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Histologic observations and biochemical composition of rachitic cartilage with special reference to mucopolysaccharides.

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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.
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2. Belanger, L. F.: Autoradiographic visualization of the entry and transit of
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1960, p. 245.
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David S. Howell, M.D., Associate Professor of LMedicine, Department of Medicine, University of Miami, Miami, Florida.
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