Epiphyseal Plate Cartilage Research Articles

A critical re-evaluation, of the anisotropic index used in polarization microscopy: A study on epiphyseal cartilage

Topo-optical reactions specific for collagen and glycosaminoglycans were performed on epiphyseal cartilage plates which differed in age, in chemical composition, and in structure. Retardation values were measured with the polarization microscope and anisotropic index values were calculated after each reaction. It was found that the anisotropic index proposed earlier for quantitative evaluation of topo-optical reactions did not refer either to the submicroscopic orientation pattern or to the quantity of cartilage matrix components. It is suggested that retardation values of induced birefringence may be only used to characterize the spatial orientation of these components in the cartilage matrix.

Resistant proteoglycans in epiphyseal plate cartilage. Variations in their distribution in relationship to age and species.

The localizations of resistant proteoglycans (RPGs) in the epiphyseal plates of rats, dogs, and humans are similar. In the epiphysial plates from young rats, dogs, and humans, the RPGs form a stratum at the junction of the zones of resting and proliferating cells. Non-calcified cartilage RPGs are associated with cells which have the potential for proliferation or column organization. As the individuals age, RPGs are found in intercolumnar regions or at times are even absent. There is also a type of RPGs in calcified cartilage, including the calcified cartilage subjacent to the articular surface, in all species. In human epiphyseal plates, looser fibrillar RPGs change abruptly to a more condensed type in the zone of provisional calcification. Calcified cartilage RPGs stain more intensely with toluidine blue and may represent a different type of RPGs.

Phosphomonoesterases in growth cartilages of the rat

Epiphyseal plate cartilage, epiphyseal cartilage, synchondroseal cartilage and mandibular condylar cartilage were studied morphologically and histochemically in 14 days old rats. Ordinary decalcified paraffin sections were stained with hematoxylin & eosin, van Giesons connective tissue stain, or toluidine blue, and used for morphological studies of the different cartilaginous structures. Undecalcified cryostat sections were used for demonstration of acid and alkaline phosphatase. The enzyme activity was tested for at regular intervals during incubation from 15 sec to 120 min. The morphologic study revealed that a marked similarity of construction exists between epiphyseal plate cartilage and synchrondroseal cartilage. The construction of epiphyseal and condylar cartilage differ from that of the other two structures and also differ mutually. With small variations the reaction for both alkaline and acid phosphatase was found to be identical in the zones of erosion, hypertrophy and maturation of the four structures. Intercellularly, acid phosphatase is present in all zones in the synchondroseal and the epiphyseal plate cartilage, while in the epiphyseal and condylar cartilages it is only present in the zones of erosion, hypertrophy and maturation. The identical reaction for acid phosphatase in the epiphyseal and the condylar cartilage is thought, in all likelihood, to be accidental. When kinetic conditions are taken into account, epiphyseal cartilage seems to react like epiphyseal plate and synchondroseal cartilage, while the condylar cartilage takes up an exceptional position among growth cartilages.

Influence of Growth Hormone and Thyroxine on Endochondral Osteogenesis in the Mandibular Condyle and Proximal Tibial Epiphysis

Interrelationships of growth hormone and thyroxine effects on endochondral osteogenesis have been investigated. Evidence is offered that thyroxine augments the effect of growth hormone on the mandibular condyle just as at other osteogenetic centers, for example proximal tibial epiphysis in hypophysectomized rats. This contrasts with previous reports of a unique thyroxine antagonism to the effect of growth hormone on condylar development. In a further study on tibial epiphysis, special attention was given to assure absence of the endogenous thyroid hormone by adding thyroidectomy to hypophysectomy and reducing the possibility of thyroteopin residues in growth hormone. By histochemical tests, the responses of both calcified and noncalcified regions of the epiphyseal cartilage plate were measured. Graded growth hormone doses stimulated graded response in a noncalcified region. Thyroxine augmented this response eightfold. The width of the calcified region of the plate did not vary with treatment.

Topographic Analysis of the Microdistribution of 241 Am in the Rat Femur as Influenced by DTPA Treatment

The influence of growth and Na/sub 3/Ca-DTPA treatment on the microscopic distribution of Am(III) in the distal femora of 8-week-old female rats were examined by means of solid-state nuclear-track detectors and an electronic scanning technique. The iv dose of (/sup 241/Am)citrate was 30 ..mu..Ci/kg; Ca-DTPA was administered ip once weekly in a dose of 30 ..mu..mole/kg, and the first dose was injected 1.5 hr after the /sup 241/Am. The animals were sacrificed 7, 14, 28, and 56 days after the /sup 241/Am injection. The distribution of /sup 241/Am was presented as computer-generated scan plots giving quantitative information about radiation dose rates. The redeposition of /sup 241/Am in newly formed bone tissue between the zone of heavy initial uptake and the epiphyseal cartilage plate was absent in the case of DTPA treatment. There was a diminution of activity by DTPA in the metaphyseal band to 40 percent of the control values on the 7th day and to 26 percent on the 56th day. While DTPA does not significantly affect removal of the epiphyseal deposit of /sup 241/Am, there is a continuing effectiveness in the metaphysis and diaphysis, yielding a reduction to 70 percent of the control radioactivity at the 7th day,more » and 28 percent at the 56th day. Bone remodeling tended to increase the nonuniformity of the dose-rate distribution, which, for example, led to an enhancement of the maximum dose rates in the metaphyseal band. This increase of the nonuniformity of the dose-rate distribution is even enhanced by DTPA treatment. The information contained in the dose-rate scans was reduced to probability density curves and compared for the effects of aging and DTPA.« less

The influence of 241-Am and DTPA on morphometric parameters of the rat femur.

Microradiographically detectable alterations of the bone structure in the femur of young rats induced by monomeric 241-Am(III) (i.v., 30 muCi/kg) were studied. The morphometric and dosimetric measurements were carried out by means of an electronic image analyzer. 8 weeks after injection of 241-Am a characteristic alteration of the frequency distribution of the chord lengths over the trabeculae in the epiphysis and over the metaphyseal marrow spaces was found. The structure of the spongiosa is irregular with both large, coarse and small fragmented trabeculae. The complexity of the bone architecture and the area of the endosteal surfaces is reduced. The surface/volume ratio in control animals varies between 36 mm-1 in the epiphysis and 64 mm-1 in the region of the epiphyseal cartilage plate. From the specific surface burden (pCi/mm2) the average dose rates were determined. There is no significant difference between the calcified tissue fraction in controls and animals with 241-Am, with the exception of the metaphyseal band where the locally high dose rates cause a devitalization of the tissue with inhibition of bone resorption as well as an abnormal trabeculation in the metaphysis. Treatment by Ca-DTPA reduces the 241-Am deposition nonuniformly and the pathological manifestations are markedly less pronounced. The mean trabecular width is about 100 mum in the epiphysis and has a minimum of 40 mum in the central part of the epiphyseal plate. The mean chord length over the marrow spaces varies between 90 and 210 mum.

An autoradiographic investigation of 3H-uridine utilization by aging mouse cartilage cells

The uptake, turnover and utilization of H 3-uridine were assessed at the epiphyseal plate and articular cartilage of the femur. A total of 144 mice, 5–78 weeks of age were subcutaneously injected with 5 μCi of H 3-uridine per gram of body weight. Animals were killed from 15 min to 30 days later. Autoradiographs were prepared from 5 μm sagittal sections of femora using NTB-3 emulsion and an exposure of 36 days. Grain counts were made over the nucleus and cytoplasm of 1200 labeled articular and epiphyseal cells per time period. Nuclear uptake was initially rapid attaining peak levels 1 to 8 hours after injection depending upon the cell type and age. Cytoplasmic peak activity occurred at 1–2 days. Epiphyseal cells displayed almost twice the activity of articular cartilage cells, especially between 1 hr and 2 days when age changes were most evident. With increasing age, peak values were significantly reduced and flattened, indicating a decline in the rate and magnitude of radiotracer incorporation. Relative to maintenance values of uridine utilization, epiphyseal labeling was remarkably higher at younger ages and once linear growth had subsided, suffered a far greater decline than articular cartilage cells.

EFFECT OF STRONTIUM ON THE EPIPHYSEAL CARTILAGE PLATE OF RAT TIBIAE—HISTOLOGICAL AND RADIOGRAPHIC STUDIES

Following dietary administration of strontium carbonate, histological and radiographic changes in the epiphyseal cartilage plate of the rat tibiae were examined in the present study. The weight gain of the rat fed a strontium diet was less than that of the control rats. Longitudinal growth of tibiae and endochondral ossification were inhibited by strontium administration. The widths of both proximal and distal cartilage plates increased enormously as has also been shown by other investigators. Sizes of chondroblasts in columns of proximal cartilage plate in rats fed a strontium diet were smaller than those of the control rats and were not different between upper and lower parts. It is suggested that strontium inhibits bone growth through the inhibitory action on the maturation process of chondroblasts and the succeeding endochondral ossification.

Chemical and morphologic alterations of rabbit bone induced by adriamycin.

Long term, low dose administration of adriamycin (ADR) to young growing rabbits resulted in significant alterations in bone structure and chemistry. Morphologic changes were most pronounced at epiphyseal and metaphyseal areas of long bones. Epiphyseal cartilage plates were thin and there was derangement of growth zones. Areas of primary and secondary spongiosa were deficient in trabeculae, osteoblasts and osteoclasts. Analysis of femora, humeri and lumbar vertebrae from ADR-treated rabbits revealed increased water and fat content and significant decreases in bone density compared to age-matched controls. Cortices of long bones were roentgenographically thin and contained large irregular spaces evident microscopically. Evaluation of bone ash from ADR treated rabbits revealed significant increases in the percentage of calcium and phosphorus, although Ca/P ratios were not different from controls. Results of in vitro studies indicate that ADR binds readily to nondemineralized, but not demineralized, fresh cortical bone powder. The findings of decreased bone density, histopathologic alterations, and a paucity of osteogenic cells in ADR treated rabbits are interpreted as retardation of bone maturation. It is suggested that ADR affects adversely both the organic and inorganic fractions of bone. Due to its unique characteristics of cytostatic action, binding to metal cations and orange-red fluorescence, ADR is a novel chemical agent that may be useful in experimental bone studies.

Traumatic Separation of the Distal Femoral Epiphyseal Cartilage Plate

Twenty patients with fractures of the distal femoral epiphyseal cartilage were followed for an average of four years and three months. At the time of follow-up fifteen patients were skeletally mature. In four of the remaining five patients premature epiphyseal closure had developed. The more severe injuries progressed to premature closure of the growth plate. Five of fifteen Salter and Harris Type-II injuries resulted in shortening which averaged 2.3 centimeters. In four patients this occurred despite anatomically maintained reductions. This also occurred in one patient with a Type-I injury. Although Type-I and II injuries generally have an excellent prognosis for normal growth, this is not uniformly true of injuries to the distal femoral epiphyseal cartilage plate. Close follow-up of patients with these injuries is advocated.

Spezielle Entwicklungsstörungen des Rattenskeletts nach Verabreichung von DL-Serin-(2,3,4-trihydroxybenzyl)-hydrazid

Introduction:In humans all epiphyseal growth zones are closed after puberty. In rats some of the epiphyseal cartilage plates remain open for a lifetime. Noxious agents can therefore give rise to alterations in these structures in the rat not only until the end of puberty, but during an entire lifespan. Consequently, it is possible that very severe pathologic changes can develop in this species. Material and methods:Several hundred male and female rats (of the Füllinsdorf Albino or Charles River strain), weighing between 30 and 300 g, were treated with daily oral doses of DL-Serine-(2,3,4,-trihydroxybenzyl)- hydrazide (DL-SH) varying between 25 and 1000 mg/kg. The time of application ranged from 3 days to 18 months. The drug was given by gastric tube or as feed additive. In addition to X-ray and histological investigations, hematological and blood-chemistry studies were carried out, as well as histological evaluations of approximately 30 organs per animal. Results:The principal feature was a derangement in the transformation of chondral into bony tissue in the epiphyseal plate. The cartilaginous tissue loses its columnar arrangement in the hypertrophic zone and takes on a lather-like appearance. The invasion of the cartilage by elements of the bony tissue is impeded by the abundant cartilaginous intercellular substance. Only a few, crippled primary bony trabeculae are formed. The epiphyseal plates have doubled in width after one week of treatment with high dosages and are 7 times broader than normal after four weeks. The demarcation line between chondral and osseous tissue becomes very irregular. In some areas chondral tissue extends deeply into the osseous tissue. Following tensile and compression stress dislocations of the epiphysis occur. In the later course of the experiment the epiphyseal plate becomes narrower following transformation processes (endochondral ossification, proliferation of fibroblasts) which take place in and around the chondral tissue. But the alterations generally are so severe, and the rats are so immobilized, that the general condition cannot be ameliorated. These changes are observed in all of the epiphyseal zones which are not closed before senility. They are especially well-marked in the proximal epiphysis of the tibia, in the head of the humerus, and in the distal epiphyses of radius and ulna. In addition lordosisscoliosis in the spine and marked dislocations of the sternal segments are found. Under a dosage of 100–150 mg/kg/day these findings become markedly apparent even within a few weeks whereas it takes months until they can be detected in animals treated with 25 mg/kg/day. The intensity with which the alterations develop is determined by the intensity of growth. In older animals and in slowly growing young rats (due to a limited feed intake) they appear slowly and are not pronounced. In young and rapidly growing animals rapid and pronounced changes are observed. When very young rats are included in the experiment, irregularities occur also in those epiphyseal growth plates which are closed at puberty (distal tibial epiphysis and metatarsal bones). The described alterations could not be demonstrated in the dog even when 8-week-old puppies were treated with 25 mg/kg/day for more than one year. Discussion:The alterations appearing in the epiphyseal plates of rats after treatment with DL-Serine-(2,3,4-trihydroxybenzyl)-hydrazide cannot be classified precisely as rachitic or osteolathyrogenic changes, although to a certain extent many similarities do exist. These alterations in rats cannot be correlated with lesions in adult humans, since in humans cartilage is not found any more in the epiphyseal plates after puberty.

The use of thin specimens for X‐ray microanalysis in biology

SUMMARYThe quantitative X‐ray microanalysis of ultrathin biological sections is exemplified by a recent study of the distribution of calcium in mineralizing cartilage and bone. The determination of calcium mass‐fraction in one microarea of a vesicle within a section of rabbit epiphyseal plate cartilage is presented in detail in order to display all steps of the processing of the data. Mass fractions are obtained from an equation which is approximate but which is adequately accurate for most cases of interest, specifically for ultrathin biological sections where the analysed area consists predominantly of organic matrix. We shall examine the physical assumptions behind the computation, and the extent to which these assumptions have been verified in the analytical microscope.The purpose of this paper is to describe in detail the scheme of data collection and processing, which has now been in use for some years in the Cavendish Laboratory, for the measurement of local elemental mass fractions in thin biological specimens in instruments like the EMMA. After an outline of the physical theory we shall describe the handling of the data by means of working through an example of an actual measurement, and finally we shall add some comments, mainly precautions and reservations.

Ultrastructural effects of estrogen on epiphyseal cartilage.

Ten male weanling Holtzman rats, injected intraperitoneally with aqueous estradiol (Progynon, Schering), in daily doses of 1 μg. per g body weight, were sacrificed, simultaneously with controls receiving an equivalent amount of diluent, at intervals ranging from one hour to six days. Upper tibial epiphyseal cartilage plates (ECP), procesed for electron microscopy, revealed, as early as three hours after injection, appreciable enhancement of secretory activity, evidenced, in the zone of matrix secretion, by the abundance in Golgi cisternae of stippled material representing proteinpolysaccharide complexes. Disintegration of the lining membrane of individual Golgi vesicles was advanced after twenty-four hours; following three days of treatment, few vesicles remained intact, and pools of initially intravacuolar material were observable in the gound plasm. Long filaments, suggestive of primary or precursor collagen fibrils were apparent in this secretion. After six days, virtual lakes of this substance filled cells in the zone of prehypertophy, with consequent displacement of the rough endoplasmic reticulum against the cell periphery. Cytoplasmic vacuoles, containing mateerial similar to that found in the lacunar moat, and displaying finely beaded, radially arrayed filaments on the lining membrane were frequently encountered. Our observations suggest an initial acclleration of chondrocytic secretory activity, with subsequent retardation of transport. The resultant retention and intracellular polymerization of precollagenous products accelerates hypertrophy, thereby promoting early degeneration of chondrocytes. These ultrastructural alterations are apparently estrogen-specific.

An autoradiographic study of H 3-proline utilization by aging mouse skeletal tissues—II. Cartilage cell compartments

In an autoradiographic study the uptake, turnover and distribution of H 3-proline was assessed at the femoral epiphyseal plate and articular cartilage of 5 to 52 weeks old mice. Ninety-nine shortlived BNL mice were given H 3-proline in a single dose of 2 μCi/gm body weight (Sp. Act. = 1 Ci/mM) and killed between 15 min to 14 days later. NTB-3 autoradiograms which were exposed for 16 days were prepared and stained with hematoxylin. Grain counts were made over epiphyseal and articular cartilage cell compartments and the data statistically evaluated. The results showed significant uptake and utilization of H 3-proline by both cell types, although the activity of epiphyseal cartilage was initially higher. H 3-proline was utilized extensively for matrical precursor formation, as well as, for a variety of intracellular substances necessary for cellular viability. The uptake and turnover of H 3-proline was diminished in both cell compartments by 26 weeks of age, revealing peak activity shifts. At 52 weeks the uptake by epiphyseal cartilage cells was insignificant, whereas, articular cartilage activity was retained. The significantly diminished grain counts observed over epiphyseal plate cells with increasing age, reflect release of physiological demands for matrical precursor formation, closure of the plates and termination of longitudinal growth. The continued significant H 3-proline uptake exhibited by articular cartilage was a reflection of the physiological requirement for maintenance of normal joint function throughout the life-span of the organism. The maintenance of cell viability against accumulating age changes is essential to the integrity of the articular cartilage.

An Autoradiographic Study of <sup>3</sup>H-Proline Utilization by Aging Mouse Skeletal Tissues

Autoradiographic data of <sup>3</sup>H-proline turnover by mouse skeletal cell compartments of different age were subjected to graphical analysis for the further characterization of the effects of age on skeletal cell metabolic behavior. Periosteal osteoblasts showed early significant age changes by 26 weeks of age, paralleling the previously reported histocytomorphological changes. Age changes appeared to be nonsignificant in metaphyseal osteoblasts at 52 weeks of age, however, the total turnover of <sup>3</sup>H-proline was adjusted to the reduced functional demands associated with trabecular remodeling and closure of the epiphyseal plate. The dramatic changes observed in <sup>3</sup>H-proline uptake and turnover of the epiphyseal plate cartilage cells coincided with the closure of the plate and termination of matrix formation. In addition, highly significant age changes occurred in response to the cessation of functional demands. Residual articular cartilage cells appeared to be capable of retaining their viability, staving off early age changes, thus insuring a reasonably healthy joint function in older animals. The effects of age on the skeletal cell utilization of <sup>3</sup>H-proline were best expressed as a series of steady-state events of diminishing activity. The time table for significant age changes varied in different cell compartments and also in similar cell types located at different anatomical sites. Modulation of the time table for skeletal cell aging appeared to be dictated by functional demands.

A comparison of circulatory and calcification changes induced in the mandibular condyle, tibial epiphyseal and articular cartilages of the guinea pig by the onset and healing of scurvy

The changes in the capillary and calcification patterns of the mandibular condyle, tibial epiphyseal plate and articular cartilage have been compared in scorbutic guinea pigs. In the scorbutic tibial epiphyseal cartilage the vascular picture was one of progressive disorganization and disruption with an overall reduction in both the metaphyseal and epiphyseal blood supply of the cartilaginous plate. In the articular and condylar cartilages the onset of scurvy brought on a similar but less drastic disruption of the organized capillary patterns. The mineralization of the scorbutic epiphyseal and articular cartilages included the entire hypertrophic zone while this did not appear to be the case in the scorbutic condylar cartilage. In the tibias of the ascorbic acid-treated animals there was a gradual recovery of the normal capillary and calcification patterns, and the reconstruction process was completed by 72-hr post-injection. The articular and condylar cartilages showed a similar but faster repair of the capillary patterns and coincident with these changes was the return of the normal calcification picture. It is considered that the absence of the gerüstmark and trummerfeld zones beneath the condylar and articular cartilages is responsible for the less drastic changes as well as the faster repair in these areas. It is suggested the capillary endothelium gives rise to the chondroclasts responsible for the erosion of the condylar cartilage and that ascorbic acid has an action on the differentiation of these cells.

The action of vitamin D deficiency on bone tissue and the epiphyseal plate in rats given adequate amounts of calcium and phosphorus in the diet

The action of vitamin D on the histological appearance of bone tissue and the epiphyseal plate cartilage was studied in young rats fed a diet containing adequate amounts of calcium and phosphorus. The purpose of the investigation has been to see if vitamin D deficiency had a direct action on bone and cartilage or whether its action was secondary to changes in serum Ca and serum P accompanying vitamin D deficiency. The Ca × P product was probably above the minimum value considered necessary for mineralization. The histological investigation demonstrated some deviations from the normal in the epiphyseal plate of the vitamin D-deficient rats. Whereas the femoral plate thickness in every case was normal, that of the tibia varied from normal to a marked widening. The internal and external morphology of the bones did not deviate much from the normal. The microradiographs showed that mineralization of bone tissue and cartilage had taken place as expected in the vitamin D-deficient rats. However, in some places in the hypertrophied cartilage, corresponding to the expanded regions, a delay in the process of mineralization was found. Beneath such areas some disorganization of the metaphyseal trabeculae was observed. Both osteoblasts and osteoclasts were observed in seemingly normal functional states. No accumulation of osteoid or other abnormal features could be observed in the bone tissue. It may be concluded that the histological and radiological methods applied in the present investigation did not reveal any differences between bone tissue from vitamin D-treated and vitamin D-deficient rats. However, changes were found in the epiphyseal plates in some of the tibiae, indicating a delay in the process of mineralization of the hypertrophic cartilage. It is reasonable to assume that this limited deviation from the normal may be a direct effect of vitamin D deficiency on the cartilage, and not mediated through changes in serum Ca or serum P.

The protein-polysaccharides of articular, epiphyseal plate and costal cartilages

1. 1. The yields of protein-polysaccharides obtained from bovine fetal articular, epiphyseal plate and porcine costal cartilage and their content of uronic acid, hexosamine, sialic acid, protein hydroxyproline were determined. The ability of the protein-polysaccharides to inhibit the precipitation of calcium phosphate in vitro was also studied, both before and after degradation of the protein-polysaccharides by enzymes extracts of epiphyseal plate cartilage. 2. 2. Yields and compositions of protein-polysaccharides from the diverse hyaline cartilages were similar, indicating that there are, in the narrow age range studied, in the different cartilages and in the different species of animals, remarkable similarities in the chemical nature of the hyaline cartilages with respect to their content of protein-polysaccharides. Differences in composition are manifest, however, when compared with calf nasal cartilages or nucleus pulosus. 3. 3. Most of the protein-polysaccharides inhibited the precipitation of calcium phosphate from solution. If the protein-polysaccharides are first degraded by enzyme extracts obtained from cartilage, the inhibitory activity of the protein-polysaccharides is abolished in the range of concentrations studied. These phenomena are discussed in relation to calcification of cartilage.

Epiphyseal growth zones in oestradiol-treated rabbits.

ABSTRACT Rabbits weighing about 800 g were treated with oestradiol monobenzoate during 30 days. At the end of the treatment, the mean body weight of the experimental animals was significantly lower than that of the control animals. Grossly, a narrowing of the epiphyseal cartilage plate and a reduction of the height of the metaphysis was observed. In cartilage, the content of water, hexosamine, uronic acid, sodium, potassium and chloride decreased. The content of hydroxyproline was unchanged. In metaphyseal bone, the water content increased and the mucopolysaccharide content tended to an increase, while the concentration of hydroxyproline and of electrolytes remained unchanged. The uptake of 35SO4 was depressed in both tissues.

Chemical Studies on the Ground Substance of Human Epiphyseal-Plate Cartilage

Six samples of human epiphyseal cartilage obtained from skeletally normal children, from one day to thirteen years old, were studied. The collagen and mucopolysaccharide content was rather constant in epiphyseal-plate cartilage of older children, but in the cartilage from the new born infant the amount of galactosamine was significantly increased. Light protein polysaccharides were extracted from three of the samples. In each case, both chondroitin sulphate and keratosulphate were present in the light protein-polysaccharide complex. The ratio of these substances seemed to be age-dependent. The amino acid composition was very similar to that of other light protein-polysaccharide preparations described in the literature. By electrophoresis, the light protein-polysaccharide fraction did not behave as an homogenous compound. Prior to any enzymatic digestion or alkaline degradation, the light protein-polysaccharide complex in each case could be resolved in two well defined bands by electrophoresis. The heavy protein-polysaccharides complex had a very low amino sugar content, which increased slightly in the samples obtained from older children.