The hypertrophic scar: Location of glycosaminoglycans within scars

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Burns, 4. 14-I 9 Printed in Great Britain

The hypertrophic scar: location of glycosaminoglycans within scars M. R. Shetlar and C. L. Shetlar Departments of Biochemistry and Dermatology, School of Medicine, Lubbock, Texas

Texas Tech University

H. A. Linares Division of Pathology,

Shriners Burns Institute, Galveston, Texas

Summary

Parallel chemical and histochemical studies were made on several hypertrophic scars in order to identify the localization of the elevated chondroitin-4-sulphate found in hypertrophic scars. For chemical and electrophoretic studies, the nodular areas were dissected from the rest of the scar and studied separately using electrophoretic methods of separation with and without chondroitinase AC and chondroitinase ABC. For histochemistry, glycosaminoglycan-specific stains were used before and after use of hyaluronidase, chondroitinase AC and chondroitinase ABC. In active hypertrophic scars the histochemical studies indicated that either chondroitin-4-sulphate or chondroitin-6-sulphate was localized in the nodular area. Electrophoretic studies indicated that chondroitin-dsulphate was not present to an appreciable extent and that the nodular areas were much higher in chondroitin-4-sulphate. Hypertrophic scars undergoing maturation had lesser amounts of chondroitin4-sulphate, but the nodular areas were higher in this

glycosaminoglycan than was the respective total scar. One scar which was most mature, by histological examination, had essentially the same chondroitin-4sulphate levels in both nodular areas and total scar tissue areas. These results suggest that the nodular regions are the actively proliferating areas in hypertrophic scars and that the presence of chondroitin-4-sulphate is related to this proliferation. PREVIOUSwork has shown that the amount of glycosaminoglycan (mucopolysaccharide) material increases in hypertrophic scars which occur as a result of burn injury as compared with normal skin and mature (non-hypertrophic)

scars (Shetlar et al., 1971) and that the distribution of the different glycosaminoglycans found in skin is also altered in the hypertrophic state (Shetlar et al., 1972). Although one may speculate that the higher glycosaminoglycan content and quantitative change in distribution are related to tissue proliferation and increase of the amount of collagen in the hypertrophic scar, there is no direct evidence for this speculation. In further testing this postulation, it would appear that knowledge of the geographic distribution of mucopolysaccharides in the hypertrophic scar would be of value. A distinctive morphological pattern as compared with normal skin or a mature scar has been described (Linares et al., 1972). This pattern, found in the active hypertrophic scar, contains nodules of collagen material. These nodules are highly compact and often appear to have a capsule-like band about their periphery. These nodules can often be seen by gross visual examination and can then be separated from the rest of the surrounding tissue. This paper relates to chemical studies of the separated nodules as compared with the whole scar and with histochemical studies of similar sections of hypertrophic scars. MATERIALS AND Chemical studies

METHODS

Five scar tissues were obtained from patients whose initial injuries had been thermal burns; two of the scars (726 and 727) occurred after

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Shetlar et al: Hypertrophic Scars surgical removal of hypertrophic scars; two samples (K-76-l and K-76-2) were originally described as keloids and the nature of the original injuries was not known. Because of the difficulties of distinguishing keloids from hypertrophic scars, we shall consider these samples as hypertrophic scars. All samples were selected from excess material removed during plastic surgery procedures. The tissues, when visually examined, contained numerous nodules of a size suitable for separation by dissection. The tissues were cut into strips approximately 4 mm thick with a razor blade. Nodules were dissected from the tissue with a surgical knife and collected until samples of 15-100 mg were obtained. The total scar analysis was done on a 4-mm slice of scar representing a transverse section through the entire scar sample. All samples were weighed and then placed in a 13 x 100 mm test tube with a screw cap and digested with papain (Sigma) by the method of Mier and Wood (1969). After digestion, aliquots of the supernatant solution were taken for cellulose acetate electrophoresis using the Beckman microzone equipment. A zinc sulphate electrolyte (0.25 M, pH 5.1) (Breen et al., 1970) and a calcium acetate electrolyte (0.3 M, pH 7.25) (Seno et al., 1970) were used. The calcium acetate electrolyte will separate chondroitin-4-sulphate from chondroitin-6-sulphate. No chondroitin-6-sulphate was detected in any of the samples by this procedure, consequently the patterns using 0.25 M zinc sulphate were used for quantitation. A standard containing hyaluronic acid, dermatan sulphate and chondroitin-Csulphate was placed on each pattern. All patterns were stained for IO min with alcian blue solution (1 g dissolved in 50 ml 95 per cent ethanol, diluted with 50 ml of 0.2 M acetate buffer, pH 5.6), washed repeatedly with 5 per cent acetic acid, air dried and quantitated without clearing with the microzone densitometer (Beckman Instrument Co.). The results were recalculated on the basis of the densitometer readings for the standard glycosaminoglycans. The electrophoretic bands were further identified by treatment of the papain digests with chondroitinase AC and chondroitinase ABC as described by Yamagata et al. (1968). Fig. I shows typical results with these enzymes on one of the samples. It is apparent that the major fractions present are hyaluronic acid, dermatan sulphate. and chondroitin-4-sulphate. Histological methods

Transverse

and histochemical

sect ions of approximately

l-2 mm

in thickness were made of all of the burn scars with a razor blade; consecutive sections were fixed in chilled (4 “C) 2 per cent calcium acetateformalin, 1 per cent cetylpyridinium chlorideformalin or IO per cent buffered formalin. All the sections were embedded in paraplast and cut at 6 urn in thickness. The following staining methods were used for histological morphology: haematoxylin and eosin for general morphology; Masson’s trichrome for collagen patterns; Verhoeff’s stain and orcein for elastic fibres (Putt, 1972). Alcian blue, azure A and colloidal iron staining techniques were used for demonstrating acid proteoglycans (Pearse, 1968). Prior to staining, some sections, similar to those used for histochemistry, from each scar were subjected to digestion for 4 h at 37°C with testicular bovine hyaluronidase, chondroitinase ABC or chondroitinase AC (all from Sigma Chemicals, St Louis, MO.) (Yamagata et al., 1968). The following dilutions were used: (a) testicular hyaluronidase was dissolved in a 0.1 M phosphate buffer at pH 6.5 in a concentration of 0.5 mg/ml ; (b) chondroitinase ABC was dissolved in a 0.1 M Tris-HCI buffer, pH 8.0, in a concentration of 5 units/ml; (c) chondroitinase AC was dissolved in a 0.1 M Tris-HCI buffer, pH 3, in a concentration of 5 units/ml. Control sections containing no enzymes or heat-inactivated enzymes were run simultaneously. RESULTS Chemical

A summary of the analytical results of the glycosaminoglycan studies is presented in Tab/e I. In 6 instances the nodule samples were strikingly higher in chondroitin-4-sulphate than in the total scar sample. The seventh scar (672) was quite different in that the chondroitin-4-sulphate values were essentially the same for the nodules and total scar samples. The result for the total scar was typical of a mature scar rather than a hypertrophic scar. Samples 726 and 727 differed from samples 517 and 531 in that the total scars had chondroitin-4-sulphate levels which were within the range of those found in mature scars. However, the nodules from these samples were definitely higher in chondroitin-4-sulphate than the respective total scars. The nodule level of chondroitin-4-sulphate for sample 726 was in the range considered hypertrophic while that for 727 was in the upper range of the mature scars. Thus several gradations of glycosaminoglycan distribution between nodules and total scar are noted, with samples 517, K-76-l and K-76-2 being

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Standard

K-76-2

Nodules

Nodules Chondroitinase

K-76-2

after

Fig. 1. Densitometer tracings of cellulose acetate patterns of glycosaminoglycan extracts of nodular tissue from a keloid before and after digestion with chondroitinase AC (above, calcium acetate electrolyte, 0.3 M, pH 7.25) and chondroitinase ABC (below, zinc sulphate electrolyte, 0.25 M, pH 5.1). Upper tracing is of a pattern from a standard mixture containing equimolar amounts of hyaluronit acid (HA), dermatan sulphate, chondroitin-4-sulphate (Ch-4-SO& and chondroitin-6-sulphate (Ch-6-SO,).

the most hypertrophic and sample 672 the least hypertrophic. It is obvious from the results that the nodular areas from hypertrophic scars contain a distribution of glycosaminoglycans differing from the non-nodular portion in that they contain relatively more of the chondroitin-4-sulphate components. This is the same alteration previously reported for hypertrophic scars (Shetlar et al., 1972). In other words, the nodular areas appear

to be more hypertrophic than the total scar. This is also true for samples 726 and 727 where the total scars have chondroitin-4-sulphate levels in the mature range, but the nodules still have levels characteristic of hypertrophic scars. Histology and histochemistry Specimens 517 and 531 had the typical histomorphology of active hypertrophic scars (Linares et al., 1972). The collagen fibres of the dermis

Shetlar et al : Hypertrophic

17

Scars

Table 1. Glycosaminoglycan distribution in nodular areas of scars as compared in terms of percentage of the total glycosaminoglycan)

Specimen

Age of patient at injury (yr)

Histologically hypertrophic 517 3 531

4

Age of patient at sampling (yr)

6 7

K-76-l

13

K-76-2

24

Histologically 726

Fraction

Nodules Total Scar Nodules Total Scar Nodules Nodules Total Scar Nodules Total Scar

hypertrophic in regression 6 11

Nodules Total Scar 11 Nodules 727 8 Total Scar Histologically formerly hypertrophic in regression 10 Nodules 672 6 Total Scar Ranges of total scar analyses* Hypertrophic scars Mature scars Normal skin *Ranges

of values found

in studies of 21 hypertrophic

ran in a whorl-like arrangement with a predominant nodular pattern. Most of the hypertrophic nodules were clearly delineated and appeared surrounded by thicker collagen fibres which formed a septal-like capsule. No elastic fibres were present in the nodular areas. The histochemical methods for acid proteoglycans stained all of the nodules strongly; in contrast, the rest of the tissue stained only weakly. A typical section stained with alcian blue is shown in Fig. 2. Sections of specimens 517 and 531 when digested with chondroitinase AC, which degrades chondroitin-4 or -6-sulphate but not dermatan sulphate almost completely abolished the alcian blue staining material as shown in Fig. 3. Similar results were obtained after digestion with testicular hyaluronidase. Digestion with chondroitinase ABC did not seem to remove any more material which stained with alcian blue than did the chondroitinase AC. These results indicate that the nodular areas contain a large amount of chondroitin-4-sulphate or chondroitin-6-sulphate and only a small amount of dermatan sulphate. Similar results

Hyaluronic acid

with the total area (expressed

Dermatan solphate

Chondroitin4-sulphate

3.4 12.3 29.1 27.0 23.3 21 .9 22.8 33.9 32.8

49.4 67.8 34.9 59.9 50.9 41.3 57.7 38.5 52.5

47.1 19.9 36.0 13.1 26.8 36.8 19.5 27.6 14.7

34.7 44.9 48.6 43.2

46.7 48.3 37.1 47.2

18.6 6.8 14.2 9.6

28.6 34.3

62.7 57.5

8.7 8.1

12-27 15-30 38-59

39-68 64-77 40-57

14-41 4-l 3 2-8

scars, 18 mature scars and 17 normal skin samples.

were obtained using the enzyme digestions and staining with azure A or colloidal iron. Specimens 726 and 727 showed the histomorphology characteristic of hypertrophic scars in regression. In sample 726 the collagen fibres of the dermis are predominantly in parallel orientation with large areas of nodular elongated patterns. The nodular areas are located largely in the reticularis layer of the dermis. Sample 727 has some collagen fibres in the dermis which are of nodular pattern with many areas of parallel patterns relative to the surface. Effects of the enzymes were not studied in these scar samples. Histological and histochemical results obtained from studies of specimen 672 indicated a formerly hypertrophic scar in regression. The collagen fibres of the dermis ran in a predominantly parallel orientation with respect to the epidermis. Some hypertrophic nodules with a marked elongated pattern were present, indicating that the maturation of the scar was in progress. The elastic fibres were present in most of the tissue. The histopathological diagnosis of this specimen corresponds to that of a non-active hypertrophic

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Fig. 2. Histological section from scar 517 showing a hypertrophic nodule (n) intensively stained for acid proteoglycans before enzymatic digestion (dark grey in the picture). (Alcian blue. x 20.)

scar in advanced process of maturation. histochemical staining procedures utilizing

The alcian

blue, azure A or colloidal iron were weakly positive, indicating a small amount of acid proteoglycans in the tissues. In contrast to specimens 517 and 531, the alcian blue staining material was still present after treatment with chondroitinase AC, consequently this material must be largely dermatan sulphate. The chemical and electrophoretic studies indicate that the nodules of hypertrophic scars are higher in chondroitin-4-sulphate than is the surrounding tissue. The histochemical studies indicate that chondroitin-4-sulphate or chondroitin-6-sulphate is elevated in the nodules as compared with the surrounding tissue. As the electrophoretic studies indicate that chondroitin4-sulphate is present in hypertrophic scars and to a lesser extent in mature scars and skin while no chondroitin-6-sulphate has been observed in any of the samples, the increase of alcian blue staining material noted by histochemistry which is degraded by chondroitinase AC must be chondroitin-4-sulphate. It is possible, however,

Fig. 3. Adjacent

showing glycans affinity (Alcian

section to that depicted in Fig. 1, the same nodule (n) stained for acid proteoatter chondroitinase AC digestion. The for alcian blue has almost disappeared. blue. x 20.)

for some chondroitin-6-sulphate to be present as a part of a mixed proteoglycan as the original proteoglycan is not completely digested by papain. In any case the combined evidence presented in this paper leads to the conclusion that the nodules found in hypertrophic scars contain much of the chondroitin-4-sulphate present in these tissues and that as the hypertrophic scar matures these nodular areas lose their high content of chondroitin-4-sulphate. Since much higher levels of chondroitin-4sulphate are found in hypertrophic scars and in granulation tissue (Shetlar et al., unpublished data) and since the levels of this glycosaminoglycan decrease as scars mature or become less hypertrophic, it seems reasonable to relate chondroitin-4-sulphate to proliferation of connective tissue in dermal scars. Furthermore, since the nodular areas of the scars are higher in chondroitin-4-sulphate, it is suggested that these areas are the active sites of proliferation in the hypertrophic scar.

Shetlar

: Hypertrophic

et al.

Scars

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REFERENCES

Breen M., Weinstein H. G., Andersen M. et al. (1970) Microanalysis and characterization of acidic glycosaminoglycans Anal. in human tissues. Biochem.

35, 146.

Linares H. A., Kischer C. W., Dobrkovsky M. et al. (1972) The histiotypic organization of the hypertrophic scar in humans. J. Invest. Dermatol. 59, 323-331.

Mier P. D. and Wood M. (1969) A simplified technique for the analysis of tissue acid mucopolysaccharides. Clin. Chim. Acta 24, 105. Pearse A. G. E. (1968) Histochemistry: Theoretical and Applied, 3rd ed. Baltimore, Williams & Wilkins, pp. 667, 671, 672. Putt F. A. (1972) Manual of’Histopathologica1 Staining Methods. New York, Wiley, pp. 89, 98, 126, 249.

Requests

for

reprints

should

Lubbock. Texas 79409. USA.

be addressed

to:

Seno N., Anno K., Kondo K. et al. (1970) Improved method for electrophoretic separation and rapid quantitation of isomeric chondroitin sulfates on cellulose acetate strips. Anal. Biochem. 37, 197. Shetlar M. R., Dobrkovsky M., Linares H. A. et al. (1971) The hypertrophic scar. Glycoprotein and collagen components of burn scars. Proc. Sot. Exp. Biol. Med. 138, 298.

Shetlar M. R., Shetlar C. L., Chien S. F. et al. (1972). The hypertrophic scar. Hexosamine containing components of burn scars. Proc. Sot. Exp. Bid. Med. 139,544.

Yamagata T., Saito H., Habuchi 0. et al. (1968) Purification and properties of bacterial chondroitinases and chondrosulfatases. J. Biol. Chem. 243. 1523.

M. R. Shetlar, Department

of Dermatology,

Texas Tech University

School of Medicine,