electron microscopic studies on chick limb cartilage...

/ . Embryol. exp. Morph. Vol. 34, 2, pp. 327-337, 1975 3 2 7

Printed in Great Britain

Electron microscopic studies on chick limbcartilage differentiated in tissue culture

By SURESH C. GOEL1 AND A. JURAND2

From the Institute of Animal Genetics, Edinburgh

SUMMARYThe hind limb-bud mesenchyme of chick embryos 4-4£ days old was cultured in Eagle's

Minimum Essential Medium supplemented with both horse serum and fresh chick embryoextract. Whereas no differences are seen at the light-microscope level, at the electron-micro-scope level the chondroblasts differentiated in tissue culture are noticeably different from thosedifferentiated in vivo, particularly in the possession of some cytoplasmic fibrils and vacuoles.It is proposed that the secretion of the extracellular matrix alone is not sufficient to accountfor the pattern of cellular arrangement in a cartilaginous condensation.

INTRODUCTION

Studies on cartilage in tissue culture started with the work of Strangeways &Fell (1926) and Fell (1928). A number of workers have since been using cartilageor presumptive cartilage tissue in culture (a) to evaluate the potency of meso-dermal cells to form cartilage independent of the influences operating in vivo(Zwilling, 1966; Hamburgh, 1971; Searls, 1973), (b) to test the effect of variouschemicals on physiology, morphology and differentiation of chondroblasts(Medoff, 1967; Reynolds, 1967; Fell & Dingle, 1969; Glauert, Fell & Dingle,1969; Levitt & Dorfman, 1974), and (c) to study the effects of genetic behaviourof cells (Ede & Agerbak, 1968; Ede & Flint, 1972).

The cartilage differentiated in tissue culture is hardly distinguishable fromthat differentiated in vivo at the light microscope level (Ede & Agerbak, 1968;Goel, 1969). It is, however, known that tissues developed in tissue culture areusually under certain stress from the environmental conditions (Levitt & Dorf-man, 1974). The present communication reports the ultrastructural differencesbetween the cartilage cells differentiated in tissue culture and in vivo in thedeveloping hind limb-buds of chick embryos (Goel, 1970).

MATERIALS AND METHODS

The hind-limb buds from 4- to 4^-day-old chick embryos of Brown Leghornvariety were used. The excised buds were washed twice in Hanks' balanced salt

1 Author's address: Department of Zoology, Poona University, Poona, India.2 Author's address: Institute of Animal Genetics, Edinburgh, EH9 3JN, U.K.

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328 SURESH C. GOEL AND A. JURAND

solution (Paul, 1970) and twice in calcium- and magnesium-free balanced saltsolution (CMF; Moscona, 1961) and incubated in CMF for 15 min at 38 °C.This was followed by further incubation for 7-10 min in CMF containing 1 %trypsin (crystallized and lyopholized, from Worthington Biochemical Co.:minimum activity 150 units/mg.*LU.B. system). After washing in Hanks'balanced salt solution the buds were transferred to standard culture medium,which consisted of Minimal Essential Medium of Eagle (with Earle's salts)supplemented with 10 % horse serum (Flow Laboratories, Irvine, Scotland)and 10-50% fresh chick embryo extract (from 8- to 10-day-old embryos in50:50 Tyrode's solution; Paul, 1970). The ectodermal jacket was removed withthe help of tungsten needles and chondrogenic mesoderm was cut into smallfragments, approximately 0-75 mm3, with cataract knives or dispersed into singlecells by passing through a micropipette. Suspension cultures in plastic Petridishes or hanging drop cultures using depression slides were set up and culturemedium changed every 48 h. The pH of the medium was maintained at 6-8-7-0.The cartilage nodules developed in about 40-48 h (see also Jackson, 1965;Ede & Flint, 1972). The cultures were incubated up to 6 days at 37-5 ±0-5 °C.

The material was fixed in 1 or 2% osmium tetroxide in veronal buffer at0-4 °C for 1 h. It was routinely processed and embedded in Araldite epoxy-resin. For electron microscopy 70-90 nm thick sections were stained withsaturated solution of uranyl acetate or 20% uranyl nitrate (15 min) followed bylead citrate (15 min), and viewed under an AEI EM 6 electron microscope. Forlight microscopy Araldite sections 1 /im thick were stained with 0-5 % toluidineblue (in 1 % solution of sodium tetraborate) at 38 °C for 15 mins (see Goel &Jurand, 1972, for further details).

The light microscope autoradiography experiments using [3H]proline(15/*Ci/ml of culture medium; specific activity 720mCi/mM; from Radio-

FIGURES 1-5

All the figures are from cartilage developed in culture for two or three days.Fig. 1. Cartilage allowed to differentiate in tissue culture for two days and incubatedwith [3H]proline (15/*Ci/ml of culture medium; specific activity 720 mCi/mvi) for2 h. In the autoradiograph the grains are not present on the extracellular phase oron a non-cartilaginous cell, x 1050.Fig. 2. Cartilage differentiated in tissue culture, and incubated with [3H]proline (as inFig. 1) for 24 h. In the autoradiograph the grains are present on the extracellularphase as well as on the cartilage cells, x 1600.Fig. 3. Cartilage differentiated in tissue culture. The whorl-like arrangement of thecells, as characteristic of limb cartilage (see Fig. 5), is seen, x 250.Fig. 4. Cartilage differentiated in tissue culture. In this cartilaginous nodule theamount of extracellular phase towards the centre is high, x 375.Fig. 5. Epiphyseal cartilage from the third toe of hind limb-bud of stage-31 embryo.The characteristic arrangement and appearance of cells in 'condensation' is evident.Compare with the tissue differentiated in culture (Figs. 3 and 4). x 270.

Chick limb cartilage in tissue culture 329


chemical Centre, Amersham) indicate that the cartilage cells differentiated inthe tissue culture were healthy (Goel, 1969). Such cells actively synthesize andexport proteins into the extracellular phase (Figs. 1, 2).

RESULTS

The cartilage nodules are usually spheroidal structures. The general arrange-ment of the cells in the nodule is comparable to that in the cartilage condensa-tion in the limbs in vivo (Figs. 3-5). The cells in the centre, like typical chondro-blasts, have a scalloped outline, but may sometimes be rounded. Towards theperiphery of the nodule, in the sections, the cells are usually thin, elongated,closely packed and concentrically arranged. These cells could be designatedas the perichondrial cells. The extracellular phase is sometimes extensive, par-ticularly in the centre of the nodule, and stains metachromatically with toluidineblue. The chondroblasts in the process of mitotic division are also noticeablein some cases.

The nucleus of the chondroblasts is eccentrically placed and is enclosed in anuclear envelope 25-70 nm thick which is frequently studded with the ribosomeson its outer surface (Figs. 6, 8). The nuclear pores, around 70 nm in diameter,are common. The nuclear matrix is largely homogeneous and consists ofgranules and fibrils. Some chromatin material is also present and a part of itforms a very thin layer adjacent to the inner nuclear membrane. Usually there isonly one nucleolus with morphology similar to the nucleolus of the chondro-blasts differentiated in vivo; the more electron-dense fibrillar areas of it arecompletely enclosed in less electron-dense particulate areas (Fig. 7).

In the cytoplasm the ribosomes are distributed either singly or as polysomes.The endoplasmic reticulum is granular and consists mainly of elongated cisternalprofiles around 120 nm in diameter. The dilated saccular cisternae are infrequent

FIGURES 6-10

Fig. 6. Cartilage differentiated in tissue culture. The matrix in the extracellular phaseis on the average not as extensive as in the in vivo cartilage, x 4400.Fig. 7. Cartilage differentiated in tissue culture. The cell shows saccular cisterna ofendoplasmic reticulum (er), some perinuclear cytoplasmic fibrils and a characteristicappearance of nucleus with nucleolus. x 20700.Fig. 8. Cartilage differentiated in tissue culture. The Golgi apparatus lamellae (C),cilium (c/) and centriole (c) are seen. The small vesicles below the plasmalemma arepossibly pinocytotic in origin, x 20700.

Fig. 9. Cartilage differentiated in tissue culture. Note the three vacuoles of dif-fering sizes (arrows) and heterogeneous contents in one of them (v). x 25700.Fig. 10. Cartilage differentiated in tissue culture. The Golgi apparatus and varioustypes of vacuoles are seen. The boundary membrane of one vacuole (v) is incomplete;the small vesicles in the extracellular phase (arrow) are perhaps due to the openingout of a vacuole. x 20700.


and measure up to l-lxO-9/tm. The vesicular profiles of the endoplasmicreticulum are seldom seen (Fig. 8). The contents of the reticulum are homo-geneous, amorphous and moderately electron-dense. The Golgi apparatus, likethat of the chondroblasts differentiated in vivo, consists of a very few lamellae,some Golgi vacuoles and many vesicles (Fig. 10). The vacuoles may be as largeas 0-7 jiim in diameter and are usually electron-translucent but sometimescontain a small quantity of materials which resemble the electron-dense granulesof the extracellular phase. It appears as if the larger Golgi vacuoles are formedby the fusion of smaller ones. The area of the Golgi vacuoles and the saccularcisternae of the endoplasmic reticulum can be easily distinguished from eachother, since, as in the chondroblasts differentiated in vivo, the area occupiedby the Golgi vacuoles is electron-translucent in appearance. Mitochondria arefrequent and vary in shape and size from oval structures, about 0-5 //m in di-ameter, to elongated ones about 3-0x0-7 jam in size. They have a moderatelyelectron-dense matrix with a few mitochondrial granules and many mito-chondrial cristae. Certain vesicles located below the plasmalemma, probably ofpinocytotic origin, are of common occurrence (Figs. 8, 11). A centriole as wellas a cilium can be seen in Fig. 8, though the cilia are uncommon. Glycogen wasnever observed in the chondroblasts.

Sometimes groups of fibrils, around 5-8 nm thick, can be seen in the peri-nuclear cytoplasm (Fig. 7). Moreover, in a few cases even bigger groups ofslightly thicker fibrils, around 12-16 nm thick, fill a considerable part of thecytoplasm of the cells situated on the periphery of the condensation (Fig. 12).

A variety of vacuoles of varying sizes and contents are frequently present(Figs. 9, 10). They are usually around 0-5-0-8/on in diameter although occa-sionally they may be as large as 1-6 x 2-5 /tm. A large number of them containsmall vesicles (50-70 nm in diameter); some, in addition, contain an amorphousmoderately electron-dense mass and various ill-formed membranous structures,while a few are lipid droplet-like structures. Most of these structures are boundby a single membrane, around 14 nm thick, but some are delimited by double

FIGURES 11-15

Fig. 11. Cartilage differentiated in tissue culture. The vesicle subjacent to plasma-lemma is possibly due to pinocytosis. x 31000.Fig. 12. Cartilage differentiated in tissue culture. A chondroblast showing a largeamount of cytoplasmic fibrils; such chondroblasts are found near the peripheryof the nodule. The arrows point to the plasmalemma. x 20700.Fig. 13. Cartilage differentiated in tissue culture. The extracellular phase hasbundles of thin fibres of collagen, x 20700.Fig. 14. Cartilage differentiated in tissue culture. Some of the fibres of extra-cellular phase are very close to the plasmalemma and appear to merge with it.x 91000.Fig. 15. Cartilage differentiated in tissue culture. The fibres of the extracellularphase are straight and sometimes show an incipient banding (arrow); the electron-dense granules (g) are few compared with the in vivo differentiated cartilage.


membranes. Vesicles around 5C-70 nm in diameter and similar to those in thevacuoles are also present free in the cytoplasm, especially in the area of theGolgi apparatus, suggesting the origin of vacuoles from this organelle. More-over, the presence of vacuoles with incomplete boundary membranes in theGolgi area also indicates their Golgi origin, though the variation in their struc-ture suggests that they may be formed in more than one way. In one case it isnoted that the vacuole is opening to the extracellular phase (Fig. 10).

The process of 'ecdysis' or 'excortication' is more frequently found in thesecells as compared to those differentiated in vivo. The fibres of the extracellularphase are often very near to or confluent with the plasmalemma (Fig. 14), andsometimes enter in the cortical cytoplasm, and run a short distance subjacentto the plasmalemma before merging into the cytoplasm. At such points of con-tact, however, the plasmalemma seems discontinuous or indistinct, possiblybecause of tangential sectioning.

The extracellular phase contains numerous fibres embedded in a homo-geneous amorphous mass (Fig. 13). The fibres, usually in bundles, are about15 nm thick, short and straight (Fig. 15). The presence of a relatively large num-ber of fibres towards the periphery, rather than in the centre, of the condensationis noticeable. The electron-dense granules of the extracellular phase are neitheras frequent nor as well developed as in the cartilage differentiated in vivo; andare usually completely missing in the cartilage from younger cultures.

DISCUSSION

The cytoplasmic fibrils, reported in the present study in the chondroblastsdifferentiated in tissue culture, are not described in the chick hind-limb cartilagedifferentiated in vivo (Goel, 1970). However, Searls, Hilfer and Mirow (1972)report the occurrence of the perinuclear and cytoplasmic fibrils of similar dia-meter in the cartilage cells from chick wing-bud. Similar fibrils are also describedin the articular cartilage of rabbit (Palfrey & Davies, 1966), man (Meachim &Roy, 1967), and mice (Silberberg, 1968), in amphibian limb cartilage (Revel &Hay, 1963) and in other types of cells, especially those grown in tissue culture(Spooner, Yamada & Wessells, 1971).

The nature and function of the cytoplasmic fibrils is uncertain but they havebeen considered instrumental in cytokinesis (Schroeder, 1968), as part of cyto-skeleton and important in cell locomotion and maintenance of cell shape(Spooner et ah 1971; Searls et al. 1972), as signs of cellular degeneration andageing (Barnett, Cochrane & Palfrey, 1963), or as indicating metabolic distur-bance (Silberberg, 1968). Our observations are in accord with the view of Mea-chim & Roy (1967) that the presence of fibrils in small quantities is not evidenceof chondrocyte degeneration but that the accumulation of large quantities isindicative of degenerative changes. It is important to point out that none ofthese authors interpret the fibrils to be precursors of collagen.


The chondroblasts differentiated in tissue culture, compared with thosedifferentiated in vivo (Goel, 1970), have a higher frequency of vacuoles (see alsoJackson, 1964; Eguchi & Okada, 1971). The vacuoles are reported in chondro-cytes from a variety of animals (Silberberg, 1968; Serafini-Fracassini & Smith,1974), but a very high frequency of vacuoles is a symptom of unhealthy stateof the cell (Glauert et al. 1969). It is therefore possible that the increased fre-quency of the vacuoles is the response of cells to the conditions prevailing intissue culture, and that some of the vacuoles are lysosomes (Norrevang, 1968;Glauert et al. 1969) and may be autophagic in nature. It is plausible that thepresence of vacuoles escapes notice in the light-microscope observations unlessthe vacuolar frequency is very high.

The glycogen has not been observed in the chondroblasts differentiated intissue culture (Eguchi & Okada, 1971), as also in the chick chondroblasts invivo (Goel & Jurand, 1972). On the other hand, Levitt & Dorfman (1974)report the presence of numerous glycogen lakes in chick hind limb-bud cellsgrown in tissue culture, and Searls et al. (1972) indicate the presence of glycogenin undifferentiated mesenchyme cells of the chick wing-bud; but Searls et al.(1972) do not comment on the presence of glycogen in chick limb cartilage.Moreover, unfortunately, none of the authors presents electron micrographsshowing any definite glycogen granules in the cells. For this reason we retainthe view that in the chick limb epiphyseal cartilage glycogen is not present andthat the presence of glycogen is not essential for cartilage differentiation (Goel& Jurand, 1972).

In the extracellular phase the 15 nm thick fibres are in all probability colla-genous in nature. Similar fibres are reported in the chick hind limb-bud cartilage(Goel, 1970), and wing-bud cartilage (Searls et al. 1972) differentiated in vivo.Searls et al. (1972) consider the fibres to be [al(II)]3 form of collagen, whereasLinsenmayer, Toole & Trelstad (1973) consider the chick cartilage collagen atthis stage to be largely [al]3; however, there is evidence to the effect that the[al]3 collagen is very similar to, if not identical with, the [al(Il)]3 collagen(Linsenmayer, 1974). The thick collagen fibres with a periodicity of about 64 nm,as reported by Searls et al. (1972) in the chick wing-bud cartilage, have notbeen seen in the present study or earlier studies (Goel, 1970; Seegmiller, Fraser& Sheldon, 1971) and it is generally believed that the acid mucopolysaccharidesof the cartilage matrix somehow interfere with the formation of collagen fibreswith a 64 nm periodicity (see Seegmiller et al. 1971; Serafini-Fracassini &Smith, 1974).

The present observations on the arrangement and appearance of cells in thecartilage differentiated in tissue culture shed light on the acquisition of patternof arrangement and appearance of the cartilage cells. Gould, Selwood, Day &Wolpert (1974) suggest a simple model in which they consider the secretion ofthe extracellular matrix as the main cause of orientation and flattening of thecells. The present study as well as earlier in vivo results (Goel, 1970) indicate


that the secretion of the matrix is very likely responsible for the scalloped appear-ance of the cartilage cells, and also for their typical randomly spaced arrange-ment in the central region (Gould et al. 1974). On the other hand it seemsunlikely that the shape and arrangement of the more peripheral or perichondrialcells is also due to the same factor. This suggestion is supported by our observa-tions both in vivo and in tissue culture. In the tissue culture the cartilage acquiresa spherical shape, and concentric arrangement and flattened appearance of theperipheral cells is produced (see also Ede & Flint, 1972) despite the fact that theperipheral cells have practically no space restriction and can move outwardsrather than become flattened, if and when the synthesis of matrix exerts pressureon them, as suggested by Gould et al. (1974). Moreover, the morphology andorganization of the cartilage nodule in tissue culture is a function of densityof inoculum and medium composition, including the presence of factors likeascorbic acid (see Levitt & Dorfman, 1974). Even in vivo (Goel, 1969; Gouldet al. 1974) the concentric arrangement of the cells near the periphery of cartilagecondensation precedes the secretion of appreciable amounts of the matrixwhich may be able to exert such a pressure. It is very likely that the arrangementand possibly also the appearance of the cartilage cells is to a marked degreeinfluenced by the inherent pattern and shape of the cartilage concerned; forexample, the arrangement and the appearance of the cartilage and perichondrialcells in a more or less spheroidal mesopodial element may not be the same as inan elongated cylinder-shaped femur.

The authors wish to thank Dr D. A. Ede for reading the manuscript. Thanks are also dueto Mr F. M. Johnston for photographic assistance and to Mr E. D. Roberts for mounting themicrographs.

REFERENCES

BARNETT, C. H., COCHRANE, W. & PALFREY, A. J. (1963). Age changes in articular cartilageof rabbits. Ann. rheum. Dis. 22, 389-400.

EDE, D. A. & AGERBAK, G. S. (1968). Cell adhesion and movement in relation to the develop-ing limb pattern in normal and talpid* mutant chick embryos. / . Embryol. exp. Morph.20, 81-100.

EDE, D. A. & FLINT, O. P. (1972). Patterns of cell division, cell death and chondrogenesis incultured aggregates of normal and talpid3 mutant chick limb mesenchyme cells. / . Embryol.exp. Morph. 27, 245-260.

EGUCHI, G. & OKADA, T. S. (1971). Ultrastructure of the differentiated cell colony derivedfrom a singly isolated chondrocyte in in vitro culture. Development, Growth and Differentia-tion 12, 297-312.

FELL, H. B. (1928). Experiments on the differentiation in vitro of cartilage and bone. Part I.Arch. exp. Zellforsch. 7, 390-412.

FELL, H. B. & DINGLE, J. T. (1969). Endocytosis of sugars in embryonic skeletal tissues inorgan culture. I. General introduction and histological effects. / . Cell. Sci. 4, 89-103.

GLAUERT, A. M., FELL, H. B. & DINGLE, J. T. (1969). Endocytosis of sugars in embryonicskeletal tissues in organ culture. II. Effect of sucrose on cellular fine structure. / . Cell Sci.4, 105-131.

GOEL, S. C. (1969). Studies on developing limbs in chick and mouse embryos. Ph.D. Thesis,University of Edinburgh.

Chick limb cartilage in tissue culture 337GOEL, S. C. (1970). Electron microscopic studies on developing cartilage. I. The membrane

system related to the synthesis and secretion of extracellular materials. / . Embryol. exp.Morph. 23, 169-184.

GOEL, S. C. & JURAND, A. (1972). Electron microscopic studies on developing cartilage.II. Demonstration and distribution of glycogen in chick and mouse epiphyseal cartilage./ . Embryol exp. Morph. 28, 153-162.

GOULD, R. P., SELWOOD, L., DAY, A. & WOLF-ERT, L. (1974). The mechanism of cellularorientation during early cartilage formation in the chick limb and regenerating amphibianlimb. Expl Cell Res. 83, 287-296.

HAMBURGH, M. (1971). Theories of Differentiation. London: Edward Arnold.JACKSON, S. F. (1964). Connective tissue cells. In The Cell, vol. vi (ed. J. Brachet & A. E.

Mirsky). London: Academic Press.JACKSON, S. F. (1965). Antecedent phases in matrix formation. In Structure and Function of

Connective and Skeletal Tissue (ed. S. F. Jackson et al.). London: Butterworths.LEVITT, D. & DORFMAN, A. (1974). Concepts and mechanisms of cartilage differentiation.

Curr. Top. Devi Biol. 8, 103-149.LINSENMAYER, T. F. (1974). Temporal and spatial transitions in collagen types during embryo-

nic chick limb development. II. Comparison of the embryonic cartilage collagen moleculewith that from adult cartilage. Devi Biol. 40, 372-377.

LINSENMAYER, T. F., TOOLE, B. P. & TRELSTAD, R. L. (1973). Temporal and spatial transitionsin collagen types during embryonic chick limb development. Devi Biol. 35, 232-239.

MEACHIM, G. & ROY, S. (1967). Intracytoplasmic filaments in the cells of adult human articu-lar cartilage. Ann. rheum. Dis. 26, 50-58.

MEDOFF, J. (1967). Enzymatic events during cartilage differentiation in the chick embryoniclimb bud. Devi Biol. 16, 118-143.

MOSCONA, A. (1961). Rotation-mediated histogenetic aggregation of dissociated cells. Aquantifiable approach to cell interactions in vitro. Expl. Cell Res. 22, 455-475.

N0RREVANG, A. (1968). Electron microscopic morphology of oogenesis. Int. Rev. Cytol. 23,113-186.

PALFREY, A. J. &DAVIES,D. V. (1966). The fine structure of chondrocytes./. Anat. 100,213-216.PAUL, J. (1970). Cell and Tissue Culture. Edinburgh: Livingstone.REVEL, J.-P. & HAY, E. D. (1963). An autoradiographic and electron microscopic study of

collagen synthesis in differentiating cartilage Z. Zellforsch. mikrosk. Anat. 61, 110-144.REYNOLDS, J. J. (1967). The synthesis of collagen by chick bone rudiments in vitro. Expl Cell

Res. 47, 42-48.SCHROEDER, T. E. (1968). Cytokinesis: filaments in the cleavage furrow. Expl Cell Res. 53,

272-275.SEARLS, R. (1973). Chondrogenesis. In Developmental Regulation (ed. S. J. Coward). London:

Academic Press.SEARLS, R., HILFER, S. R. & MIROW, S. M. (1972). An ultrastructural study of early chondro-

genesis in the chick wing bud. Devi Biol. 28, 123-137.SEEGMILLER, R., FRASER, F. C. & SHELDON, H. (1971). A new chondrodystophic mutant in

mice. Electron microscopy of normal and abnormal chondrogenesis. / . Cell Biol. 48,580-593.

SERAFINI-FRANCASSINI, A. & SMITH, J. W. (1974). The Structure and Biochemistry of Cartilage.Edinburgh: Churchill Livingston.

SILBERBERG, R. (1968). Ultrastructure of articular cartilage in health and disease. Clin.Orthop. 57, 233-257.

SPOONER, B. S., YAMADA, K. M. & WESSELLS, N. K. (1971). Microfilaments and cell division./ . Cell Biol. 49, 593-613.

STRANGEWAYS, T. S. P. & FELL, H. B. (1926). Experimental studies on the differentiation ofembryonic tissue growing in vivo and in vitro. I. The development of the undifferentiatedlimb-bud (a) when subcutaneously grafted into the post-embryonic chick and (b) whencultivated in vitro. Proc. R. Soc. B 99, 340-366.

ZWILLING, E. (1966). Cartilage formation from so-called myogenic tissue of chick embryolimb buds. Annls Med. exp. Biol. Fenn. 44, 134-139.

(Received 2 February 1975)

electron microscopic studies on chick limb cartilage...

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