 5 3 c:. Higher magnification of areas in b, showing acid phosphatase-positive osteoclasts (arrows) and bone (J3). x 340. Fig. 2. Periosteal surface of a mandible from a rat killed 1week after surgery. Tetracycline was administered at surgery and at 36 hr before sacrifice. UV light, ~ 5 3 . and molar socket, respectively. One week after sympathectomy, all measures of bone resorption-length of bone occupied by osteoclasts (active resorbing surface); inactive resorbing surface; number of osteoclasts per incisor socket-were significantly higher in mandibles of sympathectomized rats than in the controls (Table 2). However, the number of nuclei per osteoclast as well as the area of osteoclasts were significantly lower in sympathectomized rats (Table 2). Two weeks after sympathectomy, no differences in bone resorption in the incisor socket were apparent between control and experimental mandibles (Table 2). At the molar root socket, the length of bone surface was quite variable; therefore, the percentage of total bone surface lined by osteoclasts was obtained. This percentage of bone was not statistically different between the controls and the sympathectomized rats (Table 3). In sympathectomized rats, the number of osteoclasts per 1 mm of bone surface was significantly higher; however, the number of nuclei per osteoclast was 35 BONE REMODELING FOLLOWING SYMPATHECTOMY TABLE 1. Bone formation in control and sympathectomized rats 1 week following sympathectomy Controls Measurement Linear periosteal apposition (pdday) Linear endosteal apposition (pdday) Rate of periosteal mineralization (% of maximumlhr) Rate of endosteal mineralization (% of maximumihr) Mean Percentage SEM Sympathectomized of controls' 6.315 & 0.58 5.117 _+ 0.41 81.02 3.267 f 0.45 2.753 f 0.17 84.26 2.54 f 0.22 1.85 f 0.16 72.80 1.94 5 0.05 1.39 & 0.08 70.10 'Control and experimental values are significantly different (P measurements on several tissue sections of five rats. < 0.05). Each value is derived from TABLE 2. Bone resorption parameters at the incisor socket following sympathectomy 1 Week after sympathectomy, mean f SEM Measurement Bone length of incisor socket (mdsection) Length of bone occupied by osteoclasts (mm)or active resorbing surface Length of inactive resorbing surface reversal lacunae (mm) Number of osteoclasts/ incisor socket Number of nuclei/osteoclast Area of osteoclasts (mm2) 2 Weeks after sympathectomy, Controls Sympathectomized 3.48 f 2.08 3.58 f 0.17 NS 3.41 11.0 k 0.873 14.3 f 1.23 0.01 0.56 f 0.143 5.95 k 3.12 34.1 f 2.34 2.75 f 0.059 0.017 f 0.001 P1 Controls mean SEM Sympathectomized P' 3.79 k 0.321 NS 13.0 f 4.45 14.5 f 2.95 NS 0.05 2.34 2.28 f 0.665 NS 45.4 f 4.74 0.01 38.0 f 12.0 46.0 f 8.16 NS 2.06 & 0.079 0.014 5 0.001 0.01 NS 2.52 f 0.11 0.017 5 0.001 2.95 f 0.14 0.014 & 0.001 0.01 NS 0.74 0.47 Each mean and its SEM is estimated from measurements on several tissue sections from five rats, 'Differences between experimental and control values were tested by Student's t statistic; NS = not significant. significantly lower (Table 3). At 2 weeks after sympathectomy, the molar socket bone of control and experimental animals showed no differences (Table 3). DISCUSSION The present study clearly demonstrated a reduction in periosteal bone apposition at the end of the first week following sympathectomy. Three weeks after sympathectomy, these differences in bone apposition between experimental and control animals were not apparent; these findings support our earlier suggestions of a possible time-dependent compensatory mechanism (Singh et al., 1982). The reversible nature of these effects after only 21 days, however, is different from our earlier results of autoradiographic studies of osteoblastic activity (Singh et al., 1981, 1982; Herskovits and Singh, 1984). In those autoradiographic studies, osteoblastic activity and bone formation did not reach normal levels until about 90 days after sympathectomy. Possibly the tetracycline labeling technique used in the present study is not sensitive to the small differences in cell activity that can be assessed by autoradiography. Bone apposition is a direct function of osteoblasts and is dependent on a) the number of differentiated osteo- blasts and b) the activity of osteoblasts. The possibility remains that the number of differentiated osteoblasts may also be affected by sympathectomy. The concomitant decrease in the rate of mineralization along with the reduction in the rate of bone apposition is in accord with the view that rate of mineralization may be controlled by osteoblasts (Baylink et al., 1970). Although it was not clear whether the deficit was only sensory, or both autonomic and sensory, denervation reportedly resulted in lower glycosaminoglycan (GAG) synthesis in newts (Mescher and Munaim, 1986). It has also been shown that the glycoproteins and GAGS are involved in mineralization (Arsenault and Ottensmeyer, 1983). It is possible, therefore, that the greater reduction in mineralization compared to that in bone apposition (71% of controls vs. 81% of controls) noted in our data may be due to denervation-related reduction in GAG levels. It is also clear that, during the first week following sympathectomy, there was an increase in bone resorption with a concomitant decrease in endosteal bone a p position as well as periosteal and endosteal rates of initial mineralization. These findings suggest the presence of sympathetic innervation of both periosteal and endosteal bone surfaces. It is interesting that the nerve 36 H.S. SANDHU, M.S. HERSKOVITS, AND I.J. SINGH TABLE 3. Bone resorption parameters at the molar root following sympathectomy 2 Weeks after sympathectomy, mean f SEM 1Week after sympathectomy, Measurement % Bone length occupied by osteoclasts (active bone resorbing surface) Length of inactive resorbing surface reversal lacunae (mm) Number of osteoclasts/mm bone surface of molar root socket Number of nuclei/osteoclast Area of osteoclasts (mm2) Controls 19.38 2.7 0.11 k 0.042 9.10 k 1.06 3.13 k 0.110 0.018 k 0.001 mean f SEM Sympathectomized 20.32 +_ 1.10 0.519 k 0.494 11.6 * 2.14 2.53 f 0.159 0.019 k 0.002 P1 Controls Sympathectomized NS 12.0 f 3.78 11.8 5 2.47 NS NS 1.04 1.14 f 0.425 NS P1 * 0.51 0.05 19.65 f 6.32 22.71 0.01 2.97 k 0.134 0.017 k 0.001 2.95 & 0.118 0.019 k 0.001 NS 6.72 NS NS NS Each mean and its SEM is estimated from measurements on several tissue sections from five rats. 'Differences between experimental and control values tested by Student's t statistic; NS = not significant. processes in Haversian canals have been shown to terminate in the endosteum of the cranial marrow spaces (Retzlaff et al., 1981). Some role of sympathetic neurons in bone marrow cell dynamics and hemopoiesis has also been shown (Webber et al., 1970; Miller and McCuskey, 1973). Although Fell (1949) observed increased resorption spaces in bones following sympathectomy, the present investigation presents the first direct in vivo quantitative evidence that bone resorption was increased after sympathetic denervation. The number of osteoclasts as well as the total resorption surface was significantly greater in the incisor socket of sympathectomized rats than in the controls. The number of osteoclasts and their activity are influenced by systemic factors such as parathyroid hormone, calcitonin, and vitamin D3 metabolite 1,25-(OH)2D3(Roberts, 1975; Thompson et al., 1975; Luben et al., 1976; Holtrop et al., 1979; Warshawsky et a1.,1980; Liu et al., 1982; Chambers et al., 1985) Prostaglandins, Osteoclast Activating factor and Interleukin-I are believed to be local regulators of the number and activity of osteoclasts (Klein and Raisz, 1970; Tashjian et al., 1973; Horton et al., 1974; Luben et al., 1977; Holtrop and Raisz, 1979). Recently, some vasoactive peptides of sympathetic origin have been shown to modulate osteoclastic function (Hohmann et al., 1983, 1986). It is possible that the sympathetic innervation acts through modulation of parathyroid activity, thus accounting for a time-dependent compensatory mechanism in the bone changes. Osteoclasts are not known to divide; however, it is believed that new nuclei from monocytic fusion can be added to osteoclasts under systemic stimuli (Jaworski et al., 1981; Scheven et al., 1986). Our investigation showed a significant decrease in the number of nuclei per osteoclast following sympathectomy, possibly reflecting a reduced systemic need for bone resorption. Increased pO2, resulting from sympathectomy, is known to cause inhibition of osteoblastic activity, of growth of epiphyseal plate, and of cartilage calcification (Brighton et al., 1969; Brighton and Heppenstall, 1971; Brooks, 1972). The effects of increased pO2 on bone resorption, however, are controversial. A decrease in bone resorption under hyperoxic tissue culture conditions has been reported (Gray and Hamblen, 1976); other investi- gators believe that pO2 has no effect on bone resorption (Asher and Sledge, 1968; Melcher and Hodge, 1968).An increase in bone resorption following sympathectomy in cats (Fell, 1949) and following a n increase in pO2 has also been reported (Goldhaber, 1966; Gray et al., 1978, 1980). The reversible nature of the effects of sympathectomy points towards transient vascular changes as the cause. However, no changes in blood flow could be detected following lumbar sympathectomy (Hohmann et al., 1986). Furthermore, one cannot overlook the possibility of indirect sympathetic influences on bone cell compartments as the surgical removal of a single superior cervical ganglion unilaterally certainly does not destroy the whole adrenergic system, which may compensate for the local deficit caused by the ganglionectomy. Pharmaceutical sympathectomy, which would destroy all of the sympathetic system, has been shown to result in a n inhibition of osteogenesis (Singh et al., 1981, 1982; Herskovits and Singh, 1984); however, the effects on bone resorption parameters were not investigated in those studies. It has also been reported that the periodontal fibroblasts and the odontoblasts of the rat incisor do not respond to a pharmaceutical sympathectomy (Klein et al., 1981; Herskovits and Singh, 19861, whereas surgical sympathectomy resulted in a delay in the proliferative response of dental pulpal cells to trauma (Chiego et al., 1986). It is not at all clear whether the sympathetic modulation of bone remodeling is directly locally mediated or if it is the result of systemic effects. A differential, function-dependent response to systemic perturbations has been shown in trabecular bone in a series of excellent reports (Frost, 1973a,b, 1983) as well as in alveolar bone (Saffar and Baron, 1977; Vignery and Baron, 1978, 1980; Liu and Baylink, 1984). Our study showed that even sites with similar functions (incisor socket and molar socket) may respond differently to systemic or local stimuli. The higher response of osteoclasts at the incisor socket compared to the molar socket could be attributed to a more rapid remodeling of bone at the continually erupting incisor. It appears reasonable to assume that the osteoclasts were present on the acid phosphatase-positive bone surfaces sometime prior to the sacrifice of the rats. An experiment with several shorter observation periods BONE REMODELING FOLLOWING SYMPATHECTOMY (hours rather than days) would be needed to further elucidate the role of adrenergic neurons in the spatial dynamics of osteoclasts, as well as the sequence of events following surgical sympathectomy that lead to increased bone resorption. Of particular interest would be the regulation of osteoclastic movement and the role of vasoactive peptides in the modulation of osteoclastic function. ACKNOWLEDGMENTS This research was supported by a grant from Ontario Ministry of Health (H.S.) and grant RR 05332 to New York University (I.J.S.).We thank Mrs. Linda Monteith and Mr. Stephen Waldrup for skillful secretarial assistance and Mr. Lynden Keeping for his technical help. LITERATURE CITED Arsenault, A.L., and F.P. Ottensmeyer (1983) Quantitative spatial distribution of calcium, phosphorous and sulfur in calcifying epiphysis by high resolution electron spectroscopic imaging. Proc. Natl. Acad. Sci. USA, 80:1322-1326. Asher, M.A., and C.B. Sledge (1968) Hyperoxia and in vitro bone resorption. Clin. Orthop., 61:48-51. Basle, M.F., A. Rebel, and J.C. Reinier (1983) Bone tissue in reflex sympathetic dystrophy syndrome-Sudeck’s atrophy: Structural and ultrastructural studies. Metab. Bone Dis. Rel. Res., 4:305-311. Baylink, D., M. Stauffer, and J. Wergedal (1970) Formation, mineralization and resorption of bone in vitamin D deficient rats. J. Clin. Invest., 49:1122-1134. Brighton, C.T., and R.B. Heppenstall(1971) Oxygen tension of healing fractures in the rabbit. J. Bone Joint Surg., 54A:323-332. Brighton, C.T., R.D. Ray, L.W. Soble, and K.E. Kuettner (1969)In vitro epiphyseal plate growth in various oxygen tensions. J. Bone Joint Surg., 51A:1383-1396. Brooks, M. (1972) Blood supply of developing bone and its possible bearing on malformations of the limb and face in congenital haemangiomatous disorders. Proc. R. Soc. Med., 6.5~597-599. Chambers, T.J., P.M.J. McSheehy, B.M. Thomson, and K. Fuller (1985) The effect of calcium-regulating hormones and prostaglandins on bone resorption by osteoclasts disaggregated from neonatal rabbit bones. Endocrinology, 116:234-239. Chiego, D.J., R.M. Klein, J.K. Avery, and I.M. Gruhl (1986) Denervation-induced changes in cell proliferation in the rat molar after wounding. Anat. Rec., 214:348-352. Duncan, C.P., and S.S. Shim (1977) The autonomic nerve supply of bone. J. Bone Joint Surg., 59B:323-330. Evans, R.A., C.R. Dunsten, and D.J. Baylink (1979) Histochemical identifications of osteoclasts in undecalcified sections of human bone. Miner. Electrolyte Metab., 2:179-185. Fell, W.A. (1949)The effect of sympathectomy on the size of Haversian canals of the cat. J. Anat., 83~67-68. Frost, H.M. (1973a) Bone Remodeling and Its Relation to Metabolic Bone Disease. Charles C. Thomas, Springfield, IL. Frost, H.M. (1973b)The origin and nature of transients in human bone remodeling dynamics. In: Clinical Aspects of Metabolic Bone Disease. B. Frame, A.M. Parfitt, and H. Duncan, eds. Excerpta Medica, New York. Frost, H.M. (1983) Bone histomorphometry: Choice of marking agent and labeling schedule. In: Bone Histomorphometry: Techniques and Interpretation. R.R. Recker, ed. CRC Press, Boca Raton FL, pp. 37-52. Goldhaber, P. (1966) Remodelling of bone in tissue culture. J. Dent. Res., 45490-499. Gray, D.H., and D.L. Hamblen (1976) The effects of hyperoxia upon bone in organ culture. Clin. Orthop., 119:225-230. Gray, D.H., J.M. Katz, and K.S. Speak (1978) The effects of varying oxygen tension upon bone resorption in vitro. J. Bone Joint Surg. 6OB~575-578. Gray, D.H., J.M. Katz, and K.S. Speak (1980) The effect of varying oxygen tension on hydroxyproline synthesis in mouse calvaria in vitro. Clin. Orthop., 146:276-281. Herskovits, M.S., and I.J. Singh (1984) Effect of guanethidine-induced sympathectomy on osteoblastic activity in the rat femur evaluated by 3H-proline autoradiography. Acta Anat., 120:151-155. 37 Herskovits, M.S., and I.J. Singh (1986)Autoradiography of 3H-proline uptake by fibroblasts of the periodontal ligament and odontoblasts of the rat incisor after guanethidine-induced sympathectomy. Arch. Oral Biol., 31:415-417. Hohmann, E.L., and A.H. Tashjian, Jr. (1984) Functional receptors for vasoactive intestinal peptide on human osteosarcoma cells. Endocrinology 114:1321-1327. Hohmann. E.L., L. Levine, and A.H. Tashjian, Jr. (1983) Vasoactive intestinal peptide stimulates bone resorption via a cyclic adenosine 3’, 5‘ monophosphatedependant mechanism. Endocrinology, 112:1233-1239. Hohmann, E.L., R.P. Elde, J.A. Rysavy, S. Einzig, and R.L. Gebhard (1986) Innervation of periosteum and bone by sympathetic vasoactive peptides containing nerve fibres. Science, 232:868-871. Holtrop, M.E., and L.G. Raisz (1979) Comparison of the effects of 1,25(OH)&, prostaglandin E2 and osteoclast-activating factor with parathyroid hormone on the ultrastucture of osteoclasts in cultured long bones of fetal rats. Calcif. Tissue Int., 29:201-205. Holtrop, M.E., G.J. King, K.A. Cox, and B. Reit (1979) Time-related changes in the ultrastructure of osteoclasts after injection of parathyroid hormone in young rats. Calcif. Tissue Int., 27:129-135. Horton, J.E., J.J. Oppenheim, S.E. Mergenhagen, and L.G. Raisz (1974) Macrophage-lymphocyte energy in the production of osteoclasts activity factor. J. Immunol., 113:1278-1287. Jaworski, Z.F.G., B. Duck, and G. Sekaly (1981)Kinetics of osteoclasts and their nuclei in evolving secondary Haversian system. J. Anat., 133:397-405. Klein, D.C., and L.G. Raisz (1970)Prostaglandins: Stimulation of bone resorption in tissue culture. Endocrinology, 86:1436-1440. Klein, R.M., D.J. Chiego, and J.K. Avery (1981) Effects of guanethidine-induced sympathectomy on cell proliferation in the progenitive compartments of neonatal mouse incisor. Arch. Oral Biol., 26:319-325. Liu, C.C., and D.J. Baylink (1984) Differential response in alveolar bone osteoclasts residing at two different bone sites. Calcif. Tissue Int., 36:397-405. Liu, C.C., J.I. Rader, H. Gruber, and D.J. Baylink (1982) Acute reduction in osteoclast number during bone repletion. Metab. Bone Dis. Relat. Res., 4:201-209. Luben, R.A., G.L. Wong, and C. Cohn (1976) Effects of parahormone and calcitonin on citrate and hyaluranate metabolism in cultured bone. Endocrinology, 98:413419. Luben, R.A., G.L. Wong, and C. Cohn (1977) Parathormone-stimulated resorption of devitalized bone by cultured osteoclast-type bone cells. Nature, 265:629-630. Melcher, A.H., and G.M. Hodge (1968) In uitro culture of an organ containing mixed epithelial and connective tissues on a chemically defined medium. Nature, 219:301-302. Mescher, A.L., and S.E. Munaim (1986) Changes in the extracellular matrix and glycosaminoglycan synthesis during the initiation of regeneration in adult newt limb. Anat. Rec., 214t424-431. Miller, M.L., and R.S. McCuskey (1973)Innervation of bone marrow in the rabbit. Scand. J. Haematol., IOr17-23. Retzlaff, E., F. Mitchell, J. Upledger, J. Vredeveoogd, and J. Walsh (1981) Light and scanning microscopy of nerve fibres within the parietal bones of primates. Anat. Rec., 199:201A. Roberts, W.E. (1975)Cell population dynamics of periodontal ligament stimulated with parathyroid extract. Am. J. Anat., 143:363-370. Saffar, J.L., and R. Baron (1977) A quantitative study of osteoclastic bone resorption during experimental periodontal disease in Golden Hamster. J. Periodont. Res., 12:387-394. Sandhu, H.S., and S.S. Jande (1982) Effects of beta-aminopropionitrile on formation and mineralization of rat bone matrix. Calcif. Tissue Int., 34:80-85. Scheven, B.A., W.M. Elizabeth, E.W. Kawileranag-DeHaas, A. Wessenaar, and P.J. Nijweide (1986) Differentiation kinetics of osteoclasts in the periosteum of embryonic bones in vivo and in vitro. Anat. Rec., 214~418-423. Singh, I.J., R.M. Klein, and M. Herskovits (1981) Autoradiographic assessment of 3H-proline uptake by osteoblasts following guanethidine-induced sympathectomy in the rat. Cell Tissue Res., 216:215220. Singh, I.J., M.S. Herskovits, D.J. Chiego, Jr., and R.M. Klein (1982) Modulation of osteoblastic activity by sensory and autonomic innervation of bone. In: Factors and Mechanisms Influencing Bone Growth. A.D. Dixon and B.G. Sarnat, eds. Alan R. Liss, New York, pp. 534-551. Tashjian, A.H., E.F. Voelkel, P. Goldhaber, and L. Levine (1973)Prostaglandins, bone resorption and hypercalcaemia. Prostaglandins, 3:515-524. 38 H.S. SANDHU, M.S. HERSKOVITS, AND I.J. SINGH Thompson, E.R., D.J. Baylink, and J.E. Wergedal (1975) Increase in number and size of osteoclasts in response to calcium or phosphate deficiency in rat. Endocrinology, 97:283-289. Vignery, A., and R. Baron (1978) Effects of parathyroid hormone on the osteoclastic pool, bone resorption and formation in rat alveolar bone. Calcif. Tissue Res., 26:23-28. VignerY, A., and R. Baron (1980) Dynamic histomorPhometrY of ahalar bone remodeling in the adult rat. Anat. Rec., 196:191-200. Warshawsky, H., D. Goltzman, M.P. Rouleau, and J.J.M. Bergeron (1980) Direct in vivo demonstration by autoradiography of specific binding sites for calcitonin in skeletal and renal tissues of the rat. J. Cell Biol., 85:682-694. Webber, R.H., J.R. Dedice, J.R. Ferguson, and J.P. Powell (1970) Bone marrow response to stimulation of the sympathetic trunks in rats. Acta Anat., 77:92-97. Westen, H., K.F. Muck, and L. Post (1981) Enzyme histochemistry on bone marrow sections after embedding in methacrylate at low temperature. Histochemistry, 70:95-105.