Differentiation kinetics of osteoclasts in the periosteum of embryonic bones in vivo and in vitro.код для вставкиСкачать
THE ANATOMICAL RECORD 214:418-423 (1986) Differentiation Kinetics of Osteoclasts in the Periosteum of Embryonic Bones In Vivo and In Vitro BEN A.A. SCHEVEN, ELISABETH W.M. KAWILARANG-DE HAAS, ANNE-MARIE WASSENAAR, AND PETER J. NIJWEIDE Laboratory of Cell Biology and Histology, University of Leiden, 2333 AA Leiden, The Netherlands ABSTRACT Osteoclast progenitors are seeded via the blood stream in the mesenchyme surrounding embryonic long bone models long before the appearance of multinucleated osteoclasts. The proliferation and differentiation of these progenitors in embryonic mouse metatarsal bones was studied with acid phosphatase (AcP) histochemistry and 3H-thymidine autoradiography. In vivo, tartrate-resistant, acid phosphatase-positive, mononuclear cells appear in the periosteum (AcPP-P cells) at the age of 17 days (after conception). On day 18, AcP-positive, multinucleated osteoclasts invade the bone rudiment and start resorbing the calcified cartilage matrix, resulting in the formation of the marrow cavity. The kinetics of osteoclast formation in vitro was studied in metatarsal bones of embryonic mice of different ages cultured in the continuous presence of 3H-thymidine. In young bones (15 days), mainly proliferating, 3H-thymidine-incorporating progenitors gave rise to AcPP-P cell and osteoclast formation. In older bones (16 and 17 days) osteoclasts were progressively more derived from postmitotic, unlabeled precursors. Irradiation of the metatarsal bones with a radiation dose of 5.0 Gy prior to culture resulted in a selective elimination of the proliferating progenitors, whereas the contribution of postmitotic precursors in AcPP-P cell and osteoclast formation remained unchanged. The results demonstrate that in the periosteum of embryonic metatarsal bones a shift occurs from a population composed of proliferating osteoclast progenitors (15 days) to a population composed of postmitotic precursors (17 days) before multinucleated osteoclasts are formed (18 days). Obviously, postmitotic AcP-negative precursors, already present in 16-day-old bones, differentiate into precursors characterized by tartrate-resistant AcP activity, the preosteoclasts (17 days), which in their turn fuse into osteoclasts. In long bone development the primitive marrow cavity is formed by resorption of the calcified cartilage matrix in the center of the long bone model. The multinucleated giant cell responsible for cartilage and bone resorption, the osteoclast, belongs to a n extraskeletal, hematogenous cell lineage (Marks, 1983). Its progenitors are transported via the blood stream and seeded in the periosteum of long bones at a n early stage of embryonic development, long before the actual formation of the marrow cavity (Thesingh and Burger, 1983; Burger et al., 1984). Only little information is available about the processes of proliferation and differentiation, i.e., osteoclast formation kinetics, which take place between the homing of progenitors and the appearance of multinucleated osteoclasts. In a n in vitro study of embryonic mouse long bones we recently found that the radiosensitivity of osteoclast formation is age-dependent (Scheven et al., 1985). Our results suggested that in the periosteum of long bones radiosensitive osteoclast progenitors proliferate and differentiate into postmitotic preosteoclasts before the first multinucleated osteoclasts are 0 1986 ALAN R. LISS. INC formed by fusion. In order to obtain more insight into the proliferation and differentiation kinetics of the osteoclast and its progenitor population, we studied the appearance, in vivo, of tartrate-resistant acid phosphatase-positive (AcPP) mononuclear and multinucleated cells in the periosteum and marrow cavity of metatarsal bones of different ages. Subsequently we investigated the 3H-thymidine (3H-TdR)incorporation into periosteal osteoclast progenitors of embryonic long bones by assessing the number of labeled nuclei in histochemically (acid phosphatase) recognizable osteoclasts and preosteoclasts. Finally we determined the effects of ionizing irradiation on the incorporation of 3H-TdFt in these cells. MATERIALS AND METHODS Tissue Preparation The middle three metatarsal bones of the hind limbs of Swiss albino mouse embryos in the age range of 1519 days (day of vaginal plug discovery was equated with Received July 24, 1985; accepted November 18, 1985. DIFFERENTIATION OF OSTEOCLAST PROGENITORS 419 day 0, day 19 is day of birth) were dissected and either immediately processed for the demonstration of acid phosphatase (AcP) activity (see below) or cultured (Fig. 1). Culture Procedure After dissection, the bone rudiments were transferred to a culture dish containing 1ml semisolid medium that consisted of 60% Hanks’ balanced salt solution, 10% embryonic extract of 10-day-oldchick embryos, 10% rat serum, and 20% cock plasma. The bones of one of the hind limbs were irradiated with a dose of 5.0 Gy by a Philips RT 250 X-ray generator at 200 kV (HVL: 1.5 mm Cu, dose rate of 1.18 Gy/min). The corresponding bones of the other hind limb were used as nonirradiated controls. Subsequently, the explants were placed on fresh medium containing 0.025 pCi 3H-thymidine (3H-TdR, specific activity: 50 mCi/mmol) in 60% Eagle’s minimal essential medium with the same supplements as described above. Culturin was performed in a 5% COa/air atmosphere a t 37°C. ‘H-TW medium was refreshed every 2 or 3 days. At different times the bones were fixed and prepared for the AcP staining procedure and autoradiography (Fig. 1). Acid Phosphatase Histochemistry The en bloc AcP staining a s described here proved to be specific for the demonstration of osteoclastic, tartrateresistant AcP activity (Hammerstrom et al., 1971; Minkin, 1982) in embryonic long bones. Metatarsal bones were washed in cold Hanks’ balanced salt solution and fixed in 10% neutral buffered formalin for 16 h r a t 4°C. Then the bones were decalcified in 5% formic acid and 5% formalin for 3 hr at 4°C. After a quick wash in distilled water the specimens were incubated for AcP staining with naphthol As-B1 phosphate as substrate and hexazonium pararosanilin as coupler in Michaelis veronal acetate buffer for 1 hr a t 37 “C (Barka and Anderson, 1962). L( +)-tartaric acid W S E 15 METATARSL 16 BONES 17 18 AGE 19 (days after cmccplion) +AcP HISTOCHEMISTRY immediately after dissection Fig. 2. Parts of noncounterstained histologic sections of embryonic metatarsal bones stained for tartrate-resistant AcP activity. In the periosteum (P) of 17-day-oldbones (a) AcP-positive cells (double arrows) are visible. No osteoclasts have as yet developed. In 18-day-oldmetatarsal bones (b) AcP-positive osteoclasts (arrows) have invaded the center of the bones. Note the absence of AcP-positive cells in the periosteum. The bone collar is indicated by asterisks. The hypertrophic cartilage is marked with HC. x 200. dissolved in 0.1 M acetate buffer (pH 5.0) was added to the substrate solution to a final concentration of 10 mM. Omission of naphthol As-B1 phosphate or hexazonium pararosanilin from the incubation medium resulted in total absence of the azo dye staining. Then the bones were washed in distilled water, kept overnight in 70% ethanol, and embedded in paraffin. Sections of 5 pm were counterstained with 0.1% methyl green and 0.01% thionin in 0.02 M citrate buffer (pH 5.8) or with hematoxy lin. Light Microscope Autoradiography ‘i I i I CULTURE in the presence of 3H-TdR vL 3,i7,10 3.i7.K) 3.i.6 days Paraffin sections were dipped in Ilford L4 emulsion with a dipping apparatus and exposed for 4 weeks at 4°C. After development, the autoradiographs were examined by light microscope. Quantification and Statistical Evaluation In the metatarsal bones of different ages all nuclei of the osteoclasts in the primitive marrow cavity (if present) and of the acid phosphatase-positive cells in the LM AUTCUADIOGMWY periosteum (AcPP-P cells) were enumerated from serial sections. The AcP-positive cells (osteoclasts and AcPP-P Fig. 1. Schematic representation of the experimental set-up. The cells) were easily discriminated from the AcP-negative mesenchyme surrounding the bone models is indicated by dots. Resorption area (primitive marrow cavity) is illustrated as a light area in the cells by their specific, intense reddish azo dye staining (Fig. 2). calcified cartilage zone (black painted). AcP H15TCXHEMISTRY B.A.A. SCHEVEN, E.W.M. KAWILARANG-DE H AAS, A,-M.WASSENAAR, AND P.J. NIJWEIDE 420 In the autoradiographs all labeled and unlabeled nuclei of osteoclasts and AcPP-P cells per cultured bone were counted. Nuclei with more than 10 silver grains were regarded as positively labeled (background labeling was minimal). Paired (control-irradiated) observations were statistically compared by the Student t test. RESULTS A schematic outline of the experiments performed is given in Figure 1. The in vivo metatarsal development with respect to the appearance and kinetics of tartrateresistant AcP-positive cells is described in the first part of the results section. Subsequently, the results of the in vitro 3H-TdR labeling experiments are presented. Kinetics of Osteoclast Formation in vivo On day 17 the first AcPP cells were observed in the periosteum of embryonic mouse metatarsal bones (Fig. 2a). These AcPP-P cells were localized adjacent to the periosteal osteoblasts or the newly formed bone collar. Most cells were mononuclear but some multinucleated AcPP-P cells, apparently about to invade the bone, were also observed. In younger explants positive cells could not be demonstrated, except for a n occasional weakly colored cell in the periosteum of 16-day-oldbones. The first osteoclasts resorbing the calcified cartilage matrix appeared in metatarsal bones of 18-day-oldmouse embryos (Fig. 2b). The invasion process was accompanied with a sharp decrease in the number of AcPP-P cells (Fig. 3). On the 19th day of development the number of osteoclast nuclei had further increased, coinciding with a further decrease in the number of AcPP-P cells (Fig. 3). Kinetics of Osteoclast Formation In Vitro Metatarsal bones in which no multinucleated osteoclasts had been formed at the time of explantation (i.e., postconception age of 15, 16, and 17 days) were used for continuous in vitro 3H-TdR labeling experiments (Fig. 1).Pilot culture experiments showed that the presence of labeled thymidine did not significantly affect osteoclast formation (data not shown). Osteoclasts developed during culture possessed either labeled or unlabeled nu.- - I w Z c 40 L 0, D E 3 z 20 0 I 16 17 ia 19 AGE days after conception Fig. 3. Kinetics of AcPP-P cell ( 0 )and osteoclast (0) formation during metatarsal bone development in vivo. Bones of different ages were stained for tartrate-resistant AcP activity, and the number of nuclei of AcP-positive cells in the periosteum (AcPP-P cells), as well as in the marrow cavity (osteoclasts), were counted. Values are plotted as the mean & SD of 9-18 bones. Fig. 4. Light-microscope autoradiographs of osteoclasts developed in a control (a) and irradiated (b) 16-day-old bone labeled continuously with 3H-TdR in vitro. Note the presence of labeled (arrows) and nonlabeled (asterisks) nuclei in the same osteoclast in the control and the absence of labeled nuclei in the osteoclast formed following irradiation. X 980. clei or both. Especially in cultured 16-day-old bones, both labeled and unlabeled nuclei were found together in the same osteoclast (Fig. 4a). This was also the case in the multinucleated AcPP cells, which were occasionally observed in the periosteum. The results of a quantitative autoradiographic analysis of the sections are shown in Figures 5-7. Metatarsal bones of 15-day-oldmouse embryos demonstrate a gradual accumulation of newly formed, 3H-TG-labeled AcPP-P cells during a culture period of 7 days (Fig. 5 , hatched bars). Osteoclast formation started between days 5 and 7. Subsequently, the number of osteoclast nuclei strongly increased, whereas the number of AcPP-P cells slightly decreased. Almost all AcPP-P cells and osteoclasts were derived from proliferating progenitors. Nonlabeled AcPP-P cells or osteoclast nuclei (open bars) were not present or only in very small numbers. Correspondingly, labeling indices ranged between 86% and 100%. The kinetics of osteoclast formation in cultured 16day-old bones corresponded well with that of the AcPPP cell kinetics (Fig. 6). During the first days of culture DIFFERENTIATION OF OSTEOCLAST PROGENITORS 15-day- old bones -3 - C I C I L C I C l I C l C l 5 3 C I C C I 0 I C 3 I C l C l 5 C l C 7 l I I 10 7 0 C 16-day- o Id bo nes I T 421 l C l C C i C l 3 I C I C I C I 5 7 I0 5 7 10 C l C l 10 Culture lime (days) Fig. 5. AcPP-P cell and osteoclast kinetics in 15-day-oldmetatarsal bones in vitro. The number o f labeled and nonlabeled nuclei were enumerated in cultures labeled continuously with 'H-TdR. Open columns represent unlabeled nuclei, shaded columns represent labeled nuclei. I, irradiated bones (5.0 Gy); C, paired nonirradiated controls. Values are plotted as means (+SD) of 3-20 bones of two independent experiments. (*): P < 0.01, compared to paired control bones. 3 Culture hme (days) Fig. 6. AcPP-P cell and osteoclast kinetics in 16-day-oldmetatarsal bones labeled continuously with 'H-TdR in vitro. See also legend to Figure 3. Values are plotted as means k SD o f 6-8 bones of three independent experiments. (*) P < 0.01, compared to paired control bones. 17-day-old the AcPP-P cells and osteoclasts mainly originated from nonproliferating precursors (labeling index, day 3: 2025%). In time, the proportion of labeled nuclei increased (Fig. 6, hatched bars). The mean labeling indices on days 5, 7, and 10 of culture were respectively 42%, 55%, and 68%for the AcPP-P cells and 42%, 57%, and 58%for the osteoclast nuclei. In metatarsal bones of 17-day-oldembryos the number of osteoclasts and AcPP-P cells remained constant in the culture period from 3 to 6 days (Fig. 7). The contribution of proliferating, 3H-TdR-incorporating cells to either cell type was minimal (labeling indices: 4-7%). During the first 3 days of culture a massive decline in AcPP-P cell numbers, already present at the time of explantation, coincided with a rapid increase in osteoclast numbers (Fig. 8). I C I bones C I C I C : c I 6 4 3 Radiation Effects on Kinetics of Osteoclast Formation Irradiation of metatarsal bones with 5.0 Gy prior to culture specifically inhibited the appearance of labeled AcPP-P cells and osteoclast nuclei, leaving the kinetics of unlabeled nuclei in both cell types unchanged (Figs. 4b, 5-7). The radiation-induced inhibition in 15-day-old bones was almost absolute, resulting in complete elimination of osteoclast formation (Fig. 5). In cultured 16day-old bones differentiation of H-TdR-incorporating osteoclast progenitors was partially but significantly inhibited, causing severe interference with AcPP-P cell and osteoclast kinetics (Fig. 6). In older bones the radiation-induced reduction of the number of labeled nuclei did not markedly affect osteoclast kinetics, since the pool of nonproliferating precursors principally determined the total osteoclast nuclear population (Fig. 7). 1 C I C I 3 c i C I 4 c I C I 6 Culture time (days) Fig. 7. AcPP-P cell and osteoclast kinetics in 17-day-oldmetatarsal bones labeled continuously with 3H-TW in vitro. See also legend to Figure 3. Values are plotted as means (f SD) of 6-8 bones of two independent experiments. (*) P < 0.05, compared to paired control bones. 422 B.A.A. SCHEVEN, E.W.M. KAWILARANG-DEHAAS, A.-M. WASSENAAR, AND P.J. NIJWEIDE the findings of others (Burger et al., 1982; Seifert, 1984; Horton et al., 1984). -U 17-day-old bones The autoradiography experiments using continuous 3 3H-TdR labeling in vitro enabled us to investigate the Z proliferation of osteoclast progenitors in the periosteum of embryonic metatarsal bones. The results indicate that % 40 in embryonic long bones of different ages osteoclast proL W genitor populations, which give rise to osteoclast formaf 20 tion in vitro, are in different kinetic phases. In cultured 3 15-day-oldbone rudiments (prelosteoclasts derived from z proliferating, 3H-TdR,-incorporatingprogenitors. Radia0 tion caused complete inhibition of (pre)osteoclast forma3 0 tion and consequently of marrow cavity development. Bones of 16-day-old embryos mainly yielded unlabeled Culture time (days) (pre)osteoclast nuclei during the first days of culture, but a n increasing proportion of proliferating progenitors Fig. 8. Kinetics of AcPP-P cells ( 0 )and formation of osteoclasts (0) during the first days of culture of 17-day-oldmetatarsal bone. Bones contributed to osteoclast formation a t longer culture explanted from embryos of one mother mouse were either immediately times. This age group did not contain preexisting AcPPfixed for AcP staining (day 0)or cultured for 3 days. Means f SD of 9P cells, suggesting that the unlabeled (pre)osteoclast 12 bones. nuclei were derived from postmitotic AcP-negative precursors. Hence, tartrate-resistant enzyme activity is obtained and/or becomes operative during the postmitotic phase of the osteoclast differentiation. In older bones, a DISCUSSION pool of postmitotic precursors, mainly consisting of AcPPThe present study was designed to investigate the P cells, was involved in osteoclast formation. The contriproliferation and differentiation of periosteal osteoclast bution of proliferating progenitors to (pre)osteoclast deprogenitors during embryonic long bone development. velopment during culture of 17-day-oldbones is obviously The osteoclast progenitors originate from a hemopoietic a minor one. In this age group the number of bloodstem cell (Marks, 1983) and are deposited in the perios- borne progenitors may be lowered owing to the declining teum via the blood circulation at an early stage of em- number of circulating progenitor cells, as a result of the bryonic development (Kahn et al., 1981; Thesingh and diminishing hemopoietic activity of the liver during the Burger, 1983). The blood-borne progenitors then differ- late embryonic and neonatal development (Moore and entiate and fuse into multinucleated osteoclasts which Williams, 1973; Burger et al., 1984). Furthermore, a invade the calcified cartilage zone, resulting in the for- limitation of the proliferation ability of the present immation of a primitive marrow cavity. It should be em- mature osteoclast progenitors due to the advanced dephasized, however, that the osteoclast cell lineage, in velopmental stage of the 17-day-oldbones may in part particular a s far as the early progenitors are concerned, account for the small participation of these cells in osteohas not yet been elucidated (Marks, 1983; Chambers, clast kinetics. In any case, the role of the periosteum as 1985). Several studies have described a direct mononu- secondary source of osteoclast precursors appears to declear precursor, the so-called preosteoclast, showing cline with the formation of the marrow cavity (Fig. 3). morphological and cytochemical features that are simi- Nevertheless, the periosteum still remains a homing lar to those of the multinucleated osteoclast (Scott, 1967; place of bone marrow-derived (pre)osteoclasts involving Rifkin et al., 1980; Ejiri, 1983). In this study the appli- periosteal bone remodeling. cation of tartrate-resistant AcP histochemistry resulted In the 16- and 17-day-oldbones irradiation also inhibin specific staining of osteoclasts in the marrow cavity ited the appearance of labeled (prelosteoclast nuclei. as well as of mononuclear cell types in the periosteum. However, unlike in younger bones, radiation did not These periosteal AcPP cells are localized adjacent to the lead to total elimination of proliferating progenitors. bone collar and appear just before multinucleated osteo- This may be explained by the assumption that osteoclast clasts develop in 18-day-old bones. In vitro, this accu- progenitors in the explanted 15-day-old bones need to mulation process prior to osteoclast formation could also undergo more cell divisions to reach the mature, postbe demonstrated (Fig. 5). The kinetic pattern of the mitotic stage. AcPP-P cells appeared to be reciprocal to osteoclast forThis study also showed a discrepancy between the in mation during both in vivo and in vitro development vivo vs. in vitro differentiation kinetics of the osteoclast. (Figs. 3 and 8). Taken together, it is plausible that the In vitro, osteoclast formation appeared to be considerAcPP-P cell represents the mature, mononuclear preos- ably retarded compared to in vivo development (compare teoclast, ready to fuse into multinucleated osteoclasts Figs. 3 and 5). However, once invasion of osteoclasts into (Ejiri, 1983; Scheven et al., 1985).The corresponding 3H- the bone rudiment had started, the number of osteoTdR labeling pattern of the AcPP-P population and the clasts in the developing marrow cavity rapidly inosteoclast nuclear population is in support of this as- creased. Nonetheless, relatively high numbers of sumption. Since both mono-/and multinucleated macro- preosteoclasts and some binucleated osteoclasts remain phages do not express tartrate-resistant AcP activity in the periosteum of all age groups under the in vitro (Seifert, 1984; own observations), our results make it conditions while disappearing in vivo. Possibly, the reunlikely that osteoclasts are derived from mature mono- tardation and ultimate stunting of the bone developnuclear phagocytes. This supposition is compatible with ment in vitro reduced the stimuli necessary for .u I DIFFERENTIATION OF OSTEOCLAST PROGENITORS activation and mobilization of the resident periosteal (pre)osteoclast population. In conclusion, the results provide conclusive evidence for the earlier postulated differentiation pathway of periosteal osteoclast progenitors during metatarsal bone development (Scheven et al., 1985).Obviously, osteoclast progenitor differentiation is closely related to the development of the bone model, especially to initiation of hypertrophic cartilage matrix calcification (16 days) and the formation of the bone collar (17 days). In the periosteum mitotic active, blood-borne progenitors (15-16 days) differentiate into postmitotic osteoclast precursors (16 days). Subsequently, the postmitotic precursors obtain tartrate-resistant AcP activity (17 days). The latter identifiable cell may be called the preosteoclast, which forms multinucleated osteoclasts by fusion (18 days). It is evident that the kinetics and therefore the radiosensitivity (Scheven et al., 1985) of osteoclast differentiation in vitro is dependent upon the age of the bones. In the in vitro system using metatarsal bones of different ages, it appears to be possible to separate the mitotic phase (15 days) from the postmitotic phase (17 days) of the osteoclast differentiation pathway. The experimental set-up may therefore provide a suitable model for the study of factors influencing proliferation of osteoclast progenitors on the one hand (i.e., long-term effects) and for the study of factors regulating fusion, mobility, and activity of postmitotic preosteoclasts (i.e., short-term effects), on the other hand. ACKNOWLEDGMENTS We are greatly indebted to Mr. A. Boon for performing the irradiation procedures and to Prof. Dr. J. P. Scherft and Dr. C.W. Thesingh for critical reading of the manuscript. This study was supported by the J.A. Cohen Institute for Radiopathology and Radiation Protection, Leiden. LITERATURE CITED Barka, T., and P.J. Anderson (1962) Histochemical method for acid phosphatase using hexazonium pararosanilin as coupler. J. Histochem. Cytochem., 10:741-753. 423 Burger, E.H., J.W.M. Van der Mew, J.S. Van de Gevel, J.C. Gribnau, C.W. Thesingh, and R. Van Furth (1982) In vitro formation of osteoclasts from long-term cultures of bone marrow phagocytes. J. Exp. Med., 156:1604-1614. Burger, E.H., C.W. Thesingh, J.W.M. Van der Meer, and P.J. Nijweide (1984) Development of-osteoclasts in the mouse-localization of osteoclast precursors and role of bone-forming cells. In: Endocrine Control of Bone and Calcium Metabolism. D.V. Cohn, T. Fujita, J.T. Potts, and R.V. Talmage, eds. Elsevier, Amsterdam, pp. 125-130. Chambers, T.J. 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