Scanning Microscopy
Volume 1 Number 1 Article 34
10-12-1986
Early Scanning Electron Microscopic Studies of Hard Tissue Resorption: Their Relation to Current Concepts Reviewed
A. Boyde University College London
S. J. Jones University College London
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Recommended Citation Boyde, A. and Jones, S. J. (1986) "Early Scanning Electron Microscopic Studies of Hard Tissue Resorption: Their Relation to Current Concepts Reviewed," Scanning Microscopy: Vol. 1 : No. 1 , Article 34. Available at: https://digitalcommons.usu.edu/microscopy/vol1/iss1/34
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EARLY SCANNINGELECTRON MICROSCOPIC STUDIESOF HARDTISSUE RESORPTION: THEIR RELATIONTO CURRENTCONCEPTS REVIEWED
A Boyde * and SJ Jones
Department of Anatomy and Embryology, University College London, Gower St., London WClE 6BT, England
(Received for publication June 06, 1986, and in revised form October 12, 1986)
Abstract Introduction
This paper highlights some observations This paper was presented as the lead-off made by the authors in SEM studies of hard tissue talk at a meeting (Biomineralization: Edward J. resorption and considers their significance in Reith Memorial Meeting on Scientific Investiga relation to current concepts. All mammalian tion of Vertebrate Mineralized Tissues) held at mineralised tissues may undergo physiological New Orleans, May 5-7, 1986. This meeting was resorption, the resulting surface reflecting the organized with the purpose of reviewing density of mineralisation and the organic matrix some of the major areas in which the SEM has chemistry, organisation and orientation. contributed to understanding of hard tissue Resorption-repair coupling may follow the structure and function in vertebrates. The resorption of any tissue, but SEM studies first present paper sets out to examine one small facet noted this process in the case of the dental of this microscopic-method-well-suited-to-the tissues. The difference between fetal and adult problem area, and from one direction only. We boneformation and resorption provided evidence have taken a second look at our own earlier SEM against the concept of osteocytic osteolysis. SEM data to see how it has affected our viewpoints stereophotogrammetric methods for the quantita concerning the natural history of resorption, and tion of individual resorption lacunae are now how the SEM study slots in with other recent cell much quicker and have been extended to the study biological studies. of in vitro resorption by mammalian and avian osteocl asts isolated from bone and seeded into The morphology£!:_ resorption new substrates. Experimental studies using SEM were first The phenomenon of osteocl as tic resorption occurs in all the calcified tissues: not only conducted on the osteotropic hormonal effects on bone, and calcified cartilage, but all the dental bones forming in vivo and extended to the in tissues. The first published SEM study of vitro situation. The effects observed underlined resorption concerned the dentine and enamel of the several actions of PTH on osteoblasts and human deciduous teeth
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370 HARDTISSUE RESORPTION figure l. Resorption of human deciduous dentine maturation of the osteoid and its thickness may (top) and enamel prior to shedding a tooth. also be important. Fieldwidth = 296 ,um. The early studies also showed that most figure 2. Rat compact bone o_steocyt~ lacunae human adult bone surfaces entered a "resting" exposed by osteoclastic resorption showing reten condition, in which the collagen fibre bundles of tion of separate perilacunar matrix phase, common the matrix were fully mineralized <6, 7,10,12>. in this species. Fieldwidth = 55 ).Jm. Thus intact fibres and bundles could be demon figure 3. Human tooth root near apex showing strated in anorganic specimens (Fig. 5). This, patches of resorption and areas_of repair and the fact that much endosteal (Fig. 6) and emanating in prior resorbed areas. Fieldw1dth = trabecular (Fig. 7) resorption occurs in patches 1122 ,um. within fields of well mineralized collagen, figure 4. Horse molar enamel resorbed by osteo showed that osteocl asts do not have to contend clasts prior to cementum deposition: another with a barrier of a thin osteoid layer in order example of resorption formation coupling. to recognize real bone <13>. Close examination Fieldwidth = 172 ,um. (Stereo-pair). of our early SfM figures of anorganic prepara Figure 5. The lining surface of a Haversian canal tions <6> shows that some endosteal surfaces in adult rat mandible, made anorganic with 1,2 showed nearly, but not quite complete mineraliza ethane diamine, showing complete mineralisation tion: some resorption patches can be found ending of matrix surface fibres in this resting surface. within such areas. However, as just pointed out, Fieldwidth = 7. 3 ,A.Jm. some end in osteoid proper. It is obvious that one of the great advan Studies of human and other mammalian tages of the SfM is the apparent 3-D nature of cementum resorption <4> showed that some resorp the 2-D projection of a surface, making it abun tion of the roots of human permanent teeth is a dantly clear as to the morphological complexity perfectly normal occurrence. Indeed, the growth of a resorbing surface. However, a 2-D projection of a second-phase of cementum - which may or may can deceive, and we have always felt the need not contain included cells (cementocytes), but for a real 3-D image in our work. Stereopair which otherwise usually includes a high propor images not only convey a vivid impression of tion of extrinsic (Sharpey) fibre bundles - was relationships, but can be formally analysed to shown to occur in relation to small patches of reconstruct profile sections and contour resorption in the apical portion of the root maps <14-16>. We have continued to develop (Fig. 3). This was the first demonstration of stereophotogrammetric methods for the purpose of resorption-formation coupling in an extra providing a quantitative description of bone skeletal site. This now popular concept was first resorption <17-19>. proposed for bone on the basis of human metabolic data <5>. (Another dental example of resorption formation coupling will be considered later - see Fig. 4 ). Horses have high crowned teeth which erupt The first SEM studies of adult and fetal bone and function before a root is formed. The mecha resorption <6, 7> again showed how the nature of nism of tooth attachment is that the crown SEM observations could bring to our attention enamel is covered with cementum, which serves to some important facts. It was found that attach the fibres of the periodontal ligament. resorption lacunae in bone were much srnal ler than This route is adopted by many mammals, but it is in deciduous tooth root removal . It was noted usual for cement to attach to a naturally rough, that osteoclastic resorption left osteocyte pitted surface of enamel. In horses, however, the lacunar walls intact. This observation indicated completed enamel surface, after maturation that osteocytes survived until release by mineralization, is superficially resorbed by resorption: they did not themselves remove either osteoclasts (Fig. 4). The reduced, post mineral or matrix as was hypothesised in the then maturation ameloblasts have somehow to move or be popular concept of osteocytic osteolysis moved out of the way for this purpose. This <9,10>. Another early SfM finding was that resorption is repaired immediately by the resorbed areas not uncommonly finished within formation of cement. This is another example of fields with mineralizing fronts indicating that resorption-formation coupling. However, this is resorption had occurred into bone covered with most unusual in cement coupling to enamel <20>. osteoid <6,7,10>: the latter point was, and is sti 11, at odds with current notions of bone Hormone experiments on bone pathologists who felt, and feel, that osteoid does not resorb. The most recent studies have Experiments using the SEM to observe the confirmed the initial findings - osteoid can be effects of the administration of parathyroid resorbed byosteoclasts <11>. The most conservative hormone (as parathyroid extract, PTf) and explanation of this which we can offer today is calcitonin (CT) to rats <21> showed both the to hypothesize that osteoclasts are able to power of the SEM as an observational tool, and resorb osteoid, but do not prefer to do so. Thus the rapidity with which dramatic changes can be active activated osteoclasts continue resorbing induced in bone matrix and mineral morphology. a surf~ce, perhaps until they reach an ost~oid Resorption induced by parathyroid hormone (PTH) covered patch; when the "stimulation" to continue was observed to begin, at flat bone surfaces, resorption is reduced - but only after some close to the places where these were penetrated osteoid has been tested or tasted. The degree of by blood vessel canals (Fig. 8). A great deal of
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Figure 6. Resorbed Iamellar bone matrix surface, Bone eel ls adult rabbit scapula covering cells removed by trypsin. Fieldwidth = 110 .)Jm. The first SEM observations of bone cells Figure 7. Human femur endosteal trabecular bone were made in a convenient location where all surface showing resorption into mineralised other overlying tissue layers could be easily matrix. Some parts of the matrix were still stripped away with apparently minimal disturbance mineralising as judged by the incomplete to the osteoblast pavement <23>. Such a site is appearance of the collagen fibres in this the endocranial surface of the skul 1 cap (cal va anorganic preparation. Fieldwidth = 250 ).Jm. rium: Fig. 11). In young rats, most of this Figure 8. Endosteal surface of rat bone from surface is formative, and is covered with a tes animal given parathyroid extract 3 daily doses of selation of cells <24> which are "cuboidal" and Parathormone which stimulates resorption and in close lateral contact prior to the induction mineralisation of free matrix surfaces. of drying-associated, shrinkage artefacts <25>. Fieldwidth = 176 ,um. The shrinkage artefact problem provoked us Figure 9. Surface of bone matrix from similarly into studying its origins, which we now more treated animal showing hypermineralisation of clearly understand <26, 27>. If we use the resting bone matrix surface. Fieldwidth = 17 ).Jm. advanced drying techniques of freeze drying (FD) Figure 10. Another area from similar specimen or critical point drying (CPD), we are left, at showing cleavage of well mineralised layer of one near-fina 1 stage in either process, with a matrix from less mineralised sub surfaces layer specimen with no free water, but some firmly after the specimen has been made anorganic. bound structural water. This stage occurs after Fieldwidth = 78 ).Jm. the sublimation of ice in FD, and after the Figure 11. Tesselation of osteoblasts covering pressure is reduced after the critical point in the endocrania l surface of the rat cal varium. CPD. In both cases, the drying of the dry sample The small gaps between these cells are induced by induces separation of osteoblasts from each CPD shrinkage. Fieldwidth = 176 .AJm. other. The well mineralized bone matrix hardly shrinks at all in comparison <25-27>. evidence has since been collected which supports Measurements of the areas covered by osteo the idea that osteoclasts or their precursors are blasts showed that they fel 1 within a closely transported via the blood stream: these studies, defined range - the secretory territory of the including the key experiments of G Gothlin and osteoblast <22,23>. The same type of preparation JLE Ericsson, and SC Marks and DG Walker are was used to provide details of the normal morpho referenced and reviewed in ref. (22>. We logical polarisation of osteoblasts <24>. interpret our SEM findings as possibly indicating that osteoclastic resorption stimulated by PTH Hormone experiments on bone cells occurred with the minimum requirement for migration of these cells. After showing that bone cells in this system Secondly, it was found that when compared with would survive well in culture conditions, the control animals, large areas of bone surface were behaviour of these cells in response to physical converted to what we called the "prolonged, and hormonal stimuli was investigated. These resting" condition, identified by the progression studies showed that the effect of the resorption of mineralization beyond the limits of the super inducing hormone, PTH, was primarily on the ficial collagen fibrils and bundles of free bone osteoblast <28-31), and not on the osteoclast as surfaces; thus leaving a smooth, less detailed had been believed up to that time. A direct surface in anorganic preparation. In other words, response of osteoblasts to the vitamin D PTH in the whole animal promoted mineralization metabolite l,25(0H) 2D3 was also demonstrated in of the bone matrix, particularly at its surface this way <28,29>. (Fig. 9 ). These studies also showed that osteoblasts In some instances, clear cases were are actively migratory cells, spreading on to identified where this surface mineralization deliberately denuded portions of the bone, and on overlay areas in which the osteoid had not to surrogate substrates, such as pieces of glass mineralized fully. Upon making such specimens or tooth placed in contact with the osteoblast anorganic, patches of the superficial mineralized layer or with bone marrow <30): this tendency of layer could be seen to break away (Fig. 10). This bone cells to migrate has since been used as the confirmed the impression that the PTH effect basis of an important method for isolating bone and/or combined with the expected hypercalcaemia, cells in culture <32>. could result in a "dumping" of mineral from blood Returning to the hormonal responsiveness of into the nearest available receptive location in the bone cells as monitored by SEM (28-31>, bone, and this happened to be osteoid <21>. osteoblasts showed no response to calcitonin The mineralization of superficial osteoid (CT). The dorsal surface ruffling which is an promoted by PTH in these experiments is, of adaptation shown by osteoblasts in culture was course, abnormal and the wrong way round - unchanged by addition of CT. However, osteoblasts osteoid normally mineralizes at its deep surface. responded dramatically to PTH in several diffe However, such a mechanism would provide the means rent ways, which led to the suggestion that these for al lowing osteoclasts to "see" mineral at the are the cells which regulate bone resorption free bone surface if these eel 1 s are dependent PTH induced the formation of gaps in the for their normal function upon the recognition of otherwise nearly continuous osteoblastic pave a calcified matrix. ment, which, in the short term, were greater than
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Figure 12. Osteoblasts on endocranial surface of nodules or calcospherites of the mineralizing rat calvarium cultured with added PHI for 24 h. front exposed to view
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376 HARDTISSUE RESORPTION figure 19. Part of the ruff led border zone of an The origin of osteoclasts osteoclast peeled separated from bone. Fieldwidth = 18 )..Im. A few years ago, it was fashionable to figure 20. Surface of slice of sperm whale assume that osteoclasts arose by the fusion of dentine cultured with chick osteoclasts for 19 mononuclear cells of the monocyte-macrophage days, showing, extensive resorption which has series (44>. We used immunological marking techn occurred in this period. Note the wide range of iques to examine this hypothesis, using the SEM size of the resorption pits. Some new osteo as the means of observing whether rosettes of clasts may have differentiated from marrow cells RBCs formed in the assay system. The results were in this period. Surface decorated with 10 micron negative - they demonstrated that osteoclasts did polystyrene latex spheres. Fieldwidth = 480 ,urn. not bear the Fe or C3 receptors characteristic of figure 21. Lower part of field shows edge of all cells of the macrophage-monocyte lineage resorption bay in human compact bone made by <45,46>, suggesting a separate origin at an chick osteoclast, revealing the alternate earlier stage of stem cell differentiation. This orientation of lamellae. Fieldwidth = 48 )..Im. finding was at first treated with considerable Figure 22. Chick osteoclastic resorption of human scepticism, but has now found acceptance compact bone. This field is selected to show following recent work which has extended and apparent selection of densely mineralised areas confirmed the original observations <47,48>. showing as white in this BSE image. However, careful study shows equal selection for all The isolated osteoclast resorption assay <49>. density phases in bone and unmineralised osteoid. Fieldwidth = 450 ,um. Most previous assays of the bone resorption Figure 23. Internal surface of mollusc shell process in vitro have been based on the measure resorbed by chick osteoc 1 asts. ment of biochemical parameters based upon all Fieldwidth = 104 ,um. events affecting all parts of all the matrix and Figure 24. Sperm whale dentine resorbed by osteo all the cells in a limited organ culture system clasts originating from chick marrow in 19 day i .g. 50 53>. The measurement of the release of culture. Sample broken and tilted to show 5Ca or 3H praline from prelabelled bones is a profile of fracture line through resorption sensitive approach: results are usually expressed lacuna. Fieldwidth = 90 )..Im. as the ratio of experimental to control values <50-52>. The release of calcium can also be The osteocytic osteolysis concept measured by simple chemical, colorimetric proce dures <53>. The problems with all these methods From our own viewpoint, osteocytic are that they do not establish that the release osteolysis became an unsupportable concept when of matrix or mineral components is the result we were unable to discover degradative changes in of osteoclastic activity, or to what extent the matrix surrounding osteocyte lacunae <6>. osteoclastic activity is control led by osteo Other evidence discrediting this hypothesis was blasts or furthered by other cell types, for considered by AM Parfitt in his influential example, macrophages. Further, these assays review <43>. Every case of "osteocytic osteoly always use fetal bone, and fetal bone matrix is sis" so far described in the literature can be degraded by non-specific proteases which may traced back to the failure to recognize the spec activate la tent coll agenase <10, 36>. Thus, as trum of bone and bone cell types from woven discussed earlier, osteoblasts may themselves be (fetal) to adult (lamellar). SEM studies clearly important in the degradative process in cultured emphasize the points of difference in these fetal bone. Analysis of sections of bones tissue types: large eel ls incorporated in large prepared for light microscopy has also been used numbers, often close to each other, with the in resorption assays, but this poses a sampling mineralization of juxta-lacunar collagen often and measurement problem in a rapidly changing incomplete in the back wall of the forming osteo tissue. We therefore wished to establish a system cyte lacuna in the mineralizing front region, are in which osteoclasts could be tested in isolation the hallmarks of the rapidly forming, fetal bone and on a known starting platform (Figs.20 - 24). type. Such characters are found in various bone The system has been successful and has been diseases, particularly those in which bone adopted by other groups <49, 54-61>. turnover rate is increased. Thus irregularly The measurement of the volume of bone or mineralized lacunar walls can be found in all dentine removed per resorptive episode in vivo forms of rickets and hyperparathyroidism <40>, might in itself be a useful parameter in charact but they are all formed as such in the first erizing this process under different normal and instance. There is no established evidence for pathological circumstances, and this is where we osteocytic involvement in bone matrix destruction began using SEM stereophotogrammetry <8, 14-19>. or demineralization. The converse is not true To provide the instrumental back-up for an assay however. of bone resorption based on the activity of Rat osteocytes may make perilacunar matrix individual cells, we developed computer aided (Fig. 2) which mineralizes to a high degree. If systems for the measurement of the volume of they do not encourage the mineralization of old resorption pits <8, 18, 19>. XYZdata acquisition bone matrix, they certainly permit it to occur - from the RS3 instrument built for us by Ross as is demonstrated by the maturation (i.e. Instruments <18> was controlled by a software increase of mineralization) phenomenon found in package (OC3D) for a Sharp MZ80K microcomputer. all lamellar bone. This instrument has now been superceded by an
377 A Boyde and SJ Jones improved one called SFS3 and a new software composition of the substrate (Fig. 21) by deter Jackage (STEREOSHIPS)written for a Sirius micro mining this afterwards, primarily by the use of ~omputer <19, 49). BSE A+ B imaging of samples which were well In our earlier SEM studies of in vivo polished before initial use (Fig. 22). Reid <11> ~esorption we could assign accurate values to the has, in this way, been able to show that avian volume, areas, radius of curvature, and depth of osteoclasts have no predisposition to res rb bone individual "scoops" , but these were minimal of different density due to different degrees of ,alues because such resorption almost invariably maturation-mineralization. Nevertheless, the 3tarted in areas which had had curved surfaces. local depth of resorption is affecte by the fhere was no reference plane against which to degree of mineralization; just like the situation neasure, and the edge of the resorption pits were in tooth resorption (
378 HARD TISSUE RESORPTION culture periods (Figs. 20 & 24) are exceptionally <9> Belanger LF (1969) Osteocytic osteolysis. large, single and smooth, and therefore presumab Calcif.Tiss.Res. 4 1-12. ly single-cell origin scoops formed by the cont
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Haggis (ed.), SEM Inc: AMF O'Hare IL, 71-75. <46> Jones SJ, Hogg NM, Shapiro IM, Slusarenko <28> Jones SJ, Boyde A (1977) SEM of bone cells M, Boyde A (1981) Cells with Fe receptors in the in culture. Excerpt a Medi ca Int. Congr.Ser.421: cell layer next to osteoblasts and osteoclasts on 97-104 • bone. Met ab. Bone Dis. Rel. Res. 2: 357-362. <29> Jones SJ, Boyde A, Ness AR (1976) SEM (47> Horton MA, Rimmer EF, Chambers TJ (1986) studies of steeoblasts in organ culture. In: Bone Giant cell formation in rabbit long-term bone Histomorphometry. Meunier PJ, (ed). Paris: marrow cultures: Immunological and functional Armour-Montagu, 275-290. studies. J. Bone Min. Res. 1: 5-14. (30) Jones SJ, Boyde A (1977) The migration of <48> Horton MA, Lewis D, McNaul ty K, Pringle osteoblasts. Cell Tiss. Res.184: 179-185. JAS, Chambers TJ (1986) Characterisation of mono <31> Jones SJ, Boyde A (1976) Experimental study clonal antibodies specific for human osteoclasts. of changes in osteoblastic shape induced by J. Bone Min. Res. l: 94 (Abst. 140). ca lei tonin and parathyroid extract in an organ <49> Jones SJ, Boyde A, Ali NN, Maconnachie E culture system. Cell Tiss.Res. 169: 449-465. (1986) Variation in the sizes of resorption <32> Beresford JN, Gallagher JA, Poser JW, lacunae made in vitro. Scanning Electron Russell RGG (1984) Production of osteocalcin by Microscopy 1986; IV: 1571-1580. human bone cells in vitro. Effects of <50> Reynolds JJ, Dingle TJ 1970) A sensitive in l,25(0H) 2 D3 , 24,25(0H) 2D3 , parathyroid hormone vitro method for studying the induction and inhi and glucocorticoids. Metab. Bone Dis. Rel.Res. bition of bone resorption. Calcif. Tiss. Res. 4: 5: 229-234. 339-349. (33) Reeve J, Hesp R, Williams D, Hulme P, <51> Raisz LG, Simmons HA, Sandberg AL, Canalis Zanel l i JM, Darby AJ, Tregear GW, Parsons J.A. E (1979) Direct stimulation of bone resorption by (1976) Anabolic effect of low doses of a fragment epidermal growth factor. Endocrinology, of human parathyroid hormone on the skeleton in 107:270-272. postmenopausal osteoporosis. The Lancet, 15 May, <52> Mundy GR, Eilon G (1981) Effects of 1976, pp. 1035-1038. inhibition of microtubule assembly on bone <34> Egan JJ, Rodan GA (1986) Cell density mineral release and enzyme release by human dependent disassembly of osteoblast cytoskeleton breast cancer cells. Lab. & Clin. Invest. 67: correlates with increased cAMP levels. J. Bone 69-76. Min.Res. l: 68 (Abst. 39). <53> Ali NN, Auger DW, Bennett A, Edwards DA, <35> Vaes G (1972) The release of collagenase as Harris M. (1979) Effect of Prostacylin and its an inactive proenzyme by bone explants in cul breakdown product 6-oxo-PGF 1 on bone resorption tu re. Bi ochem J. 126: 275-289. in vitro. In Prostacylin, (eds). JR Vane, S <36) Boyde A, Banes AJ, Dillaman RM, Mechanic GL Bergstrom Raven Press: New York, 179-185. (1978) A morphological study of an avian bone <54> Boyde A, Ali NN, Jones SJ (1984) disorder caused by myeloblastosis-associated Resorption of dentine by isolated osteoclasts in virus. Met ab.Bone.Dis.Rel.Res. l: 235-242. vitro. Brit. Dent. J. 156: 216-220. <37> Eeckhout Y, Vaes G (1977) Further studies <55> Ali NN, Boyde A, Jones SJ (1984) Motility on the activation of procollagenase, the latent and resorption: Osteoclastic activity in vitro. precursor of bone coll agenase. Bio chem. J. Anat. & Embryo!. 170: 51-56. 166: 21-31. <56) Jones SJ, Boyde A, Ali NN, Maconnachie E <38) Heath JK, Atkinson SJ, Meikle MC, (1985) A review of bone eel l and substratum Reynolds JJ (1984) Mouse osteoblasts synthesize interactions. An illustration of the role of collagenase in response to bone resorbing agents. scanning electron microscopy. Scanning 7: 5-24. Biochim. Biophys. Acta. 802: 151-154. <57> Boyde A, Ali NN, Jones SJ (1985) Optical (39) Chambers TJ, Fuller K (1985) Bone cells and scanning electron microscopy in the single predispose bone surfaces to resorption by osteoclast resorption assay. Scanning Electron exposure of mineral to osteoclastic contact. J. Microscopy 1985; Ill: 1259-1271. Ce! l Sci. 76: 155-165. <58> Jones SJ, Boyde A, Ali NN (1984) The <40) Boyde A (1980) Evidence against osteocytic resorption of biological and non biological osteolysis. Metab. Bone Dis. Rel.Res. 2 Suppl: substrates by cultured avian and mammalian 239-255. osteoclasts. Anat. & Embryo!. 170: 247-256. <41> Heersche JNM (1978) Mechanism of <59> Chambers TJ, Revell PA, Fuller K, Athanasou osteoclastic bone resorption: A new hypothesis. NA (1984) Resorption of bone by isolated rabbit Cale. Tiss.Res. 26: 81-84. osteoclasts. J. Cell Sci. 66: 383-399. <42> Marotti G (1980) Three dimensional study <60> Chambers TJ, Fuller K, McSheehy PMJ, of osteocyte lacunae. Metab. Bone Dis. Rel.Res. Pringle JAS (1985) The effect of calcium 2Suppl: 223-229. regulating hormones on bone resorption by <43> Parfitt AM (1977) The cellular basis of isolated human osteoclastoma eel ls. J.Pathol. bone turnover and bone loss. Clin. Orthop. Rel. 145: 297-305. Res. 127: 236-247. <61> Chambers TJ, McSheehy PMJ, Thomson BM, <44> Chambers TJ (1980) The cellular basis of Fuller K (l9B5) The effect of calcium regulating bone resorption. Clin. Orthop. Rel.Res. 151: 283- hormones and prostaglandins on bone resorption by 293. osteoclasts disaggregated from neonatal rabbit <45> Hogg N, Shapiro IM, Jones SJ, Slusarenko M, bones. Endocrinol. 116: 234-239. Boyde A (1980) The lack of Fe receptors on <62> Reid SA (1986) A study of lamellar organisa osteoc lasts. Ce 11. Tiss. Res. 212: 509-516. tion in juvenile and adult human bone. Anat. & Embryo!., 174: 329-338.
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(63> Jones SJ, Ali NN, Boyde A (1986) Sur vi val Authors: Other connective tissue cells are able and rescrptive activity of chick osteoclasts in to dismantle the matrix which they have made. culture. Anat. & Embryo1. 174: 265-275. This is amply illustrated in many studies on the <64> Jaworski ZFG, Duck B, Sekaly G (1981) activities of fibroblasts (as an EM based Kinetics of osteoclasts and their nuclei in example, see AR Ten Cate (1972) Morphological evolving secondary Haversian systems. J. Anat. studies of fibrocytes in connective tissues 133: 397-405. undergoing rapid remodelling. J. Anat. 112: 410- <65> Loutit JF, Townsend KMS (1982) Longevity 414). Our argument is that evidence for of osteoclasts in radiation chimaeras of beige osteocytes or osteoblasts performing this and osteopetrotic microphthalmic mice. Brit. J. function during normal resorption in vivo is Exp. Pathol. 63: 214-220. lacking. (66> Marks SC (1984) Congenital osteopetrotic Lacunar margin modification by osteocytes mutations as probes of the origin, structure, and does occur, but this has only been demonstrated function of osteoclasts. Clin. Orthop. Rel. Res. in the sense of the accretion of new matrix and 189: 239-263. mineral in the walls of osteocyte lacunae. <67> Chambers TJ, Thomson BM, Fuller K (1984) Perilacunar bone was not discovered by SEM - Effect of substrate composition on bone that can be attributed to the work of IA Mjor resorption by rabbit osteoclasts. J. Cell Sci.7[J: (Bone matrix adjacent to lacunae and canaliculi. 61-71. Anat Rec. 144, 327-329, 1962) using the TEM. However, it became very much clearer from the Discussion with Reviewers earliest SEM studies that perilacunar bone was an interesting material which can be deposited by Reviewer I: What contribution could the SEMmake osteocytes in some locations and/or in some towards resolving some of the outstanding issues, species. such as resorption-formation coupling and the It remains to be discovered what is the origin of the osteoclasts? nature of the antigen discovered by Nijweide's Authors: The SEM is just one tool which can be antibody to something at the cell surface of used to focus on small facets of the bone chick osteocytes. It is as likely that this resorption problem. As regards resorption represents new synthetic activity (something to formation coupling, we have indicated that it do with peri 1 acunar bone?), as that it may enabled us to see that this was happening in the represent matrix degradative activity. extra-skeletal context of cellular cementum The examples we have chosen here from the formation on the roots of human permanent teeth. study of hard tissue resorption, we state again, It is improbable that the necessary global view are essentially chosen to illustrate the merits would have been obtained by reconstructions from of the SEM in this sphere of investigation. Of serial sections from any other microscopic course, it has also been of great utility in method. The survey potential of the SEM has not studying the formation and the structure of the yet been exploited in examining skeletal hard tissues, including many aspects of resorption-formation coupling at the local scale. mechanical and clinical interference with these As regards the origin of osteoclasts, the tissues in orthopaedics and dentistry. SEM provided the means to observe that the surface antigens of the cells of the monocyte 0. Johari: In stereo-pair (Fig. 4), how can macrophage series were not borne by osteoclasts. stereo-effect be observed? At that time it was widely assumed that the cell Authors: A few persons can detect the stereo types were at least very closely related. So, effect without the aid of any stereo-viewing at least, even if the SEM cannot solve problems, device. For others, we suggest that the page be it can raise them. rotated 90° and viewed under a stereo-viewer. However, we believe that the SEM has a role to play in investigating the function of osteoclasts. An example of this is its sensitivity to the detection of changes in the surface of a substrate using BSE imaging. This may be very important when testing the resorptive ability of putative osteoclast precursor cells, since it is a characteristic ability of an osteoclast to demineralize the tissue it contacts.
Reviewer II: It is not justified to extrapolate from the standpoint that lacunar margin modifica tion by osteoclasts does not exist, to conclude that their precursors, the osteoblasts, cannot remove osteoid. The authors have overlooked recent evidence for an osteocyte-speci fie eel l surface antigen (PJ Nijweide & RJP Mulder, A monoclonal antibody specifically directed against osteocytes. Cale. Tiss. Int. 38 suppl, Abst. 28, 1985).
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