Annals ofthe Rheumatic Diseases 1995; 54: 339-344 339

EXTENDED REPORTS Ann Rheum Dis: first published as 10.1136/ard.54.5.339 on 1 May 1995. Downloaded from Magnesium whitlockite deposition in articular cartilage: a study of 80 specimens from 70 patients

Colin A Scotchford, S Yousuf Ali

Abstract strate a pericellular distribution associated with Objective-To examine articular cartilage an extension of the calcified zone.'0 1' BCP from a number ofjoint sites, using a large crystals have been reported in the synovial fluid sample group, for the presence of mag- of approximately 30% of patients in a number nesium whitlockite crystal deposition. of studies.3 12`14 Several patterns of arthritis have Methods-Articular cartilage specimens been associated with BCP crystals, including were taken from a total of 70 patients. The primary arthritis, polyarticular arthritis and majority of specimens were taken from Milwaukee shoulder syndrome. The deposition femoral heads, with smaller numbers of BCP crystals in articular cartilage may be a from femoral condyle, tibial plateau, secondary event resulting from other changes in radius, ulna, and several small peripheral cartilage metabolism or structure. Their pres- joints. Normal and osteoarthritic articular ence, however, may induce further damage to cartilage specimens were obtained from the tissue by feedback mechanisms resulting in patients undergoing prosthesis replace- an amplification loop.'5 Support for such a ment or amputation. Specimens were relationship may be drawn from a number of resin embedded and examined using studies. The severity of knee joint degeneration transmission electron microscopy and x has been correlated with con- ray microanalysis. centration in synovial fluid'2 16 and a rapidly Results-Magnesium whitlockite crystals progressive destructive disease of the shoulder were identified, on the basis of mor- associated with hydroxyapatite deposition has phology, size and elemental composition, been described.'7 18 in articular cartilage from all sites Anomalous calcium 'cuboid' sampled. The distribution of crystals was crystals were first described in osteoarthritic similar in all samples (restricted to the and elderly human articular cartilage.' 10 19 20 superficial zone), although the density of On the basis of their calcium to phosphorus deposition was extremely variable, with ratio and morphology, they were postulated to http://ard.bmj.com/ the greatest density observed in femoral be magnesium whitlockite. 10 21 More recently, head specimens. No magnesium whit- crystals having the same morphology, size lockite crystals were observed in osteo- range and calcium to phosphorus ratio were phytic or epiphysial cartilage. reported in osteoarthritic (OA) articular Conclusions-This study demonstrated cartilage from nine of 19 patients22 and the widespread extent ofmagnesium whit- consistently, in a study of normal human lockite deposition in human articular femoral head articular cartilage, in all of 12 on September 29, 2021 by guest. Protected copyright. cartilage, albeit at much lower density patients across a broad age range.23 Crystal than previously reported in femoral head density was significantly greater in superior articular cartilage. In consideration of region samples than inferior region samples; possible roles for these crystals in this difference was smaller in older specimens. articular cartilage, it is concluded that an A band of crystals 10-20 ,um below the opportunistic mode offormation, possibly articular surface was observed in superior influenced by mechanical stresses, would region samples. Using electron and x ray be most plausible. diffraction techniques, these crystals have since Institute of been identified as magnesium whitlockite; the Orthopaedics, (Ann Rheum Dis 1995; 54: 339-344) possibility of artefactual formation was University College discounted by using a variety of tissue prep- London Medical School, aration methods.24 Magnesium whitlockite is a Royal National Arthropathies associated with monosodium basic calcium phosphate, similar to 1-tri Orthopaedic Hospital, urate (MSU) and calcium pyrophosphate calcium phosphate, in which Mg2+, H20 and Stanmore, United Kingdom dihydrate (CPPD) deposition are well HP042 play a structural role.25 C A Scotchford established.' 2 More recently, basic calcium In this study, normal articular cartilage from S Y Ali phosphate (BCP) crystals, including hydroxy- an extensive range of patients and joint sites Correspondence to: ,3 carbonated apatites4 and octocalcium has been examined for magnesium whitlockite Professor S Y Ali, Institute of Orthopaedics, phosphate,"6 have been assigned a role in crystal deposition. Royal National Orthopaedic crystal arthropathy.3 7 Hospital, Brockley Hill, BCP crystal deposition takes place at two Stanmore, main articular sites, periarticular tendon8 and Materials and methods Middlesex HA7 4LP, United Kingdom. intra-articular cartilage.3 In articular carti- SPECIMENS Accepted for publication lage, apatite crystals tend to be deposited in Articular cartilage specimens were taken from 16 December 1994 smaller amounts than in tendon,'0 and demon- a total of 70 patients. The majority of 340 Scotchford, Ali

specimens were normal femoral head articular dylate buffer containing 0-2 mol/l sucrose cartilage, resected because of tumour located (Merck) (pH 7 4) (wash buffer). The tissue

distant from the sampled cartilage, or blocks were then divided and half the blocks Ann Rheum Dis: first published as 10.1136/ard.54.5.339 on 1 May 1995. Downloaded from offemoral neck, or osteoarthritic cartilage from underwent secondary fixation with 1% osmium the same site. Cartilage was taken from other tetroxide (Agar Scientific) in 0-085 mol/l joint sites, when available, from patients having sodium cacodylate for 90 minutes at room amputation or resection for bone tumour. temperature. Buffer washing was repeated and In all cases full depth blocks of articular all tissue blocks were dehydrated through a cartilage, plus subchondral bone, were taken graded alcohol series. Specimens were trans- from sample sites. Specimens were generally ferred to propylene oxide (Agar Scientific) for obtained within 20 minutes of resection. 30 minutes before infiltration with a 1:1 Normal articular cartilage samples were taken propylene oxide:araldite CY2 12 resin (Agar from weight bearing regions of joints where Scientific) mixture for one hour, followed by possible. All tissues remained undecalcified. infiltration in neat CY2 12 resin under vacuum (150 mbar) overnight, and embedding in fresh resin at 60°C for 48 hours. CY212 resin was TRANSMISSION ELECTRON MICROSCOPY mixed according to the manufacturer's Tissue samples were subdivided to full depth instructions with the omission of methyl blocks approximately 1 mm in the remaining phthalate (plasticiser). two dimensions. A small amount of subchon- Ultrathin sections (70-100 nm) were cut dral bone was left attached to minimise tissue with diatome diamond knives (Leica, Milton distortion during processing and facilitate easy Keynes, UK) using a Reichert Ultracut E tissue orientation. The tissue blocks were fixed ultramicrotome (Leica) and floated onto for two to four hours in 1-5% glutaraldehyde 0-085 mol/l sodium cacodylate buffer before (Agar Scientific, Stansted, UK) in 0-085 mol/l collection on to G200 HS copper grids (TAAB sodium cacodylate buffer (pH 7-4) (Merck, Laboratories, Aldermaston, UK). Where Poole, UK). Specimens were then washed in necessary the grids were precoated with 0.45% three changes of 0-085 mol/l sodium caco- piolform (Agar Scientific) in chloroform (Merck). If required, ultrathin sections were Table 1 Range ofsample sitesfrom which full depth articular cartilage specimens were stained on drops of aqueous saturated uranyl taken, number ofspecimens obtainedfrom each site listed, and age range and male tofemale ratio ofpatients. The extent ofmagnesium whitlockite crystal deposition is indicated on a acetate (Agar Scientific) for 10 minutes at presence/not observed basis, with a qualitative grading scorefor the density ofdeposition, as room temperature followed by lead citrate for determined by transmission electron microscopy five minutes at room temperature in the Samnple site No of Age range M/F No ofsamples presence of sodium hydroxide pellets (Merck). samlples (yr) Crystals Crystals present (grade) not observed X RAY MICROANALYSIS Femoral head (normal) 34 10-93 14-20 34 (3-4) http://ard.bmj.com/ Femoral head (OA) 26 46-83 8/18 26 (1-2) Unstained sections were examined using a Philips Peripheral osteophyte 5 53-73 3/2 -(0) 5 CM12 transmission electron microscope with an Femoral condyle 3 5-20 2/1 3 (2) Tibial plateau 2 19-20 2/0 2 (1) EDAX PV9800 x ray microanalysis (XRMA) Radial head 1 19 1/0 1 (3) system. Spectra were recorded at 100 kV Proximal ulna 1 19 1/0 1 (3) 1st metatarsal 2 17-61 2/0 2 (1-2) (operation at maximum available kV has been Carpal 1 19 1/0 1 (1) recommended to obtain maximum peak to 2nd metacarpal 2 19-62 2/0 2 (1-2) Lunate 2 19-62 2/0 2 (1-2) background ratios26), for 200 live seconds with a Phalanges 1 17 0/1 1 (1-2) tilt angle of 20°, giving a total 'take off angle of on September 29, 2021 by guest. Protected copyright. Epiphysial growth plate 1 9 1/0 -(0) 1 400. The electron probe size was selected to encompass individual crystals and was never greater than 200 nm diameter; a 70 nm 'top hat' Ka condenser aperture was used to minimise the background effect of x rays generated higher in the column reaching the specimen area. Calcium to phosphorus ratios were calculated using the quantitative analysis of thin sections software package (EDAX PV9800) based on the ratio P Koc model.27 The synthetic hydroxyapatite sample was used as the calcium phosphate standard for these analyses.28

Results Crystals were observed in articular cartilage from all sites sampled (table 1). Identification was based on the distinctive 'cuboid' morphology, electron density, size range (50-500 nm) and XRMA spectrum, and the calcium to phosphorus 2.00 4.00 6.00 8.00 ratio generated from this. Crystals from areas of KeV deposition all generated spectra with charac- teristic calcium, phosphorus and magnesium Figure 1 Characteristic x ray microanalysis spectrum from magnesium whitlockite crystals in human articular cartilage. Peaks for calcium, phosphorus and magnesium (arrow) are peaks (fig 1). Calcium to phosphorus ratios clearly present. ranged from 1 30 to 1 47. Magnesium whitlockite deposition in articular cartilage: a study of80 specimensfrom 70 patients 341

The distribution in all cases was similar to contain characteristic granular intramatrical that described for normal femoral head lipidic debris, including membranous struc- articular cartilage.23 Crystals were present tures (fig 2A). Other specimens demonstrated Ann Rheum Dis: first published as 10.1136/ard.54.5.339 on 1 May 1995. Downloaded from either within a band running parallel to the a deepening of the band, encompassing initial articular surface from 5 ,um to a maximum of superficial zone chondrocytes (fig 2B). Such 50 ,um depth below the surface, or in close differences between specimens appeared to be proximity to chondrocytes, and occasionally in related to the age of the patient, specimens dense clusters. A band of crystals was iden- from older patients having deeper bands of tified in many specimens with variable density crystal deposition. Crystals were commonly and depth, frequently with a degree of observed deposited pericellularly to chondro- patchiness. The depth of the band in some cytes, apparently associated with intramatrical specimens was limited to extreme superficial lipidic debris (fig 2C). In many cases the articular cartilage extracellular matrix between crystal distribution was biased, with greater the articular surface and the most superficial numbers adjacent to the articular surface chondrocytes, a region frequently observed to aspect of the cells when viewed in sagittal

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Figure 2 Transmission electron micrographs ofmagnesium whitlockite crystal deposition in human articular cartilage. A: Crystals (C) are present, forming a band immediately below the articular surface, together with intramatrical lipidic debris (L). (Patient 20years;femoral condyle; section stained with uranyl acetate and lead citrate.) B: Denser crystal deposition infemoral head articular cartilage shows a broader band. (Patient 43 years; femoral head; unstained section.) C: Sparse deposition in specimens ofmetacarpal articular cartilage. (Patient 19 years; unstained section.) D: Pericellular distribution ofcrystals (C) and intramatrical lipidic debris (L) in the superficial zone ofmetatarsal articular cartilage. (Patient 61 years; unstained section.) 342 Scotchford, Ali

section. Crystals were frequently observed This work supports and broadens the within chondrocyte territorial matrix, often in original limited studies of Ali et al9 10 21 and

close proximity to the plasma membrane; Stockwell et al.'9 20 With the exception of these Ann Rheum Dis: first published as 10.1136/ard.54.5.339 on 1 May 1995. Downloaded from however, crystals were not observed in living authors, such crystal deposition in articular chondrocytes. In deeper zones, magnesium cartilage has not been previously reported, whitlockite crystals were rarely observed. although the reports of the deposition of Crystal deposition when encountered was, in calcified bodies in human meniscus by the main, perilacunar, amongst areas of lipidic Ghadially and Lalonde30 and of tricalcium debris. phosphate in articular cartilage by Bardin The density of deposition showed much et a122 are most closely related. A number of variability between joint sites, with no site points may be raised in an attempt to explain reaching the greatest densities observed in this apparent oversight. normal femoral head articular cartilage. A five The ultrastructural study of normal human point qualitative grading scale (table 2) was femoral head articular cartilage-a site where devised to compare crystal distribution in deposition has been observed at its most tissues from different sites. In the knee, whilst dense-is limited. Ghadially31 commented that articular cartilage from both the femoral knowledge of the fine structure of normal condyle (grade 2) and the tibial plateau had articular cartilage is derived largely from a crystals present, deposition in the tibial plateau study of animal material, particularly rabbit, tissue was very sparse (grade 1). A small with more limited studies of human cartilage number of specimens from several smaller from fibrillated and normal looking OA joints were examined. Crystal deposition specimens, juvenile specimens taken at autopsy density in cartilage from the proximal ulna and young adult specimens surgically and radial head was similar to that in the obtained. Many of these studies were based on femoral condyle samples (grade 2-3). In specimens from the femoral condyle a site samples from the remaining sites, deposition which, in this study, exhibited a lower was more sparse than this, with the least maximum crystal deposition density than density observed in samples of carpal and superior region femoral head specimens.23 The phalangeal cartilage (grade 1) (fig 2D). No commonly used thin section stain uranyl crystals were observed in peripheral acetate, and distilled water, routinely used to osteophytes from OA femoral heads or float thin sections away from the ultratome epiphysial growth cartilage. knife edge, both showed a tendency towards Despite occasional focal densities and dense crystal loss.32 33 Uranyl acetate and lead citrate banding of crystals in the superficial zone staining imparts greater electron density to matrix, there was no evidence of structural matrix components in thin section, possibly disruption of collagen fibril orientation by concealing the presence of crystals in cases of crystals in normal articular cartilage, nor was sparse deposition. With this in mind, re- there any evidence of chondrocyte degener- examination of early studies ofhuman articular http://ard.bmj.com/ ation induced by crystal deposition. Crystals cartilage34 reveals electron densities amongst appeared to be present in the interfibrillar stained lipid debris bearing an arguable space without disrupting the overall fibrillar resemblance to 'cuboid' crystals. arrangement. Distribution of crystal deposition in this study conformed to that described for normal femoral head articular cartilage,23 the main Discussion difference being the density of deposition at on September 29, 2021 by guest. Protected copyright. Previous studies using electron diffraction and different locations. The earlier study demon- x ray diffraction techniques have shown strated an association between load bearing crystals with the same morphology, elemental and crystal density around the femoral head.2' composition, calcium to phosphorus ratio, and Evidence for the influence of load bearing size range to be magnesium whitlockite.24 29 stresses may be extrapolated from this study. The positive identification of crystals in this Mechanical loading exerts a major influence on study using the above criteria is considered articular cartilage.35 36 It has been suggested sound. The results of this study highlight the that mechanical stress is involved in the extent of magnesium whitlockite crystal regulation of chondrocyte metabolism.37 deposition in human articular cartilage. Whilst crystals were present in cartilage from all joint sites examined, the density of such deposition was always less than in the superior Table 2 Criteriafor qualitative grading ofcrystal deposition density region femoral head as determined by the qualitative grading system. This was most Grade Definition noticeable in specimens from the smaller, more 0 Crystals not observed. peripheral joint sites which are normally 1 At least one crystal observed, searching necessary. subject to lower load bearing stresses.38 2 Crystals present in a diffuse distribution, no apparent Magnesium whitlockite deposition has been pattern, regular encounters, extensive searching not necessary. reported in a number of biological calcifi- 3 Crystals present in localised groupings, generally cations, both as calculi and in tissues. The pericellular to chondrocytes plus a diffuse most common of these are orally associated, intermittent band. with sites including dental calculi,39 arrested 4 Crystals effectively form a continuous band, running parallel with, and immediately below the articular carious dentine,40 and sialoliths.4' Whitlockite surface; local patchiness may be observed in the has also been reported in urinary calculi,42 band at greater magnifications. though this is considered a minor component Magnesium whitlockite deposition in articular cartilage: a study of 80 specimensfrom 70 patients 343

in such situations. Deposition of whitlockite of fibrillation has been reported by Clift et al;49 within tissues has been reported at various sites however, no evidence for the correlation of including aortic valvular tophi,43 pulmonary fibrillation with magnesium whitlockite crystal Ann Rheum Dis: first published as 10.1136/ard.54.5.339 on 1 May 1995. Downloaded from calcifications,44 and in cartilage from the nasal deposition was observed in this study. septum, trachea, epiglottis, and intervertebral In conclusion, magnesium whitlockite disc.45 Marked differences in magnesium crystal deposition has been demonstrated in content have been reported between whit- articular cartilage across a broad age range at lockites found in calculi and those from several joint sites. If an apparently oppor- cartilages.45 The magnesium content of the tunistic mode of crystal deposition is accepted, whitlockite crystals in articular cartilage a role as an aggravator of pathological corresponds with those of tracheal and degeneration via an amplification loop, similar intervertebral disc cartilage.24 The pathological to that described by Dieppe and Calvert,'5 nature of these depositions appears at odds would seem plausible. The potential for such with the current report of magnesium whit- a role may be clarified by fuirther study. lockite deposition in apparently normal tissue. This work was funded by the Arthritis and Rheumatism The presence of these crystals requires Council, UK. We are grateful to Action Research for provision consideration oftheir role in articular cartilage, of the transmission electron microscope. physiological or pathological. The demon- stration of crystal deposition in articular cartilage from normal adolescent to elderly 1 Freemont A J. Arthritis. In: Salisbury J R, Woods C G, Byers P D, eds. Diseases of bone and joints. London: cartilage in this study, suggests that they do Chapman and Hall, 1994; 163-210. not, per se, pose a pathological risk to the 2 Moskowitz R W. Diseases associated with the deposition of CPPD or Hydroxapatite. In: Kelley W N, Harris E D Jr, tissue. Ruddy S, Sledge C B, eds. Textbook of rheumatology, 4th This may indicate that they are the result of edn. Philadelphia: W B Saunders, 1993; 1337-54. 3 Dieppe P A, Huskisson E C, Crocker P, Willoughby D A. opportunistic deposition, with no defined Apatite deposition disease: a new arthropathy. Lancet function. Their circumstantial association with 1976; 1: 266-8. 4 McCarty D J, Lehr J R, Halverson P B. Crystal populations intramatrical lipidic debris may relate crystal in human synovial fluid. Arthritis Rheum 1983; 26: deposition to the removal ofmaterial generated 1220-4. 5 Faure G, Netter P, Malaman B, Steinmetz J, Duheille J, from chondrocyte necrosis from the extra- Gaucher A. Scanning electron microscopic study of cellular matrix to the synovial fluid, a function microcrystals implicated in human rheumatic diseases. Scanning Electron Microscopy 1980; III: 163-76. considered by Ghadially.3' This would provide 6 Faure G, Daculsi G, Netter P, Gaucher A, Kerebel B. an opportunity for further speculation on in heterotopic calcifications. Scanning Electron Microscopy 1982; IV: 1629-34. crystal deposition: first, providing possible 7 Schumacher H R, Cherian P V, Reginato A J, Bardin T, means of damping fluxes created by cell Rothfuss S. Intra-articular apatite crystal deposition. Ann Rheum Dis 1983; 42 (suppl): 54-9. necrosis, minimising the effects on the 8 McCarty D J, Gatter R A. Recurrent acute inflammation pericellular environment and second, raising associated with focal apatite crystal deposition. Arthritis the do stay at the site of Rheum 1966; 9: 804-19. question, crystals 9 Ali S Y, Griffiths S. New types of calcium phosphate crystals http://ard.bmj.com/ formation, or is there a migration to the in osteoarthritic cartilage. Semin Arthritis Rheum 1981; 11 (suppl 1): 124-6. articular surface as suggested for necrotic 10 Ali S Y. Apatite-type crystal deposition in articular cartilage. debris?3' The existence of a crystal band in the Scanning Electron Microscopy 1985; IV: 1555-66. 11 Howell D S. Crystal deposition disease. In: Wright V, ed. superficial zone may support the latter. Topical reviews in rheumatic disorders 2. Bristol: PSG The possibility of the involvement of Wright, 1982; 75-96. 12 Halverson P B, McCarty D J. Identification of hydroxy- subarticular matrical lipid in boundary lubri- apatite crystals in synovial fluid. Arthritis Rheum 1979; 22: cation-the formation - of an adsorbed 389-95. 13 Schumacher H R, Miller J L, Ludvico C, Jessar J A. Erosive on September 29, 2021 by guest. Protected copyright. protective layer on articulating surfaces-has arthritis associated with apatite crystal deposition. been raised.46 47 If lipid does act as a boundary Arthritis Rheum 1981; 42 (suppl): 54-9. an 14 Ohira T, Ishikawa K. Hydroxyapatite deposition in lubricant, involvement in crystal formation osteoarthritic articular cartilage of the proximal femoral may reduce such activity. In the presence of head. Arthritis Rheum 1987; 30: 651-60. 15 Dieppe P A, Calvert P. Crystals and joint disease. London: synovial fluid, with the potential for boundary Chapman and Hall, 1983. lubrication from hyaluranon,48 it would be 16 Paul H, Reginato A J, Schumacher H R. Alizarin red to S-staining as a screening test to detect calcium difficult envisage a significant decrease in the compounds in synovial fluid. Arthritis Rheum 1983; 26: coefficient of friction resulting from a loss of 191-200. 17 McCarty D J, Silcox D C, Coe F, et al. Diseases associated lipid as suggested. An increase in the with calcium pyrophosphate dihydrate crystal deposition: coefficient of friction of cartilage samples has a controlled study. AmJ Med 1974; 56: 704-14. 18 Dieppe P A, Doherty M, MacFarlane D G, Hutton C W, been reported in conjunction with increased Bradfield J W, Watt I. Apatite associated destructive incidence of crystal deposition, nominally arthritis. BrJ3Rheumatol 1984; 23: 84-9 1. 19 Marante I, MacDougall R, Ross A, Stockwell R A. Ultra- CPPD, and severity of tissue fibrillation, structural observations of crystals in articular cartilages of although the relative contribution of either aged human hip joints. Ann Rheum Dis 1983; 42 (suppl): was not 96-7. factor determined.49 20 Stockwell R A. Distribution of crystals in the superficial The presence of crystals in articular cartilage zone of elderly human articular cartilage of the femoral head in subcapital fracture. Ann Rheum Dis 1990; 49: may alter tissue compliance. It would seem 231-5. unlikely that the crystal deposition observed in 21 Rees J A, Ali S Y, Mason A Z. Scanning electron microscopy this and microanalysis of 'cuboid' crystals in human articular study, being primarily at the articular cartilage. In: Ali S Y, ed. Cell mediated calcification and surface, would significantly alter the load matrix vesicles. Amsterdam: Elsevier Science Publishers the BV, 1986; 365-71. spreading ability of tissue. More pertinent 22 Bardin T, Lansaman J, Bucki B, et al. may be the local differences in physical crystal identification in human cartilage. An ultra- structural elemental analysis. Arthritis Rheum 1993; 36: properties between cartilage matrix and S161. crystals, providing potential for disruption of 23 Scotchford C A, Greenwald S, Ali S Y. Calcium phosphate articular surface A crystal distribution in the superficial zone of human integrity. correlation femoral head articular cartilage. J Anat 1992; 181: between CPPD crystal deposition and severity 293-300. 344 Scotchford, Ali

24 Scotchford C A, Ali S Y. The isolation and characterization 37 Stockwell R A. Structure and function of the chondrocyte of magnesium whitlockite crystals from human articular under mechanical stress. In: Helminen H J, Kiviranta I, cartilage. Osteoarthritis and Cartilage. In press. Tammi M, Saamanen A-M, Paukkonen K, Jurvelin J, eds. 25 Elliot J C. Structure and chemistry of the apatites J7oint loading. Bristol: Wright Brothers, 1987; 126-48. and other calcium . Amsterdam: Elsevier, 38 Paul J P. Approaches to design. Force actions transmitted Ann Rheum Dis: first published as 10.1136/ard.54.5.339 on 1 May 1995. Downloaded from 1994. by joints in the human body. Proc R Soc Lond [Biol] 1976; 26 Joy D, Maher E M. Sensitivity limits for thin specimen 192: 163-72. x-ray analysis. Scanning Electron Microscopy 1977; I: 39 Sakae T, Yamamoto H, Mishima H, Matsumoto T, 325-34. Kozawa Y. Morphology and chemical composition of 27 Russ J C. The direct element ratio model for quantitative dental calculi mainly composed of whitlockite. Scanning analysis of thin sections. In: Hall T A, Echlin P, Microscopy 1989; 3: 855-60. Kaufman R, eds. Microprobe analysis as applied to cells and 40 Schupbach P, Lutz F, Guggenheim B. Human root caries: tissues. London: Academic Press, 1974; 269-76. histopathology of arrested lesions. Caries Res 1992; 26: 28 Klein C P A T, Patka P, den Hollander W. A histological 153-64. comparison between hydroxylapatite and b-whitlockite 41 Burstein L S, Boskey A L, Tannenbaum P J, Posner A S, macroporous ceramics implanted in dog femora. The Mandel I R. The crystal chemistry of submandibular and Third World Biomaterials Congress 1988; 67. parotid salivary gland stones. J Oral Pathol Med 1979; 8: 29 Scotchford C A, Ali S Y. Calcium phosphate microcrystal 284-91. deposition in articular cartilage: characterization by 42 Verplaetse H, Verbeeck R M H, Minnaert H, Oosterlinck W. XRMA. Micron MicroscopActa 1992; 23: 383-4. Solubility of inorganic kidney stone components in the 30 Ghadially F N, Lalonde J-M A. Intramatrical lipidic debris presence of acid-base sensitive complexing agents. Eur and calcified bodies in human semilunar cartilages. JAnat Urol 1985; 11: 44-51. 1981;132:481-90. 43 Gawoski J M, Balogh K, Landis W J. Aortic valvular tophus: 31 Ghadially F N. Fine structure of synovial joints. London: identification by X-ray diffraction and calcium Butterworth and Co. Ltd, 1983. phosphates. JT Clin Pathol 1985; 38: 873-6. 32 Bishop M A, Warshawsky H. Electron microscopic studies 44 Bestetti-Bosisio M, Cotelli F, Schaffino E, Sorgato G, on the potential loss of crystallites from routinely Schmid C. Lung calcification in long term dialysed processed sections ofyoung enamel in the rat incisor. Anat patients: a light and electron microscopic study. Rec 1982; 202: 177-86. Histopathology 1984; 8: 69-79. 33 Arsenault A L, Hunziker E B. Electron microscopic analysis 45 Rowles S L. The precipitation of whitlockite from aqueous of mineral deposits in the calcifying epiphyseal growth solutions. Bull Soc Chim 1968; 1797-1802. plate. Calcif Tissue Int 1988; 42: 119-26. 46 Swanson S A V. Friction, wear and lubrication. In: 34 Ghadially F N, Meachim G, Collins D H. Extra-cellular Freeman M A R, ed. Adult articular cartilage. Tunbridge lipid in the matrix of human articular cartilage. Ann Wells: Pitman Medical, 1979; 415-60. Rheum Dis 1965; 24: 136-46. 47 Little T, Freeman M A R, Swanson S A V. Experiments on 35 Sah R L Y, Kim Y J, Doong J Y, Grodzinsky A J, friction in the human hip joint. In: Wright V, ed. Plaas A H, Sandy J D. Biosynthetic response of cartilage Lubrication and wear in joints. London: Sector, 1969; explants to dynamic compression. J Orthop Res 1989; 7: 110-4. 619-36. 48 Maroudas A. Physicochemical properties of articular 36 Van Campen G P J, Van de Stadt R J. Cartilage and cartilage. In: Freeman M A R, ed. Adult articular cartilage. chondrocyte responses to mechanical loading in vitro. In: Tunbridge Wells: Pitman Medical, 1979; 215-90. Helminen H J, Kiviranta I, Tammi M, Saamanen A-M, 49 Clift S E, Harris B, Dieppe P A, Hayes A. Frictional Paukkonen K, Jurvelin J, eds. Joint loading. Bristol: Wright response of articular cartilage containing crystals. Brothers, 1987; 112-25. Biomaterials 1989; 10: 329-34. http://ard.bmj.com/ on September 29, 2021 by guest. Protected copyright.