Histologic observations and biochemical composition of rachitic cartilage with special reference to mucopolysaccharides.код для вставкиСкачать
Arthritis and Rheumatism JUNE, 1965 VOL. VIII, NO. 3 Histologic Observations and Biochemical Composition of Rachitic Cartilage with Special Reference to Mucopolysaccharides By DAVIDS. HOWELL Costal tissues were analyzed biochemically in respect to a profile of sulfated mucopolysaccharides from normal and rachitic calves which provided large samples of anatomically identifiable regions for biochemical analysis. Histologic distribution of these compounds was obtained through the use of x-ray elemental analysis for sulfur. Distinctive differences between polysaccharide content of normal and rachitic tissues were found and calcification in vitro of the rachitic cartilage was demonstrated. Tissus costal esseva analysate biochimicamente in relation a un profilo de sulfatate mucopolysaccharidas ab vitellos normal e rhachitic que provideva extense specimens de anatomicamente identificabile regiones pro le analyse biochimic. Le distribution histologic de iste compositos esseva determinate per medio del us0 del roentgeno-analyse elemental pro sulfure. Distinctive differentias inter le contento polysaccharidic de tissu normal e ill0 de tissu rhachitic esseva constatate. Calcification in vitro de cartilagine rhachitic esseva demonstrate. D URING THE PAST two decades, dentine, bone and cartilage have been studied intensively by a variety of histologic and biochemical technics with emphasis upon the role of sulfated mucopolysaccharides (SMPS) in biologic calcification. Rubin and Howard studied histological sections from v<irious sites undergoing calcification following acid demineralization and noted consistently, a histologic reaction suggestive of polyanions such as mucopolysaccharides.' Similarly, a dense silver grain reduction in S35 autoradiographs following Na2S"04 administration to animals has, in general, correlated well both temporally and histographically with the appearance of mineral deposits in various t i ~ s u c s . ~The , ~ importance of these autoradiographic studies in relation to identification of SMPS is reinforced by relevant biochemical studies. Namely, slices of calf epiphyseal cartilage incubated in This work was performed at the Institute for Cell Research, Medical Nobel Institute, Karolinska Institutet, Stockholm, Sweden. Supported b y Grant A-1155 of the National Institute of Arthritis and Metabolic Diseases tv the University of Miami; Grant C-4716 of the National Cancer Institute (U.S.A.), and Giants from the Wallenberg Foundation to the Institute for Cell Research, Stockholm, Stc eden. 337 ARTHRITISAND RHEUMATISM, VOL.8, No. 3, (JUNE), 1965 338 DAVID S. HOWELL vitro with Na2S:%504 revealed that 90 per cent or more of S35 appeared in SMPS fractions as ester sulfate4 and similar results occurred in rat epiphyseal tissues when the isotope was given in viva.'? Also, in a recent study employing x-ray microscopic elemental analysis, measurements at periosteal mineralizing sites of calf mandible revealed a high S content similar to that of epiphyseal cartilage over regions 10 to 2 0 , ~in diameter.5 Evidence from infrared microspectrophotometry suggested that the narrow periosteal zones of bone growth were abundant in organic ~ u l f a t e In . ~ studies of calf bones, chondroitin 4-sulfate, chondroitin 6-sulfate, keratosulfate as well as unidentified fractions have been isolated.6 Although the presence of an abundance of SMPS in mineralizin,g sites has received considerable support, it has not yet been possible to establish what features of local metabolism of SMPS, if any, are uniquely identified with mineral deposition per se. Elucidation of such distinctions is hampered by several problems including difficulty of separating cellular functions connected with fibillogenesis, etc., from those regulating mineral transfer and lack of suitable model systems to study bone salt deposition within tissue in vitro, wherein sufficient tissue for SMPS studies can be obtained. The purpose of the current study was two-fold. First, it was of interest to determine whether cliff erences of polysaccharide composition might be detected within a mineralizing (normal) as opposed to a growing but nonmineralizing epiphyseal matrix ( rachitic ) . Thereby a defect in mineralizing sites additional to the systemic deficiency of circulating mineral ions might be detected. Secondly, costal plates of rachitic calves might offer a model system for in vitro mineral deposition similar to that in rats, and provide much larger amounts of tissue for biochemical study of compounds difficult to isolate in correlation with histologic findings. Therefore, a preliminary appraisal of this system was made. MATERIALS AND METHODS 1. Animal Preparations Animals were prepared and the calcification experiments conducted at the Cattle Breeding Institute at Wiad, Sweden. Four calves were weaned on the 4th postpartum day; they were isolated in a dark room and placed on a daily diet of skim milk ( 1 to 5 Kg. ) , linseed oil and coconut oil, beet pulp, as well as vitamins, mineral concentrates, tornla yeast and tocophorol. Control calves received the same diet with the addition of whole milk, hay and supplementary mixture:; containing vitamin D, calcium and phosphorus. Growth and development of the experimental animals seemed grossly satisfactory upon veterinary inspection at weekly intervals for the first 3 months, but in the succeeding 1 to 3-month period, they developed lameness, ataxia, weakness and excessive epiphyseal prominences of their extremities, as anticipated from other documentation.7,b: Blood samples from 3 of the rachitic calves were obtained at the time of sacrifice and serum calcium x phosphate concentration products were less than 30 mg.2 per cent in each instance. Three calves were sacrificed 1 to 3 weeks after the first detectable rachitic signs at ages 4, 5 and 6 months respectively. Their tissues were employed for the current study. The 4th animal died from an unidentified infection at age 2-% months. All control calv-; thrived during the period of this study and were sacrificed at 5 to 6-months of age for histologic and biocheniicd control observations. HISTOLOGY AND BIOCHEMISTRY OF RACHITIC CARTILAGE 339 Fig. 1.-Costochondral junctions of control (left) a n d rachitic calf No. 2 (right). Areas of t h e cartilage a r e arbitrarily divided, based on approximated histologic composition: Area A-zones of resting a n d aggregating cells; Area B- zones of proliferating a n d hypertrophic cells; Area B'-admixture of proliferating a n d hyperosteoid a n d small amounts of trophic cells with osteoid; Area C-metaphyseal tissue of Area B; a n d Area D--maturing cancellous bone marrow. 2. Determination of Calcifinbility in Vitro Immediately after sacrifice, the costochondral tissues of the first 9 ribs, bilaterally, were dissected free and mo,t of them immersed in isopentane surrounded by liquid nitrogen for frozen storage and further biochemical studies. However, the massively enlarged costal junctions from 5 ribs of each calf were sectioned, sagitally, into 1 em. wide slices 0.5 to 1 mm. thick. divided cross-sectionally at Area B' (fig. 2 ) . These slices were incubated in a synthetic lymph (Yendt's so1ution)e for 24 hours at 37" C, beginning l/z hour after sacrifice (fig. 1). For each experiment, Yendt's solution was freshly prepared with penicillin 50 units/ml. added; final adjiistments of pH with CO, to 7.3 to 7.4 (measured on a Beckman pH meter) were made prior to all incubations. The synthetic lymph contained concentration products of calcium and phosphate varying from 0 to 50 mg.2" per cent by stepwise raising of phosphate concentration (table 1). For every level of concentration product studied. 5 to 10 slices of frozen, heated or fresh tissue were incubated in 3 separate experiments (table 1). pH was again measured on solutions at the end of the incubations and found to be within the range of 7.2 to 7.5. An overall appraisal of mineral uptake by half the slices was made by employing the silver nitrate method of Hiatt, Marks m t l Shorr,l" whereas the remaining slices were used for histologic studies described below. 3. Biochemical Preparati,ons Most of the rachitic costochondral junctions were trimmed of fibrous tissue and under a dissecting microscope they were divided into 3 fractions to include approximately ( 1) cartilage of Areas A and B (fig. 2 ) ; ( 2 ) osteoid and cartilage of Area B' and ( 3 ) o;teoid of Area C. Each fraction was chopped into fine pieces at 5" C in acetone and ground with a Virtis-45 homogenizer for 1 hour at a dial reading of 70. The acetone extracted _ _ _ _ ~ _ ~- "Values discussed here are a total figure for the component phosphates in solution. DAVID S. HOWELL 340 Fig. 2.-Histologic section of a typical untreated rachitic plate which is disorganized by cartilaginous tongues extending deeply into osteoid granulation. powders were digested with panprotease; mucopolysaccharides were isolated as calcium salts and precipitated by ethanol. Measurements of hexosamine, uronic acid, sulfate, optical rotation and infrared spectra as well as nitrogen determinations were performed according to methods previously referred to or described.11 Thereby, content of chondroitin .l-sulfate, chondroitin 6-sulfate, chondroitin sulfate B, keratosulfate, and other polysaccharides were quantitatively estimated. Results were computed as to content per unit dry mass of original whole tissue sample (table 2 ) . 4 . Histologic Methods Astrablau,'2 toluidine blue, Von Kosaa as well as hematoxylin and eosin stains were applied to freeze-dried, frozen and formalin-fixed paraffin-embedded tissues. Microradiograms of 6 to 10A wavelengths,13 as well as x-ray elemental analysis14?15for sulfur were niade on unaltered cartilage and osteoid to assess the distribution of S compounds, principally SMPS. Sudan black staining by Irving's method,lG employing 24-hour pyridine extraction at 60" C was applied to unaltered and incubated tissues. Tissues from fresh control and calcifying slices were fixed in buffered osmium tetroxide, embedded in Epon, sectioned on an LKB ultratome and photographed at 10,000 initial magnification in an electron microscope ( RCA-EMU-I1 ) . Table I.-Results in Respect to Amount and Distribution of Mineral Salt Uptake b y Rachitic Costal Slices Incubated in a Synthetic Lymph ~Miners1 Deposition Tissue Preparation Histologic Area Ca x PO4 rng.270 0 20 Heat inactivated Frozen 7 days Fresh Fresh Fresh Fresh 0 0 0 0 0 0 0 0 0 0 -t 0 Entire slice Entire slice A 30 40 +0 0 0 0 0 0 50 +" ++ + + B B' ++t C 0 0 + *In comparison to control, I + slight, 2+ moderate, 3+ advanced blackening of histologic region in silver nitrate preparations. + ++ 341 HISTOLOGY AND BIOCHEMISTRY OF RACHITIC CARTILAGE Table 2.-Principal S M P S in Normal and Rachitic Calf Bony Tissues Computed on Basis of Dry Weight of Original Tissuv. Tissue Hyaline cartilage Osteoid (demineralized bone shaft)' Hyaline cartilage Cartilage and ostcoid Ostcoid 'ma in Total Yield of SMPS Chondroitin Chondroitin 4-Sulfate 6-Sulfate Distribution according to infrared spectra % Dry Weight % of Total SMPS ( A ) Normal 17.0 80-90 3.2 ( B ) Rachitic 8.3 10.4 0.8 Keratosulfate 10-20 6 80-90 10-20 5 40 90 77 60 7 Trace <5 5 23 this line are from a previous paper6 with some additional determinations. 5. Histologic Description of Experimental Preparations The appearance of growth Tones of normal and rachitic costochondral junctions (figs. 1 and 2 ) showed striking differences. In the normal tissue these zones were ordered in respect to cellular architecture with approximately parallel growth zones and an abrupt transition between the bone marrow and the junction of primary spongiosa with epiphyseal cartilage. The rachitic tissues revealed pathologic landmarks discussed recently by Iljertquist17 and were divided for present purposes into 5 regions. Area A was composed primarily of resting cells and Area B of proliferating and hypertrophic cells. These 2 areas showed a slight diminntion of width in compariron to that of normal plates. Area B' was composed of osteoid coated projections of hypertrophic and degenerating cartilage fragments and cartilage tongues. The width of this region was greatly increased in comparison to that of the normal rib cell colunms and fanned into the metaphysis in an irregular manner. As might be expected, cartilage projections revealed intense metachromatic staining with toluidine blue and basophilia with Astrablau; which generally became progressively more intense in the direction of the diaphysis. Some islands of hypertrophic cell tissue in the metaphysis were intensely basophilic whereas other cellular masses remained essentially unstained by these dyes and gave the appearance of pseudoosteod.17~18Osteoid in unopened cartilage cell lacunae and cuffs of osteoid surrounding capillaries were found in the distal layer of hypertrophic cell cartilage and adjacent granulation. Although several small foci of calcification were detected surrounding each of several cartilage cells in calf No. 4, such endogenous deposits in costal plates of calves Nos. 1, 2 and 3., used for mineralization studies were not encountered. Area C comprised the bulk of the granulation and was composed principally of bony trabeculae coated with wide bands of unmineralized osteoid matrix (fig. 2 ) . These areas were separated by diffuse granulation and thinly scattered bone marrow elements; Area D was defined by the commencement of large cellular marrow spaces and trabeculae composed of more completely mineralized bone. Electron micrographs of samples of terminal hypertrophic cartilage and islands of osteoid and degenerating cartilage (secondary spongiosa) failed to reveal evidence of mineral nuclei in the areas which in adjacent slices showed mineral deposition in vitro.* RESULTS lncuhution of Ruchitic Tissue in Mineralizing Solutions There was a consistent pattern of mineral accumulation in areas of hyper'Not exhibited because of negative finding. 342 DAVID S. HOWELL Fig. 3.-Hypertrophic cartilage and primary spongiosa of rachitic slice from calf No. 3, incubated in a solution of Ca x PO4 product 40 mg2 per cent. Accumulations of mineral are observed throughout the primary spongiosa within the matrix between the hypertrophic cartilaginous cell columns as well as scattered transverse septa of the cells. Microradiogram 6-10A of undecalcified section 5~ thick; x146. trophic cartilage (table 2; figs. 3, 8 and 10). At concentration products of 0 and 20, no deposits of mineral salt in the cartilage in regions B and B' were noted. At products of 30 and 40 m g 2 per cent slight to moderate spread of mineral deposits were encountered in Area B' identified by x-ray micrography and silver nitrate stains (figs. 3 and 9 ) . No mineral deposits were encountered in areas consisting of resting cell and proliferating cell cartilage. Of considerable interest was the apparent failure of the wide osteoid seams in area C of all tissues to accumulate mineral at concentration products of 40 mg.? per cent (fig. 4).Aggregates of mineral salts were found to accumulate at products of 50 m g 2 per cent, along trabecular margins of area B' and C (figs. 7 and 8) whereas at this level heated and frozen slices accumulated only small amounts of mineral. The alteration in staining properties of these calcifying sites with Sudan-black was of particular interest. In scattered parts of Areas B and B' of control tissues, the dye stained the hypertrophic cells but not the matrix as noted by Irving in rachitic rat cartilage (fig. 5).19The HISTOLOGY AND BIOCHEMISTRY OF RACHITIC CARTILAGE 343 Fig. 4.-Secondary spongiosa of a slice from calf No. 3, incubated at Ca x PO4 product of 40 mg2 per cent. Low x-ray absorption in the osteoid border indicates slight or no mineral deposition there. Microradiogram 6-106 of undecalcified section Fip thick; x600. Fig. 5.-Hypertrophic cartilage and metaphyseal osteoid or rachitic slice from Calf No. 2, untreated slice. No evidence of sudanophilic material is obvious in the matrix of either the cartilage or osteoid. Sudan black stain; x100. 344 DAVID S. HOWELL Fig. 6.-Adjacent slice of the same tissue described in Fig. 3, incubated in a solution of Ca x PO, product 40 mgz per cent. Sudan black stain; x146. matrix in most regions of Area B’ was stained by Sudan black along the rims of mineralized cartilage following 48 hours incubation in 40 mg.2 per cent of Ca x PO,. An intense black stain was recorded, principally in the primary spongiosa and cartilaginous sites of calcification (fig. 6 ) . In the same slices the Sudan black stained bone mineral weakly and failed to stain osteoid seams of Area C. Von Kossa stains and x-ray measurements showed close correlation in determining the sites of calcification. Stains by Astrablau ( a t pH 0.2) were of the highest grade intensity in some fragments within the metaphysis and indicated roughly the distribution of sulfated compounds.12 Sections of untreated rachitic costal plate, Area B’ analyzed by monochromatic x-ray spectrophotometry indicated distribution of S at a range of 1.2 to 2.1 per cent, average 1.5 per cent ( n = 10) in areas of 2 0 diameter ~ measured distal over hypertrophic cell regions (fig. 6 ) compared to 0.3 per cent ( n = 5), range 0.1 to 0.4 per cent in adjacent osteoid. Biochemical Observations For rachitic tissues, the total yield of SMPS was 8.3 per cent for Areas A and B (cartilage - table 2 ) , 10.4 per cent for Area B’ (cartilage and osteoid) HISTOLOGY AND BIOCHEMISTRY OF RACHITIC CARTILAGE 345 Fig. 7.-Osteoid of a slice incubated in a solution at Ca x PO4 product of 50 mg2 per cent. Pattern of high x-ray absorption along the margins of osteoid adjacent to hbpertrophic cartilage indicates clumps of deposited mineral salt. Microradiogram 6-1OB of section 5, thick; x600. and 0.84 per cent for Area C (principally osteoid). The ratio of chondroitin 4-sulfate to chondroitin 6-sulfate was 0.67 for Area A; 3.35 for Area C; whereas Area B’ contained almost entirely chondroitin 4-sulfate. Area A contained 6.6 per cent keratosulfate and this compound was a similarly small component in the other fractions. In samples of normal costal cartilage of similarly-aged calves, total yield of SMPS was 17 per cent with 80-90 per cent chondroitin 4-sulfate; 10-20 per cent chondroitin 6-sulfate and 6 per cent keratosulfate. As previously reported for normal bone,6 there was no evidence in the rachitic tissues of heparitin or chondroitin sulfate B. In comparison to data on normal demineralized calf bone (table 2), there was only a small yield of total polysaccharide (including keratosulfate) in the rachitic osteoid. DISCUSSION Gross and histologic appearance of the rachitic epiphyseal plates and adjacent metaphyseal granulation resembled that previously reported in calves7,* and other s p e c i e ~ . ~The ~ J ~delay , ~ ~in the appearance of rickets in the current animals until 3 to 5 months after onset of the rachitogenic regimen is similar to that previously reported in calves7**and differs from the rapid induction of this metabolic defect in dogs and rats, as recently reviewed.17 Whether this delay resiilts from vitamin D storage acquired in the antenatal period is unknown. 346 DAVID S. HOWELL Fig. %-Area of hypertrophic cartilage from another slice incubated in a solution containing Ca x PO, product of 50 mgz per cent. Extensive mineral accumulation between cartilage cells and along longitudinal septa over wide regions of most sections was observed. Microradiogram 6-10A of section 5p thick; x146. In Vitro Calcification of Rachitic Tissue Results of experiments upon calcification of calf rachitic cartilage were divergent from those reported under comparable conditions of incubation in rats of tibia1 Unlike rats, mineral deposition in the tongues of hypertrophic cartilage of the calf occurred in most slices in an irregular manner; there was no line of demarcation through the hypertrophic zone as found in rat epiphyses. Also, even at a concentration product of 40 mge2 per cent, some slices of calf No. 3 failed to accumulate mineral. Rachitic cartilage from rats may also fail to accumulate mineral if the animals are kept on the regimen for 6 weeks or more,21 so that possibly metabolic alterations attending the slow development of the rachitic state in the calves may have led to a partial or scattered refractoriness of matrix calcifiability. The question arises as to whether in the current experiments, deposits of bone salt in slices of rachitic tissue incubated in solutions containing Ca x PO, products of 30 mg.2 per cent and 40 mg.2 per cent may have resulted HISTOLOGY AND BIOCHEMISTRY OF RACHITIC CARTILAGE 347 Fig. 9.-Paired, adjacent slices of rachitic tissue areas A, B and B‘ stained with silver nitrate following incubation at 37°C for 24 hours in synthetic lymph at Ca x PO4 concentration products of 20 mgz per cent (control-c) and 40 mg2 per cent (experimental-E) . The arrows point to the approximate transition from Area B’ to Area C. from aggregations on submicroscopic preformed nuclei rather than initial nacleation and epitactic crystal growth of a physiologic nature. Three separate lines of observation suggested that the cartilage of area B’ retained a native capacity to deposit mineral salts, when incubated in the above solution\. First, many new mineral deposits appeared by microradiograms to follow trabecular frameworks in an orderly manner (fig. 3 ) , rather than to collect in obviously nonspecific aggregates described by Lamm and Neuman for similar incubates of rachitic rat cartilage.23 Such aggregates as they described were also found in the present experiments, but principally in the presence of concentration products Ca x PO, of 50 mg.2 per cent, in accord with obsen~ationsof a finding which suggests that for most areas within the slices, nucleating sites prior to incubation of these samples were absent. Thirdly, as mentioned above, electron micrographs failed to show evidence of mineral clusters in sections of Area B’ closely adjacent to points which showed dense deposits of mineral following 48 hours of incubation at :t Ca x PO, product of 50 mg.2 per cent. The finding that only cartilaginous portions of the slices in calves developed new mineral deposition was similar to that observed by Robison and othersz5for rat tibia1 epiphyses. One striking histologic observation of this series of experiments was the appearance of Sudan black staining material surrounding and within areas cf cartilage which, in adjacent slices, showed new mineral deposits following 348 DAVID S. HOWELL Fig. 10.-Calf No. 2 after 4 months on the rachitic regimen. There is marked enlargement of the ankle and tarsal joints as well as widened stance. 48-hours incubation. Under a variety of experimental conditions in vivo Irving has reported the presence of a localized staining with Sudan black in mineralizing sites.19 From histologic evidence he postulated the presence of a lipid, probably linked to other compounds such as proteins.26 In studies involving this staining technique, Howell and Carlson noted a high content of S by x-ray microscopic elemental analysis in correlation with Sudan black stained areas in mineralizing sites in vivo.5 In other experimental preparations they were able to deplete S from matrix by administration of papain in vivo without destroying the strong sudanophilia (unpublished observations) . The current histologic results are suggestive of the appearance or unmasking of a factor stained by Irving’s method16 during the present in vitro incubation, but data were insufficient to relate this finding to the level of mineral ions in solution. Finally, the high content of S by x-ray analysis in the hypertrophic cartilage (abundant in SMPS analyzed in this fraction biochemically) compared to relatively little in the adjacent osteoid, indicated that the polysaccharide profile of the osteoid-cartilage fraction reflected almost completely composition of hypertrophic cell cartilage. Biochemical Analysis Apparent differences (table 2 ) among the polysaccharides studied here were found when the SMPS data of all rachitic fractions (table 2 ) were compared to that of similar regions of normal costal plates. The current data indicate a quantitative deficiency of total SMPS in calf osteoid and cartilage from ribs. Dziewiatkowski noted that incorporation of S35 from Na2S3504 HISTOLOGY AND BIOCHEMISTRY OF RACHITIC CARTILAGE 349 into chondroitin sulfate of tibial epiphyses of vitamin D deficient rats increased following administration of c a l c i f e r ~ l Also, . ~ ~ Cipera, et al. found tliat hexosamine content of chick epiphyseal plates was augmented markedly during healing by vitamin D3 treatment.'s A rise of total S content at microwopic sites in osteoid and cartilage of the rachitic rat tibial epiphyses during vitamin D treatment, suggesting accelerated synthesis or decreased degradation of S compounds-predominantly SMPS29 was noted by the author. Although there are previous data reflecting a likely deficiency of polysaccharide metabolism in nutritional rickets in certain species, evidence for specific polysaccharide deficiencies in cartilage and osteoid have not hitherto been obtained from the literature reviewed. In the present study, particular attention was also paid to whether distinctive polysaccharides were linked with a normally mineralized, as opposed to a poorly mineralized osteoid, with special reference to keratosulfate. Thus, Kaplan and Meyer found that keratosulfate content of human costal cartilage (which calcifies with aging) increased until age 30 years beyond which time the ratio continued to rise because of a fall in chondroitin sulfate con~ e n t r a t i o n Altered .~~ amino sugar ratios consistent with an expansion of the keratosulfate fractions have been noted in respect to aging articular cartilage and nucleus pulposus, both of which tissues often develop calcific deposits in the course of time.:i1-32Also, Bertolin recorded that the galactosamineglucosamine ratio of uncalcified cartilage was double that of adjacent mineralized metaphyseal tissue in 33-day old rabbits.33 However, no striking qualitative diflerences in the polysaccharides analyzed herein between normal and rachitic tissue were found, and no evidence of a unique mucopolysaccharide." Although previously a phosphorylated aminosugar was claimed in cartilage by Distephano, et al.,x4neither this nor any other compounds have yet been isolated elsewhere which are unique for mineralizing sites. Whether the high content of chondroitin 4-sulfate in Area B1 (table 2 ) has functional significance requires further exploration. What role SMPS play in calcification of various cartilages remains uncertain and may depend on histologic site and other biologic c i r c u m ~ t a n c e s . ~ 5 ~ ~ ~ A search for compounds which might aid in the nucleation of mineral salts, possibly linked to collagen, has been motivated by evidence of Sobel and others involving extensive experiments on in vitro mineral deposition. In these studies binding of polyanionic negative groups with a variety of cations blocked mineral deposition both in vitro and in vivo, but whether SMPS or other classes of compounds were critically involved remains to be elu~idated.~~~~~ Another hypothesis relates to effects of SMPS-protein compounds on mineral phase transitions. Thus Weinstein, et al. recently showed that calcium phosphate precipitation in vitro from solutions containing high products of these ions, could be held in the fluid phase against low sedimentation forces (<500 g) in proportion to content of a light proteinpolysaccharide fraction "Evidence for predominantly chondroitin 4- and 6-sulfates in epiphyseal cartilage of rachitic dogs was recently reported by S.-0. Hjertquist: Acta Soc. Med. Upsal. 69: 83. 1964. 350 DAVID S. HOWELL at low concentration ranges.37 Cliondroitin sulfate alone failed to manifest this dispersive effect on calcium phosphate in solutions at similar concentrations. It was postulated by the authors that such stabilizing influences on prevention of mineral aggregation by proteinpolysaccharides might operate in vivo, as with other linear polymers, by impedance of ion transfer to embryo crystal surfaces through entanglement or other mechanical trapping effects related to a large water domain.37 It is plausible that if degradation or removal of these polymers occurred under the proper conditions, mineral phase separation would ensue. Other hypotheses also rest upon degradative alteration of proteinpolysaccliarides but stress a loss of shielding properties at mineralizing sites with initiation of mineral deposition by release of stored calcium,42 increase of pore size in the matrix gel, with resultant acceleration of linear diffusion rates oi relevant ions, or possibly effects on water transfer out of mineralizing sites. As for one possible triggering mechanism there is evidence of protein-polysaccharide degradation at some sites of mineral deposition. Campo and Dziewiatkowski in combined biochemical and autoradiographic studies found evidence of SMPS retention in the calcifying metaphyses of estradiol-treated rats with concurrent loss of a protein moiety apparently removed by enzymatic splitting or d e g r a d a t i ~ n . That : ~ ~ the predominant S compounds of mineralizing regions are in a different physical or biochemical state from those in adjacent non-mineralizing cartilage was suggested by the altered extractability of S compounds following pyridine treatment previously reported in normal calf tissues5 Recently, hyaluronidase activity has been demonstrated in synovial and other body fluids by Bollet, et aL40 Lack postulated that where capillaries invade cartilage, such as the epiphyseal plate, enzymatic degradation of polysaccharide from protein complexes results from activated plasmin~ g e n . There ~l is evidence that some cartilage cells contain an abundance of lysosomes which store proteolytic enzymatic factors. However, specific roles for these enzymes in mineral deposition remain u n d o c ~ m e n t e d . ~ ~ Finally, studies upon reconstitution of solublized collagen in vitro indicate that slight diflerences in polysaccharide composition of the bathing medium profoundly influences electron microscopic appearance and mechanical properties of the fibrous phase.43 Consideration, therefore, must be given to whether mineral deposition in vivo might depend upon nucleation on properly polymerized fibrous constituents of the cartilage, as influenced by the polysaccharide environment. Clearly needed are experiments to discriminate between the various model systems constructed to explain a functional role for polysaccharide5 in cartilaginous mineral deposition. More specific differences of polysaccharide composition particularly in respect to their protein and lipid components might be elucidated through use of the current adaptation of in vitro incubation of cartilage slices. SUMMARY Rickets was induced consistently in 4 calves treated with a special diet and was characterized by ataxia, lameness, knobby protuberances at the knees, ankles and costochondral junctions. 351 HISTOLOGY AND BIOCHEMISTRY OF RACHITIC CARTILAGE Appendix I Nature of Daily Diet Administration Composition of Mixtures (Gm./Kg.) Water Solution Control 0.5 0.25 3.5 11.5 1.75 + + + + + Aqueous Mixtzire-A ( S O ml./day) Cobalt sulfate Copper sulfate Manganese sulfate Ferrous sulfate Sodium chloride Solid Mixture-B (SO Gm./day) Dicalciuin phosphate 1)isodium phosphatc Rachitic + + 4+ + Force Fed* + + + + + % Dry Weight 7s 25 + + + + Solid Afixtwe-Cf Oat Barley Wheat bran Soybean oil nieal Coconut cakes Linseed cakes 20 20 25 12 10 13 Vitumins Purified vitamin A (10,000 units/day) l'ocophorol (small amounts) Torula yeast, 50 Gm./day Vitamin A,D,E Mixture Other trace minerals llay Skim milk After first 6 days, 5 Kg./day$ + + 4+ + + + + + + f A 28.5 28.5 28.5 13.8 0.7 + + + + + + + + + + + + + + + Straw Whole milk-slcim milk ratio (Figures = Kg./day) First 3 postnatal days-3.0'f Next 3 days-3:1, 2:2, 1:2 + + + + + -k + + T Solid Mixture-D Oat Barley Soybean meal Beet pulp Sodium chloride Ad Libitum + + 3 + + + + + + 'If not taken voluntarily. +Intake of vegetable fat: circa 100 Gm./day administered between 1 and 12 weeks of age. 1Colostrum. $Controls received 5 Kg./day whole milk after first 6 postnatal days. 352 DAVID S. HOWELL When slices of rachitic costal plates were incubated in solutions of synthetic lymph, there was evidence of mineral uptake irregularly distributed in terminal hypertrophic cartilage, but only in the presence of calcium and phosphate at levels at or above those found in normal lymph. Total sulfated mucopolysaccharide content was higher in demineralized osteoid and epiphyseal plate cartilage of normal calves than in comparable rachitic calf osteoid and hypertrophic cartilage. Qualitative differences in polysaccharide composition between mineralizing and non-mineralizing regions were not detected. Measurements of S content by microscopic x-ray analysis of the untreated rachitic cartilage-osteoid fraction indicated most of the SMPS were in the cartilaginous regions rather than osteoid. ACKNOWLEDGMENTS The author is indebted to Dr. Karl hleyer of Columbia University, New York for the polysaccharide analyses; to Engineer Leon Carlson, Professors Hugo Theorell, T. Caspersson, and Dr. Gunnar Bloom for the use of laboratory facilities at the Karolinska Institute in Stockholm, a5 well as to Professors A. Hansson and A. Niemann-Sorensen, together with Agronomists L.-E. Liljedahl and T. Henningsson for facilities at the Animal Breeding Institute at Wiad. Ilr. Bonnie Anderson, of Dr. K. Meyer’s laboratory, performed some of the polysaccharide analyses of normal calf costal cartilage and demineralized bone matrix. REFERENCES 1. Rubin, P. S., and Howard, J. E.: Histochemical studies on the role of acid mucopolysaccharides in calcifiability and calcification. In Transactions of the 2nd Conference on Metabolic Interrelations. Reifenstein, E. C. ( e d . ) . New York, Josiah Macy, Jr., Found., 1950, p. 155. 2. Belanger, L. F.: Autoradiographic visualization of the entry and transit of S35 in cartilage, bone and dentine of young rats, and the effect of hyaluronidase, in vitro. Canad. J. Biochem. 32:161, 1954. 3. Dziewiatkowski, D. 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