1 1 . M U C O P O L Y S A C C H A R I D E S IN T I S S U E C U L T U R E
369
ated synovial cells in simplified culture medium (Castor, 1957), i.e.
80% Eagle's basal medium and 2 0 % of normal horse serum. In this
system the formation of hyaluronic acid in the medium was demonstrated both by hexosamine assay and turbidimetrically.
The production of hyaluronic acid in Tissue Culture is not of course
confined to cells of synovial origin. Indeed, in an extensive study
Grossfeld et al. (1955) found that material that gave a mucin clot was
produced in the culture medium by beef embryonic bone, by human
embryonic skin, bone, tendon, epiphyses, aorta, and pericardium, and
also by chick embryonic bone and heart. Beef embryonic skin, however,
only occasionally gave traces of a mucin clot, evidently because
epithelial growth was predominant in these cultures. These, and later
studies (Grossfeld et al., 1957) were the first to suggest that fibroblasts
from a variety of tissues produce the acid mucopolysaccharide of the
ground substance. This conclusion was supported by the work of
Gaines (1960) in which continuous mucopolysaccharide production
was observed during the period of viable growth of cultures of human
subcutaneous tissue, chick embryonic heart and skin (see Table I I ) . The
simultaneous production of collagen was regarded as evidence that the
fibroblasts, the predominant cell type in these cultures, were responsible
for mucopolysaccharide synthesis.
The first attempts to obtain information on the production of acid
mucopolysaccharides, other than hyaluronic acid, were those of Grossfeld et al. (1957; see also Grossfeld, 1957, 1958) with tissue explants of
embryonic beef bone, rat subcutaneous tissue, and human foetal skin
and bone. In order to obtain sufficient material for chemical fractionation and identification of the acid mucopolysaccharide, a mass tissue
culture method was used. In experiments with human skin, approximately 63 mg of hyaluronic acid and 25 mg of chondroitin sulphate
were isolated. From analytical data and enzymic digestion the chondroitin sulphate was identified as type C ; no B was found. Human and
bovine bone, and rat subcutaneous tissue produced, in vitro as in vivo,
about equal quantities of hyaluronic acid and chondroitin sulphate,
the latter being incompletely sulphated. The authors regard failure of
sulphation to be the probable reason for the production of undersulphated chondroitin sulphates in culture, and emphasize that in
contrast to hyaluronic acid, which occurs both in fluids and structural
elements, in the organism chondroitin sulphates seldom occur in
fluids. In tissue cultures, however, the chondroitin sulphates produced
by fibroblasts are in solution in the culture medium. These findings
and considerations are relevant both to the mechanisms of synthesis
of the sulphated mucopolysaccharides (see p. 377) and to their release
from tissues by the action of hydrolases (see p. 380).
369
ated synovial cells in simplified culture medium (Castor, 1957), i.e.
80% Eagle's basal medium and 2 0 % of normal horse serum. In this
system the formation of hyaluronic acid in the medium was demonstrated both by hexosamine assay and turbidimetrically.
The production of hyaluronic acid in Tissue Culture is not of course
confined to cells of synovial origin. Indeed, in an extensive study
Grossfeld et al. (1955) found that material that gave a mucin clot was
produced in the culture medium by beef embryonic bone, by human
embryonic skin, bone, tendon, epiphyses, aorta, and pericardium, and
also by chick embryonic bone and heart. Beef embryonic skin, however,
only occasionally gave traces of a mucin clot, evidently because
epithelial growth was predominant in these cultures. These, and later
studies (Grossfeld et al., 1957) were the first to suggest that fibroblasts
from a variety of tissues produce the acid mucopolysaccharide of the
ground substance. This conclusion was supported by the work of
Gaines (1960) in which continuous mucopolysaccharide production
was observed during the period of viable growth of cultures of human
subcutaneous tissue, chick embryonic heart and skin (see Table I I ) . The
simultaneous production of collagen was regarded as evidence that the
fibroblasts, the predominant cell type in these cultures, were responsible
for mucopolysaccharide synthesis.
The first attempts to obtain information on the production of acid
mucopolysaccharides, other than hyaluronic acid, were those of Grossfeld et al. (1957; see also Grossfeld, 1957, 1958) with tissue explants of
embryonic beef bone, rat subcutaneous tissue, and human foetal skin
and bone. In order to obtain sufficient material for chemical fractionation and identification of the acid mucopolysaccharide, a mass tissue
culture method was used. In experiments with human skin, approximately 63 mg of hyaluronic acid and 25 mg of chondroitin sulphate
were isolated. From analytical data and enzymic digestion the chondroitin sulphate was identified as type C ; no B was found. Human and
bovine bone, and rat subcutaneous tissue produced, in vitro as in vivo,
about equal quantities of hyaluronic acid and chondroitin sulphate,
the latter being incompletely sulphated. The authors regard failure of
sulphation to be the probable reason for the production of undersulphated chondroitin sulphates in culture, and emphasize that in
contrast to hyaluronic acid, which occurs both in fluids and structural
elements, in the organism chondroitin sulphates seldom occur in
fluids. In tissue cultures, however, the chondroitin sulphates produced
by fibroblasts are in solution in the culture medium. These findings
and considerations are relevant both to the mechanisms of synthesis
of the sulphated mucopolysaccharides (see p. 377) and to their release
from tissues by the action of hydrolases (see p. 380).