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The histochemistry of glycosaminoglycans within hypertrophic scars
JOURNAL
OF SURGICAL
RESEARCH
28,
171-181 (1980)
The Histochemistry of Glycosaminoglycans within Hypertrophic Scars STANLEY A. ALEXANDER, D.M.D.,*
AND R. BRUCE DONOFF, D.M.D.,M.D.t
*School of Dental Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794, and tHarvard School of Dental Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114 Submitted for publication
March 15, 1979
Hypertrophic scars were examined by histochemical methods and selective enzymatic digestions for glycosaminoglycan localization. Film substrates of hyaluronic acid were also used to demonstrate tissue hyaluronidase activity. The scars consisted of specific areas that reacted positively to enzyme treatment and indicated that hypertrophic scars can be composed of varying levels of tissue maturity. The absence of hyaluronidase activity within the tissue signifies some abnormality or inactivity of this enzyme and may be responsible for reported increases of chondroitin-4-sulfate in hypertrophic scars.
INTRODUCTION
Hypertrophic scars are morphologically distinct from normal skin or granulation tissue. Increased collagen deposition and over-abundance of chondroitin-4-sulfate have been postulated as a cause for these differences [ 12, 181. Donoff and Burke [5] have shown that the quality of glycosaminoglycans (GAGS) within the blood vessels of hypertrophic scar were different from those of mature scar and normal skin. Histological studies of GAGS in normal wounds, however, show a regular sequence of events leading to repair. Hyaluronic acid and serum glycoproteins predominate in the early wound and are then replaced with the more highly charged GAGS containing both carboxy1 and sulfate groups during the later phases of healing [ 1, 6-81. In contrast to normal granulation tissue, there is a great deal of evidence to suggest that defective wound maturation is a major factor in hypertrophic scar formation [9, 11, 12, 18, 191. Selective histochemical methods can identify individual glycosaminoglycans in tissues. The present work reports the Fesults of GAG localization in hypertrophic scar using highly selective enzyme digestions combined with a variety of staining methods 171
for the demonstration of GAGS and collagen tissue components. MATERIALS
AND METHODS
Seven samples of hypertrophic scar were obtained from burn victims. The age of the tissue ranged from 1 to 13 years. The age of the patients ranged from 3 to 28 years. Tissues were fixed for 48 hr in 1% cetylpyridinium chloride- 10% formalin. They were washed, dehydrated in a graded series of alcohol, cleared in xylene, and embedded in paraffin at 56°C. Serial sections were cut at 5 pm and mounted on acid clean glass slides and stored for subsequent staining. Prior to staining, tissue sections were treated with the following enzyme preparations. Streptomyces Hyaluronidase. Sections were incubated at 37°C for 4 hr in a 0.1 M sodium phosphate buffer at pH 5.0 containing Streptomyces hyaluronidase (Calbiothem) at a concentration of 100 TRU/ml [22, 231. Chondroitinase
AC. Tissue sections were incubated in a 0.1 M Tris-HCl buffer at pH 7.3 containing chondroitinase AC (Sigma) at a concentration of 2.5 units/ml at 37°C for 4 hr [24]. Chondroitinase ABC. Tissue sections
0022-4804/80/020171-11$01.00/o Copyright 8 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
172
JOURNAL OF SURGICAL RESEARCH: VOL. 28, NO. 2, FEBRUARY
were incubated in 0.1 M Tris-HCI buffer at pH 8.0 containing chondroitinase ABC (Sigma) at a concentration of 2.5 units/ml at 37°C for 4 hr [24]. These preparations have been shown to be free of ribonuclease, deoxyribonuclease, and protease activity [23, 241.
The enzymes were tested for substrate specificity at the concentrations used in the histochemical methods. Chondroitin-Csulfate, chondroitin-6-sulfate, dermatan sulfate, and hyaluronic acid were obtained commercially (Sigma). Solutions of each were prepared in water at a concentration of 25 &ml. Films were prepared on microscopic slides and treated as described with the enzyme solutions. Tissues were subsequently stained with the following methods: (1) 1% solution of Alcian blue 8GX at pH 1.Oand 2.5 using nuclear fast red as a counterstain in both cases [IO, 201; (2) colloidal iron and acridine orange for hyaluronic acid and sulfated GAG differentiation (Alexander and Donoff, in preparation); (3) colloidal iron and Van Gieson stain for collagen and GAG relationships [14]; and (4) Hart’s elastic tissue stain [14]. Controls consisted of tissue sections incubated in the specific buffers containing no enzyme, and incubation of tisssue sections with heat-inactivated enzymes. These results were compared to intact control tissue that was not experimentally treated.
1980
Hyaluronidase localization within the hypertrophic scars was attempted using fresh frozen sections on film substrates according to the methods of McCombs and White [15]. These tissues were subsequently stained with toluidine blue. Areas of the film substrate not staining metachromatitally indicate enzyme activity. RESULTS
The findings are summarized in Table 1. All of the scars exhibited a similar morphology. In most specimens, the epidermis was in a condition of acanthosis. Rete pegs were entirely absent. The papillary layer of the presumptive dermis consisted of loosely arranged collagen fibers and glycosaminoglycans, and closely resembled normal skin. The reticular area of the presumptive dermis differed from the papillary layer. Three distinct formations could be observed. In some areas, bundles of collagen showed a unidirectional, parallel orientation with respect to the surface. In other areas collagen bundles and glycosaminoglycans appeared disorganized and began to show whorl-like patterns (Fig. 1). Within the same tissue, the collagen and glycosaminoglycans were oriented into nodular formations (Fig. 2). These nodules appeared to be surrounded by a capsular arrangement of fibrous tissue. Elastic tissue was scanty throughout the specimens and, when observed, appeared to be in a state of fragmentation. A distinct boundary was observed between the hyper-
TABLE 1 RELATIVE
REACTIVITY FOLLOWED
OF HYPERTROPHIC SCAR TO SPECIFIC ENZYME DIGESTIONS BY ALCIAN BLUE AND COLLOIDAL IRON STAINING
Streptomyces hyaluronidase
Chondroitinase AC
Chondroitinase ABC
Papillary layer
+
++
+++
Reticular layer Parallel fibers Whorls Nodules
+ + +
+++ +++ +++
+++++ +++ +++
Tissue section
ALEXANDER
AND DONOFF:
GLYCOSAMINOGLYCANS
HYPERTROPHIC
SCARS
173
FIG.
2. Three-year-old hypertrophic scar stained with Alcian blue 8GX (pH 1.O). Note the presence of nodular formations (N) in this tissue. x 160.
a
6 r
ALEXANDER
AND DONOFF:
GLYCOSAMINOGLYCANS
trophic scar and the normal subcutaneous tissue. This appeared to be composed of a thin band of collagen fibers with normal characteristics. Histochemical and enzyme methods revealed distinct differences within the dermal layers of the hypertrophic scar. The papillary layers stained intensely with colloidal iron and with Alcian blue. When treated with Streptomyces hyaluronidase, a generalized loss of stain occurred throughout the tissue, indicating the presence of hyaluranic acid prior to enzyme treatment. When treated with chondroitinase AC, the staining reaction was reduced further. After treatment with chondroitinase ABC, Alcian blue (pH 1.0) staining was abolished, indicating the loss of dermatan sulfate. The other staining methods were consistent with these results. The papillary collagen was unaffected by these enzyme methods and stained prominantly with Van Gieson’s technique. The reticular layer reacted similarly when treated with Streptomyces hyaluronidase. A generalized loss of staining was noted throughout the tissue after exposure to enzyme. The whorl-like and nodular formations stained intensely with Alcian blue (pH 1.O)and colloidal iron methods, indicating the presence of sulfated material within these areas. Collagen was always strongly associated with this sulfated material, particularly in the nodular formations. Differences were observed between the parallel, whorl-like, and nodular areas of the reticular layer upon enzyme treatment. Chondroitinase AC and chondroitinase ABC resulted in a similar loss of staining in the whorl-like and nodular formations when compared to controls, indicating an absence of dermatan sulfate in these areas (Figs 3 and 4). The parallel arrangements of tissue reacted positively with chondroitinase ABC, indicating the presence of dermatan sulfate (Figs. 5 and 6). No hyaluronidase activity was demonstrated. Substrate films of hyaluronic acid stained metachromatically with toluidine
HYPERTROPHIC
SCARS
175
blue, indicating the absence of enzyme activity in the parallel, whorl-like, and nodular areas of the scars. DISCUSSION
The glycosaminoglycans form a part of the structural framework of normal wounds [l, 3,6-g]. Linares et al. [ll] described the formation of whorls and nodules of collagen in hypertrophic scars. Increased amounts of chondroitin-4-sulfate have been localized in the nodular areas 118, 191while increased collagen has also been demonstrated in this tissue [9]. Hypertrophic scar is composed of two general presumptive dermal components: the papillary and reticular layers. The papillary layer seems to resemble normal papillary dermis in response to enzyme histochemistry and staining methods. The reticular layer can be subdivided into three distinct areas of development: (1) parallel orientation of collagen and glycosaminoglycans; (2) a whorl-like arrangement; and (3) a nodular arrangement. Since these patterns coexist within the same tissue, it appears that the hypertrophic scar is in a state of constant flux between immature and mature stages or that this tissue represents various degrees of development during healing. These stages have been previously categorized 1111. Whorl-like and nodular formations represent immature tissue, and parallel arrangements of collagen and glycosaminoglycans are associated with tissue maturity. Treatment of tissues with chondroitinase ABC demonstrated the presence of dermatan sulfate only in areas that showed parallel fiber arrangement. Low levels of dermatan sulfate have been observed in other developmental tissues [ 131. Chondroitinase ABC was ineffective in removing additional tissue stain in other areas of the hypertrophic scar and was comparable to chondroitinase AC in its activity in these areas. The whorl-like and nodular arrangements were susceptible to chondroitinase AC
176
JOURNAL
OF SURGICAL
RESEARCH:
VOL. 28, NO. 2, FEBRUARY
1980
ALEXANDER
AND DONOFF:
GLYCOSAMINOGLYCANS
HYPERTROPHIC
SCARS
177
178
JOURNAL
OF SURGICAL
RESEARCH:
VOL. 28, NO. 2, FEBRUARY
1980
ALEXANDER
AND DONOFF:
GLYCOSAMINOGLYCANS
HYPERTROPHIC
SCARS
179
180
JOURNAL OF SURGICAL RESEARCH: VOL. 28, NO. 2, FEBRUARY
digestion, indicating the presence of chondroitin-C and/or -6-sulfate. This result was consistent with previous findings [ 181.Treatment with Streptomyces hyaluronidase, an enzyme specific for hyaluronic acid 1161, caused a generalized removal of tissue stain in all areas of the hypertrophic scar. Intrinsic wound hyaluronidase has been demonstrated in normal granulation tissue [2], which is capable of degrading hyaluronic acid and chondroitin-4-sulfate [4]. No enzyme activity was found in hypertrophic scar, which may explain the generalized action of Streptomyces hyaluronidase upon the tissue. Following the initial trauma to the tissue and prior to biopsy, wound enzyme may have been present in, and responsible for, localized organization and maturation, since the presence of hyaluronidase has been associated with differentiation in other systems 117, 211. Reports of increased nodular chondroitin-4-sulfate [ 181 may indicate the absence or abnormality of localized wound enzyme activity in this area. In conclusion, the hypertrophic scar appears to be composed of varying levels of tissue maturity. The low levels or absence of dermatan sulfate indicate the developmental immaturity of this new tissue. Consistent with these findings, Donoff and Burke 151have demonstrated low levels of dermatan sulfate in the blood vessels of hypertrophic scars when compared to mature scar. These observations indicate that the hypertrophic scar is quite different qualitatively from normal skin or mature scar tissue, particularly in the disorganized areas. ACKNOWLEDGMENTS This work was supported by United States Public Health Service-National Institutes of Dental Research Training Grant 570lDE0012-14 and National Institutes of Health Bum Center Grant GM217000.
REFERENCES 1. Ahmad, M. Glucosamine and hydroxyproline content of granulation tissue on different days of wound healing in white albino rats. Ann. Biochem. Exp. Med. 21: 295, 1961.
1980
2. Alexander, S. A., and Donoff, R. B. Localization ofwound hyaluronidase. J. Dent. Res. 57: 93,1978. 3. Bentley, J. P. Rate of chondroitin sulfate formation in wound healing. Ann. Surg. 165: 186, 1967. 4. Bertolami, C. N., and Donoff, R. B. Identification and purification of a mammalian wound hyaluronidase. J. Dent. Res. 57: 96, 1978. ,5. Donoff, R. B., and Burke, J. F. Abnormality of hypertrophic scar blood vessels. /. Surg. Res. 25: 251, 1978. 6. Dorner, R. W. Glycosaminoglycans of regenerating tendon. Arthritis Rheum. 10: 275, 1967. 7. Dunphy, J. E., and Udupa, K. N. Chemical and histochemical sequences in the normal healing wound. N. Engl. J. Med. 253: 847, 1955. 8. Flint, M. Interrelationships of mucopolysaccharide and collagen in connective tissue remodeling. J. Embryol. Exp. Morphol. 27: 481, 1972. 9. Kischer, C. W., and Shetlar, M. R. Collagen and mucopolysaccharides in the hypertrophic scar. Connect. Tissue Res. 2: 205, 1974. 10. Lev, R., and Spicer, S. S. Specific staining of sulphate groups with Alcian blue at low pH. J. Histochem. Cytochem. 12: 309, 1964. 11. Linares, H. A., Kischer, C. W., Dobrkovsky, M., and Larson, D. L. The histotypic organization of the hypertrophic scar in humans. J. Invest. Dermatol. 59: 323, 1972. 12. Linares, H. A., and Larson, D. L. Proteoglycans and collagenase in hypertrophic scar formation. Plast. Reconstr. Surg. 62: 589, 1978. 13. Loewi, G., and Meyer, K. The acid mucopolysaccharides of embryonic skin. Biochim. Biophys. Acta 27: 453, 1958. 14 Luna, L. G. Manual of Histologic Staining Methods of the Armed Forces Institute
of Pathol-
ogy, 3rd ed. New York: McGraw-Hill, 1968.P. 79. 15. McCombs, H. L., and White, H. J. Histochemistry of hyaluronidase. Amer. J. C/in. Pathol. 49: 68, 1968. 16. Ohya, T., and Kaneko, Y. Novel hyaluronidase from Streptomyces. Biochim. Biophys. Acta l998: 607, 1970. 17. Polansky, J. R., Toole, B. P., and Gross, J. Brain hyaluronidase: Changes in activity during chick development. Science 183: 862, 1974. 18. Shetlar, M. R., Shetlar, C. L., and Linares, H. A. The hypertrophic scar: Location of glycosaminoglycans within scars. Burns 4: 14, 1977. 19. Shetlar, M. R., Shetlar, C. L., Chien, S. F., Linares, H. A., Dobrkovsky, M., and Larson, D. L. The hypertrophic scar. Hexosamine containing components of burn scars. Proc. Sot. Exp. Biol. Med. 139: 544, 1972. 20. Spicer, S. S., Horn, R. G., and Leppi, J. J. Histochemistry of connective tissue mucopolysaccharides. In D. E. Smith and B. M. Williams (Eds.), The Connective Tissue. Baltimore: Williams & Wilkins, 1967. P. 251.
ALEXANDER
AND DONOFF: GLYCOSAMINOGLYCANS
21. Toole, B. P., and Trelstad, R. L. Hyaluronate production and removal during corneal development in the chick. Develop. Bid. 26: 28, 1971. 22. Yamada, K. The effect of digestion with Streptomyces hyaluronidase upon certain histochemical reactions of hyaluronic acid-containing tissues. 1. Histochem. Cytochem. 21: 794, 1973.
HYPERTROPHIC SCARS
181
23. Yamada, K., and Hirano, K. The histochemistry of hyaluronic acid containing mucosubstances. J. Histochem. Cytochem. 21: 469, 1973. 24. Yamada, K. The effect of digestion with chondroitinases upon histochemical reactions of mucosaccharide-containing tissues. J. Histochem. Cytothem. 22: 266, 1974.
OF SURGICAL
RESEARCH
28,
171-181 (1980)
The Histochemistry of Glycosaminoglycans within Hypertrophic Scars STANLEY A. ALEXANDER, D.M.D.,*
AND R. BRUCE DONOFF, D.M.D.,M.D.t
*School of Dental Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794, and tHarvard School of Dental Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114 Submitted for publication
March 15, 1979
Hypertrophic scars were examined by histochemical methods and selective enzymatic digestions for glycosaminoglycan localization. Film substrates of hyaluronic acid were also used to demonstrate tissue hyaluronidase activity. The scars consisted of specific areas that reacted positively to enzyme treatment and indicated that hypertrophic scars can be composed of varying levels of tissue maturity. The absence of hyaluronidase activity within the tissue signifies some abnormality or inactivity of this enzyme and may be responsible for reported increases of chondroitin-4-sulfate in hypertrophic scars.
INTRODUCTION
Hypertrophic scars are morphologically distinct from normal skin or granulation tissue. Increased collagen deposition and over-abundance of chondroitin-4-sulfate have been postulated as a cause for these differences [ 12, 181. Donoff and Burke [5] have shown that the quality of glycosaminoglycans (GAGS) within the blood vessels of hypertrophic scar were different from those of mature scar and normal skin. Histological studies of GAGS in normal wounds, however, show a regular sequence of events leading to repair. Hyaluronic acid and serum glycoproteins predominate in the early wound and are then replaced with the more highly charged GAGS containing both carboxy1 and sulfate groups during the later phases of healing [ 1, 6-81. In contrast to normal granulation tissue, there is a great deal of evidence to suggest that defective wound maturation is a major factor in hypertrophic scar formation [9, 11, 12, 18, 191. Selective histochemical methods can identify individual glycosaminoglycans in tissues. The present work reports the Fesults of GAG localization in hypertrophic scar using highly selective enzyme digestions combined with a variety of staining methods 171
for the demonstration of GAGS and collagen tissue components. MATERIALS
AND METHODS
Seven samples of hypertrophic scar were obtained from burn victims. The age of the tissue ranged from 1 to 13 years. The age of the patients ranged from 3 to 28 years. Tissues were fixed for 48 hr in 1% cetylpyridinium chloride- 10% formalin. They were washed, dehydrated in a graded series of alcohol, cleared in xylene, and embedded in paraffin at 56°C. Serial sections were cut at 5 pm and mounted on acid clean glass slides and stored for subsequent staining. Prior to staining, tissue sections were treated with the following enzyme preparations. Streptomyces Hyaluronidase. Sections were incubated at 37°C for 4 hr in a 0.1 M sodium phosphate buffer at pH 5.0 containing Streptomyces hyaluronidase (Calbiothem) at a concentration of 100 TRU/ml [22, 231. Chondroitinase
AC. Tissue sections were incubated in a 0.1 M Tris-HCl buffer at pH 7.3 containing chondroitinase AC (Sigma) at a concentration of 2.5 units/ml at 37°C for 4 hr [24]. Chondroitinase ABC. Tissue sections
0022-4804/80/020171-11$01.00/o Copyright 8 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
172
JOURNAL OF SURGICAL RESEARCH: VOL. 28, NO. 2, FEBRUARY
were incubated in 0.1 M Tris-HCI buffer at pH 8.0 containing chondroitinase ABC (Sigma) at a concentration of 2.5 units/ml at 37°C for 4 hr [24]. These preparations have been shown to be free of ribonuclease, deoxyribonuclease, and protease activity [23, 241.
The enzymes were tested for substrate specificity at the concentrations used in the histochemical methods. Chondroitin-Csulfate, chondroitin-6-sulfate, dermatan sulfate, and hyaluronic acid were obtained commercially (Sigma). Solutions of each were prepared in water at a concentration of 25 &ml. Films were prepared on microscopic slides and treated as described with the enzyme solutions. Tissues were subsequently stained with the following methods: (1) 1% solution of Alcian blue 8GX at pH 1.Oand 2.5 using nuclear fast red as a counterstain in both cases [IO, 201; (2) colloidal iron and acridine orange for hyaluronic acid and sulfated GAG differentiation (Alexander and Donoff, in preparation); (3) colloidal iron and Van Gieson stain for collagen and GAG relationships [14]; and (4) Hart’s elastic tissue stain [14]. Controls consisted of tissue sections incubated in the specific buffers containing no enzyme, and incubation of tisssue sections with heat-inactivated enzymes. These results were compared to intact control tissue that was not experimentally treated.
1980
Hyaluronidase localization within the hypertrophic scars was attempted using fresh frozen sections on film substrates according to the methods of McCombs and White [15]. These tissues were subsequently stained with toluidine blue. Areas of the film substrate not staining metachromatitally indicate enzyme activity. RESULTS
The findings are summarized in Table 1. All of the scars exhibited a similar morphology. In most specimens, the epidermis was in a condition of acanthosis. Rete pegs were entirely absent. The papillary layer of the presumptive dermis consisted of loosely arranged collagen fibers and glycosaminoglycans, and closely resembled normal skin. The reticular area of the presumptive dermis differed from the papillary layer. Three distinct formations could be observed. In some areas, bundles of collagen showed a unidirectional, parallel orientation with respect to the surface. In other areas collagen bundles and glycosaminoglycans appeared disorganized and began to show whorl-like patterns (Fig. 1). Within the same tissue, the collagen and glycosaminoglycans were oriented into nodular formations (Fig. 2). These nodules appeared to be surrounded by a capsular arrangement of fibrous tissue. Elastic tissue was scanty throughout the specimens and, when observed, appeared to be in a state of fragmentation. A distinct boundary was observed between the hyper-
TABLE 1 RELATIVE
REACTIVITY FOLLOWED
OF HYPERTROPHIC SCAR TO SPECIFIC ENZYME DIGESTIONS BY ALCIAN BLUE AND COLLOIDAL IRON STAINING
Streptomyces hyaluronidase
Chondroitinase AC
Chondroitinase ABC
Papillary layer
+
++
+++
Reticular layer Parallel fibers Whorls Nodules
+ + +
+++ +++ +++
+++++ +++ +++
Tissue section
ALEXANDER
AND DONOFF:
GLYCOSAMINOGLYCANS
HYPERTROPHIC
SCARS
173
FIG.
2. Three-year-old hypertrophic scar stained with Alcian blue 8GX (pH 1.O). Note the presence of nodular formations (N) in this tissue. x 160.
a
6 r
ALEXANDER
AND DONOFF:
GLYCOSAMINOGLYCANS
trophic scar and the normal subcutaneous tissue. This appeared to be composed of a thin band of collagen fibers with normal characteristics. Histochemical and enzyme methods revealed distinct differences within the dermal layers of the hypertrophic scar. The papillary layers stained intensely with colloidal iron and with Alcian blue. When treated with Streptomyces hyaluronidase, a generalized loss of stain occurred throughout the tissue, indicating the presence of hyaluranic acid prior to enzyme treatment. When treated with chondroitinase AC, the staining reaction was reduced further. After treatment with chondroitinase ABC, Alcian blue (pH 1.0) staining was abolished, indicating the loss of dermatan sulfate. The other staining methods were consistent with these results. The papillary collagen was unaffected by these enzyme methods and stained prominantly with Van Gieson’s technique. The reticular layer reacted similarly when treated with Streptomyces hyaluronidase. A generalized loss of staining was noted throughout the tissue after exposure to enzyme. The whorl-like and nodular formations stained intensely with Alcian blue (pH 1.O)and colloidal iron methods, indicating the presence of sulfated material within these areas. Collagen was always strongly associated with this sulfated material, particularly in the nodular formations. Differences were observed between the parallel, whorl-like, and nodular areas of the reticular layer upon enzyme treatment. Chondroitinase AC and chondroitinase ABC resulted in a similar loss of staining in the whorl-like and nodular formations when compared to controls, indicating an absence of dermatan sulfate in these areas (Figs 3 and 4). The parallel arrangements of tissue reacted positively with chondroitinase ABC, indicating the presence of dermatan sulfate (Figs. 5 and 6). No hyaluronidase activity was demonstrated. Substrate films of hyaluronic acid stained metachromatically with toluidine
HYPERTROPHIC
SCARS
175
blue, indicating the absence of enzyme activity in the parallel, whorl-like, and nodular areas of the scars. DISCUSSION
The glycosaminoglycans form a part of the structural framework of normal wounds [l, 3,6-g]. Linares et al. [ll] described the formation of whorls and nodules of collagen in hypertrophic scars. Increased amounts of chondroitin-4-sulfate have been localized in the nodular areas 118, 191while increased collagen has also been demonstrated in this tissue [9]. Hypertrophic scar is composed of two general presumptive dermal components: the papillary and reticular layers. The papillary layer seems to resemble normal papillary dermis in response to enzyme histochemistry and staining methods. The reticular layer can be subdivided into three distinct areas of development: (1) parallel orientation of collagen and glycosaminoglycans; (2) a whorl-like arrangement; and (3) a nodular arrangement. Since these patterns coexist within the same tissue, it appears that the hypertrophic scar is in a state of constant flux between immature and mature stages or that this tissue represents various degrees of development during healing. These stages have been previously categorized 1111. Whorl-like and nodular formations represent immature tissue, and parallel arrangements of collagen and glycosaminoglycans are associated with tissue maturity. Treatment of tissues with chondroitinase ABC demonstrated the presence of dermatan sulfate only in areas that showed parallel fiber arrangement. Low levels of dermatan sulfate have been observed in other developmental tissues [ 131. Chondroitinase ABC was ineffective in removing additional tissue stain in other areas of the hypertrophic scar and was comparable to chondroitinase AC in its activity in these areas. The whorl-like and nodular arrangements were susceptible to chondroitinase AC
176
JOURNAL
OF SURGICAL
RESEARCH:
VOL. 28, NO. 2, FEBRUARY
1980
ALEXANDER
AND DONOFF:
GLYCOSAMINOGLYCANS
HYPERTROPHIC
SCARS
177
178
JOURNAL
OF SURGICAL
RESEARCH:
VOL. 28, NO. 2, FEBRUARY
1980
ALEXANDER
AND DONOFF:
GLYCOSAMINOGLYCANS
HYPERTROPHIC
SCARS
179
180
JOURNAL OF SURGICAL RESEARCH: VOL. 28, NO. 2, FEBRUARY
digestion, indicating the presence of chondroitin-C and/or -6-sulfate. This result was consistent with previous findings [ 181.Treatment with Streptomyces hyaluronidase, an enzyme specific for hyaluronic acid 1161, caused a generalized removal of tissue stain in all areas of the hypertrophic scar. Intrinsic wound hyaluronidase has been demonstrated in normal granulation tissue [2], which is capable of degrading hyaluronic acid and chondroitin-4-sulfate [4]. No enzyme activity was found in hypertrophic scar, which may explain the generalized action of Streptomyces hyaluronidase upon the tissue. Following the initial trauma to the tissue and prior to biopsy, wound enzyme may have been present in, and responsible for, localized organization and maturation, since the presence of hyaluronidase has been associated with differentiation in other systems 117, 211. Reports of increased nodular chondroitin-4-sulfate [ 181 may indicate the absence or abnormality of localized wound enzyme activity in this area. In conclusion, the hypertrophic scar appears to be composed of varying levels of tissue maturity. The low levels or absence of dermatan sulfate indicate the developmental immaturity of this new tissue. Consistent with these findings, Donoff and Burke 151have demonstrated low levels of dermatan sulfate in the blood vessels of hypertrophic scars when compared to mature scar. These observations indicate that the hypertrophic scar is quite different qualitatively from normal skin or mature scar tissue, particularly in the disorganized areas. ACKNOWLEDGMENTS This work was supported by United States Public Health Service-National Institutes of Dental Research Training Grant 570lDE0012-14 and National Institutes of Health Bum Center Grant GM217000.
REFERENCES 1. Ahmad, M. Glucosamine and hydroxyproline content of granulation tissue on different days of wound healing in white albino rats. Ann. Biochem. Exp. Med. 21: 295, 1961.
1980
2. Alexander, S. A., and Donoff, R. B. Localization ofwound hyaluronidase. J. Dent. Res. 57: 93,1978. 3. Bentley, J. P. Rate of chondroitin sulfate formation in wound healing. Ann. Surg. 165: 186, 1967. 4. Bertolami, C. N., and Donoff, R. B. Identification and purification of a mammalian wound hyaluronidase. J. Dent. Res. 57: 96, 1978. ,5. Donoff, R. B., and Burke, J. F. Abnormality of hypertrophic scar blood vessels. /. Surg. Res. 25: 251, 1978. 6. Dorner, R. W. Glycosaminoglycans of regenerating tendon. Arthritis Rheum. 10: 275, 1967. 7. Dunphy, J. E., and Udupa, K. N. Chemical and histochemical sequences in the normal healing wound. N. Engl. J. Med. 253: 847, 1955. 8. Flint, M. Interrelationships of mucopolysaccharide and collagen in connective tissue remodeling. J. Embryol. Exp. Morphol. 27: 481, 1972. 9. Kischer, C. W., and Shetlar, M. R. Collagen and mucopolysaccharides in the hypertrophic scar. Connect. Tissue Res. 2: 205, 1974. 10. Lev, R., and Spicer, S. S. Specific staining of sulphate groups with Alcian blue at low pH. J. Histochem. Cytochem. 12: 309, 1964. 11. Linares, H. A., Kischer, C. W., Dobrkovsky, M., and Larson, D. L. The histotypic organization of the hypertrophic scar in humans. J. Invest. Dermatol. 59: 323, 1972. 12. Linares, H. A., and Larson, D. L. Proteoglycans and collagenase in hypertrophic scar formation. Plast. Reconstr. Surg. 62: 589, 1978. 13. Loewi, G., and Meyer, K. The acid mucopolysaccharides of embryonic skin. Biochim. Biophys. Acta 27: 453, 1958. 14 Luna, L. G. Manual of Histologic Staining Methods of the Armed Forces Institute
of Pathol-
ogy, 3rd ed. New York: McGraw-Hill, 1968.P. 79. 15. McCombs, H. L., and White, H. J. Histochemistry of hyaluronidase. Amer. J. C/in. Pathol. 49: 68, 1968. 16. Ohya, T., and Kaneko, Y. Novel hyaluronidase from Streptomyces. Biochim. Biophys. Acta l998: 607, 1970. 17. Polansky, J. R., Toole, B. P., and Gross, J. Brain hyaluronidase: Changes in activity during chick development. Science 183: 862, 1974. 18. Shetlar, M. R., Shetlar, C. L., and Linares, H. A. The hypertrophic scar: Location of glycosaminoglycans within scars. Burns 4: 14, 1977. 19. Shetlar, M. R., Shetlar, C. L., Chien, S. F., Linares, H. A., Dobrkovsky, M., and Larson, D. L. The hypertrophic scar. Hexosamine containing components of burn scars. Proc. Sot. Exp. Biol. Med. 139: 544, 1972. 20. Spicer, S. S., Horn, R. G., and Leppi, J. J. Histochemistry of connective tissue mucopolysaccharides. In D. E. Smith and B. M. Williams (Eds.), The Connective Tissue. Baltimore: Williams & Wilkins, 1967. P. 251.
ALEXANDER
AND DONOFF: GLYCOSAMINOGLYCANS
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