Characterization of the collagen of human hypertrophic and normal scars

Biochimiea et Biophysica Acta, 405 (1975) 412--421

© Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands BBA 37164 C H A R A C T E R I Z A T I O N OF T H E C O L L A G E N OF H U M A N H Y P E R T R O P H I C A N D N O R M A L SCARS *

A. J. BAILEYa, S. BAZINb, T. J. SIMS a, M. LE LOUSb, C. NICOLETIS c and A. DELAUNAY b aAgricultural Research Council, Meat Research Institute, Langford, Bristol (U.K.), bUnitd de Recherches de G(ndtique M(dicale, Hopital des Enfants Malades, Paris XV, and cCentre de Etudes et de Recherches sur les Maladies de la Cicatrisation, Hopital d'Ivry 94230, Ivry (France)

(Received March 28th, 1975)

SUMMARY The collagen produced in response to an injury of human skin is initially stabilized by a cross-link derived from hydroxyallysine, and characteristic of embryonic skin. In normal healing there is a change over with time to the cross-link derived from allysine, which is typical of young skin collagen. In contrast, hypertrophic scars fail to follow the time-related changes of normal skin, but retain the characteristics of embryonic collagen, indicating a continued rapid turnover of the collagen. This is further supported by the high proportion of the embryonic Type III collagen present in hypertrophic scars.

INTRODUCTION The large amounts of granulation tissue synthesised in response to skin injury are normally resorbed and the scar tissue reverts to a composition and structure similar to that of the original tissue [1]. In contrast, some individuals, particularly after burns or deep trauma, form excess scar tissue in the middle and deep layers of the dermis; such hypertrophic scars are especially frequent in negroes and some caucasians who can develop these abnormal scars after any kind of wound. Bazin et al. [2] have shown that the biochemical composition of these hypertrophic scars reveals a similarity, based on the glycosaminoglycan content, to newly formed granulation tissue even in scars 10-15 years old. Other workers [3, 4] have confirmed an increased chondroitin 4-sulphate content over normal skin. Evidence for increased collagen synthesis has been based on high solubility values [5, 6] and on high proline hydroxylase levels [7], whilst increased or normal [6] neutral collagenase levels indicate that catabolism is not inhibited. It is clear that to understand wound healing adequately as many parameters as possible must be investigated. Studies on the nature of the collagen in scars are

* A brief account of part of this work was presented at the 1st International Symposium on Wound Healing, Rotterdam, April 1974.

413 just beginning [2, 9] and the chemistry of the cross-links has not yet been thoroughly investigated. Although the presence of dihydroxylysinonorleucine has previously been reported in borohydride-reduced granulation tissue from early wounds in guinea pigs [10], no time-related changes were reported. In this paper we present evidence that there is a change over in the nature of the cross-links with time in normal human scar tissue, similar to that occurring in early post-natal growth l11] followed by normal maturation. In contrast, no such maturation process occurred in the hypertrophic scars, the collagen retaining the characteristics of embryonic collagen. MATERIALS AND METHODS All samples of human tissue were excised during operation for plastic surgery. The specimens were immediately placed in refrigerated containers and maintained at --20 °C until required. Special care was exercised to separate the scar tissue from adjacent normal tissue. Age and site matched control tissues were obtained whenever possible. Water content. The water content of the tissue was determined by drying at 100 °C to a constant weight. Total collagen content. The total collagen content of the tissue was determined from the collagen extracted by hot 5 ~ trichloroacetic acid [12]. Insoluble collagen. The proportion of insoluble collagen was determined by gelatinization, in hot 5 ~ trichloroacetic acid, of the collagen remaining in the homogenates following exhaustive extraction with 0.066 M phosphate buffer, pH 7.8, and then with 0.10 M citrate buffer, pH 3.5. Reducible cross-links. The nature of the reducible cross-links was determined by reaction with tritiated KBH4 and subsequent separation of the components in the acid hydrolysate on ion-exchange columns using volatile buffers. The tritium radioactive components were determined with Brays solution using a Packard Scintillation counter as described previously [13]. Confirmation of the identity of the reduced cross-links was achieved by comparison with authentic standards using the extended basic columns of the Beckman amino acid analyzer [13]. Periodate oxidation of hydroxylysinonorleucine from scar tissue. The hydroxylysinonorleucine isolated from 10-month- and 10-year-old hypertrophic scar tissue was subjected to a Smith degradation [14]. The cross-link in pH 5.3 citrate buffer (1 ml) was treated with 0.01 M NaIO4 at room temperature for 5 min. The reaction was stopped and the products reduced by adding 3 M NaOH and KBH4 (2.5 mg). After 30 min the solution was adjusted to pH 2 by the addition of 2 M HC1 and the solution analysed for [aH]proline and [3H]lysine on the Locarte amino acid analyser. Types of collagen. The homogenized tissue samples were solubilized by digestion with pepsin and the collagen types I and III separated by fractional precipitation according to the procedure of Chung and Miller [15] and Epstein [16]. Briefly, the tissues were extracted with 1 M NaCl, then 0.5 M acetic acid and the insoluble residue incubated with pepsin at a substrate/enzyme ratio of 10:1 for 24 h at 5 °C. The solubilized collagen was then subjected to fractional precipitation to obtain type III at 1.8 M and type I at 2.5 M NaCl, 0.05 M Tris.HCl. Sodium dodecyl sulphate acrylamide gel electrophoresis. The Type I and Type III collagen precipitates were redissolved, denatured in 2 ~ sodium dodecyl sulphate

414 at 38 °C and analyzed for a-chain composition by acrylamide gel electrophoresis using the flat-bed technique previously described [17]. Identification of elastin. Samples of the scar tissue were extracted with 2 M NaCI at 90 °C for 2 h and the insoluble residue weighed. The purity of the elastin residue was determined by the desmosine and isodesmosine content [18] of an acid hydrolysate analysed on a Jeol amino acid analyzer. RESULTS

Water content, total collagen, insoluble collagen The ages of the patients ranged from 6 to 47 years. No correlation could be noted between the age of the patients and the age of the scars. The results of these analyses were therefore combined within specific groups depending on the age of the scar irrespective of the age of the donor. The hypertrophic scars were divided into six groups and the normal scars into two groups (Table I). TABLE I COMPARISON OF WATER CONTENT, TOTAL COLLAGEN, AND INSOLUBLE COLLAGEN IN HYPERTROPHIC SCARS, NORMAL SCARS, AND NORMAL SKIN Tissue Group (age in years) Water (~, w) Total collagen (rag hydroxyproline/g dry tissue) Insoluble collagen (percent (w) of total collagen)

Hypertrophic scars

Normal scars

Normal skin

0.25-0.5 0.8-1.25 1.5-2.5 3-5 75.8 75.8 74.8 72.4

6-8 10-15 0.25-2 72.8 72.3 73.8

3-10 65.8

65.2

74.8

76.3

77.6

76.8

78.2 75.3

82.5

69.6

72.8

36.3

40.6

38.8

45.7

34.3 40.7

46.1

36

50.5

From these results it can be seen that the water content of hypertrophic scars decreases little with ageing of the scars, whilst in normal scars the value decreases to a water content comparable with normal skin. The collagen content of hypertrophic scars is not significantly different from normal skin. In contrast the collagen content of normal scars is initially higher than normal skin but reverts to the same level as normal skin in old scars. Similar results for normal scars have been reported by other workers [19-21]. As might have been expected from a comparison of newly formed collagen and that from mature skin, the proportion of soluble collagen (i.e. difference between total and insoluble collagen) was higher in hypertrophic and normal scars than the normal skin. Similar observations have been reported by other workers [5, 6].

Reducible cross-links Individual scar samples were analysed for cross-links covering a wide range in both the age of the subject and of the scar (Table II).

415 TABLE II DISTRIBUTION OF SAMPLES OF SCAR TISSUE FOR CROSS-LINK STUDIES WITH RESPECT TO AGE OF THE SCAR AND OF THE DONOR Age of donor (years) 14 15 16 19 20 21 25 26 27 28 30 36 47 56 62

Age of scar (years) Normal scar Hypertrophic scar 3 1.5 0.75 0.25 3 10 5 4 0.25 10 2 I 1 3 2

(i) Normal scar. The major reduced cross-link in early wounds is dihydroxylysinonorleucine (Lys(OH)2-Nle) but after a few months the major reduced components are hydroxylysinonorleucine (Lys(OH)-Nle) and histidino hydroxymerodesmosine (His-Mdes(OH)) (Figs 1 and 3). Other components identified were the hexosyl-lysines normally present in old dermal collagen, and reduced desmosine from elastin. (ii) Hypertrophic scars. The major reduced cross-links present in these scars are Lys(OH)2-Nle and Lys(OH)-Nle. Initially the proportion of the Lys(OH)2-Nle is higher than Lys(OH)-Nle (about 3:1) but rapidly plateaus to a ratio of about 1:1.3 (Figs 2 and 3).

Periodate oxidation of cross-links The proportion of Lys(OH)-Nle in the keto form, based on the yield of tritriated proline after a Smith degradation was determined as 18 or 14~o for the 10year-old and 10-month-old hypertrophic scars, respectively. These values are significantly higher than normal dermis, in which only 3-7 ~ of the Lys(OH)-Nle is in the ketoform, based on the analysis of six subjects in the age range 12-18 years. Type of collagen Hypertrophic scar. The fractional salt precipitation of the pepsin solubilized collagen yields a precipitate at 1.8 M NaC1 and a second precipitate at 2.5 M NaCI. These two precipitates were shown to be Type III and Type I collagen, respectively, by sodium dodecyl sulphate gel electrophoresis, CM-cellulose chromatography (Fig. 4) and amino acid analyses (Table III). Based on the weights of the precipitates obtained a ratio of Type I to Type III of 2:1 was obtained. The amount of Type III present in hypertrophic scar is therefore slightly higher than normal human dermis

416 30

2 MONTHNORMALSCAR ...Lys[oh)2-Nle

2'0

His-Mdes(oh 1'0

J b

5 YEARNORMALSCAR

20

•. L y s

(oh)

-Nle

3H Activity (cpm x 103)

His -Males(oil 1'0

2'0

~.~

C .

.

.

.

5 YEAR NORMALSKIN .

.

_

10

PHE

TYR

HYL

LYS

Fig. l. Elution chromatogram showing the radioactive components from an acid hydrolysate of collagen reduced with tritiated borohydride and separated on a Technicon auto analyzer using volatile buffers, a, 2-month-old normal scar; b, 5-year-old normal scar; c, normal skin collagen from a 5year-old subject.

o f the same age (ratio 3.5:1), but n o t as high as t h a t o f e m b r y o n i c h u m a n skirt (ratio 1:1).

Elastin content The elastin contents o f n o r m a l skin, m a t u r e scar, a n d h y p e r t r o p h i c scar were d e t e r m i n e d as 5, 2 and less t h a n 0.1 ~ . As in the case o f n o r m a l skin the p r o p o r t i o n o f elastin in n o r m a l scars increases with age. DISCUSSION

Nature of the cross-links I n i t i a l l y the collagen o f b o t h the h y p e r t r o p h i c scar a n d the n o r m a l scar possess d i h y d r o x y l y s i n o n o r l e u c i n e as the m a j o r reduced cross-link, but after a few m o n t h s there is an a p p r o x i m a t e l y equal p r o p o r t i o n o f m o n o h y d r o x y l y s i n o n o r l e u c i n e . Subsequently the two types o f scar follow a different course. T h e 1:1 ratio o f

417 3'0

2 MONTHHYPERTROPHICSCAR ...I..,ys(oh)2-Nle

2'0

1-0

His-Mdes(oh

3H Activity (cpm x 10-3) 30

5 YEAR HYPERTROPHICSCAR

b

•. Lys(oh)- Hie 2'0

...'Lys (oh)- Nle His-Mdes(oh',

PHE

TYR

HYL

LYS

Fig. 2. Elution chromatograph of acid hydrolysates of hypertrophic scar collagen reduced with tritiated borohydride, a, 2-month-old hypertrophic scar; b, 5-year-old hypertrophic scar.

_J Z .J i

~'0

i

i

i

i

i

! 3-0

.d Z .J i

-r 20 O

•

Hypertrophic scar

O

~,

1'0

\

% %o,,,,,~

..Normal scar

I

I

2

4

O--~

I ~ m m ' ~ Q ~ ,

6

8

O~

10

Age of s c a r ( y r s )

Fig. 3. Changing ratio of Lys(OH)z-Nle to Lys(OH)-Nle with age of the scar tissue, irrespective of the age of the donor.

the two cross-links is retained in the hypertrophic scar even after 10 years. Comparison o f the cross-link pattern with the reducible components in n o r m a l h u m a n skin reveals an absence o f the hexosyl-lysines even in 10-year-old scars. In addition the absence o f reduced desmosine indicates that hypertrophic scars synthesise little, if any, elastin. These results clearly confirm that new collagen is being continually laid d o w n in old hypertrophic scars. These results are analogous to those found in sponge implants in rats [22], there being a constant turnover o f the collagen embedded in the poly-

418 (a)

E

1.c

c

5

®

Q~ o .13

<

5OO

250 Effluent volume (ml)

(b) (---FY~

I ppt-"9

(-

i'Y]~:~~ I Z I

peaks

1

2

pp%

peaks

3

/l

5

tt *

5 ~

5

ot t 6,,,) m ~ (')

¢.2(') .

.

.

. %,

* without

mercaptoeth~nol

Fig. 4. (a) Comparison of the elution profiles of Type I and Type III collagen, obtained by fractional precipitation, after separation by CM-cellulose chromatography. -- -- , type I precipitated at 2.5 M N aC1; , Type III precipitated at 1.8 M NaCI. (b) Electrophoresis in sodium dodecyl sulphateacrylamide gels (6 %) of the components under the peaks from CM-cellulose chromatography. All samples except tracks 6 and 7 were reduced with 2 % mercaptoethanol prior to electrophoresis. (i) Tracks 1,2, 3 : Type I collagen, peaks 1, 2 and 3 show a 1 (I), fin and a 2 (I), respectively. (ii) Tracks 4 and 5 : Type III collagen, peaks 4 and 5 show a 1 (I) and a 1 (III), respectively. (iii) Tracks 6, 7, 8 : Type III collagen, peak 4 without mercaptoethanol shows c~ 1 (I); peak 5 without mercaptoethanol shows 7(III); peak 5 shows al(IIl). N o t e the slightly slower mobility of ctl(III) compared to al(1).

vinyl sponge. Sponge implants may therefore be good models for studying the turnover mechanism in hypertrophic scars. However, in the case of the hypertrophic scar the "trigger" causing the continued rapid turnover is not known. In contrast, the cross-link pattern of normal scar gradually reverts to that of normal young dermal collagen, i.e. there is a virtual disappearance of Lys(OH)z-NIe and replacement by Lys(OH)-Nle and His-Mdes(OH).

419 TABLE III AMINO ACID COMPOSITIONS OF TYPE I AND TYPE III COLLAGEN EXTRACTED FROM HYPERTROPHIC SCARS

Hydroxyproline Aspartic Threonine Serine Glutamic Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Hydroxylysine Lysine Histidine Arginine Cysteine*

al(I)

al(lID

99 44 17 33 76 128 323 117 21 6 7 19 2 13 6 33 2 49 --

113 48 16 39 74 107 340 94 17 5 13 21 2 9 9 37 9 46 2

* Determined as cysteic acid after oxidation with performic acid.

A significant difference from the hypertrophic scar was the gradual increase in desmosine from elastin and hexosyl-lysines from collagen with age of the normal scar. These components are a feature of mature skin, thus supporting other evidence that normal scars slowly approach the composition of normal skin. This result was unexpected as regard to the elastin since histologically no elastin fibres can be detected in mature scar tissue; therefore, the biosynthesis of elastin was thought to be impaired in scars. However, the presence of elastin in mature normal scars was confirmed by the presence of desmosine and isodesmosine in a direct acid hydrolysate. During normal maturation of skin the proportion of reducible cross-links decreases such that they are virtually absent at about 20 years of age, and that of the reduced desmosines (from elastin) and hexosyl-lysines increases. Similarly, in normal scars the proportion of reduced desmosine and hexosyl-lysines increase and that of hydroxylysinonorleucine decreases, although insufficient specimens were available to accurately assess the rate of maturation of the scar compared to normal skin. The collagen of granulation tissue appears to be similar to that of embryonic collagen since an analogous change in the nature of the cross-links occurs from embryonic to early post-natal skin. The Lys(OH)2-Nle has virtually disappeared after a few months. Unfortunately insufficient specimens of scar tissue were available to make an accurate comparison of the two rates of changeover in the cross-links. Of course, it is possible that scar tissue in young skin may revert to normal skin, based on type of collagen and cross-links, at a faster rate than scar tissue in older animals. However, the point clearly demonstrated here is that there is a time-dependent changeover in normal scar to a cross-linking pattern comparable with that of normal skin.

420 In addition to the higher proportion of Lys(OH)z-Nle in early normal and hypertrophic scars a significant proportion of the Lys(OH)-Nle in old hypertrophic scars is also derived from hydroxyallysine, and therefore stabilized by undergoing the Amadori rearrangement [23]. The Lys(OH)-Nle of older scars is like normal dermis in being almost entirely derived from allysine and exists in vivo as an aldimine cross-link rather than as the keto form. Thus, extensive hydroxylation of the lysine residue in the non-helical telopeptide must take place in early scar tissue when rapid remodelling is taking place, at a moderate level in old hypertrophic scars where remodelling is still taking place, and virtually not at all in older normal scars where the turnover time is very long and comparable with normal tissue. It is noteworthy that the hydroxylysine content of granulation tissue collagens is not high, suggesting that the extra hydroxylation is confined to the telopeptide. Since this overall pattern is complicated by the post-natal changeover from Type III to Type I collagen the mechanism regulating the extent of hydroxylation is not clear. It is possible that it is simply related to a high lysyl hydroxylase activity in rapidly proliferating tissues and a low level in normal tissues, although it is possible that a more subtle mechanism is involved.

Type of collagen In a recent report by Epstein [16] it was demonstrated that there is a changeover during the latter part of pregnancy and early post-natal period of the type of collagen present in infant human dermis. The ratio of Type I to Type llI changed from 0.8 at 15 weeks foetus, to 3.6 at 3 months after birth. It was therefore considered of some importance to determine whether human scar granulation tissue contained both types of collagen and if a similar change in the ratio occurred. The difficulty of obtaining normal human scars at the early stage of development precluded the examination of this tissue. Further, scars of long standing may contain a small proportion of Type III but the possibility of contamination from normal skin cannot be excluded. However, previous studies have demonstrated the presence of a high proportion of Type III collagen in the granulation tissue of open wounds on the limbs of horses [24] and in acute granulation induced in rats by subcutaneous injection of turpentine (Bailey, A. J. and Bazin, S., unpublished). Effort was therefore concentrated on early (10 month) hypertrophic scar collagen. The presence of Type III collagen in this tissue indicated that synthesis of "embryonic" collagen was occurring. In the light of these findings it is noteworthy that in normal scars from guinea pig Type III collagen is not present [25]. The significance of this difference is not at present clear. Since fibroblasts in early wound healing and in granulation tissue have been shown to be immature fibroblasts developing a contractile apparatus which make them similar to smooth muscle cells [26] it is possible that the initial stimulus to produce collagen results in the formation of an embryonic collagen. Whether this represents a conversion in cell type or cell selection remains to be seen. In the case of hypertrophic scars this stimulus is maintained by some unknown mechanism whilst in normal scar the conditions gradually approach that of normal skin. It is obvious that the metabolic activity of the local fibroblasts is strictly controlled in normal scars. Such control has been disturbed in hypertrophic scars, as revealed by the present studies, the abnormal proportions of chrondroitin 4-sulphate [27] synthesised in this

421 tissue, a n d a p p a r e n t collagen-mucopolysaccharide interactions observed in the elect r o n microscope [28]. ACKNOWLEDGEMENT The authors t h a n k N. Avery and J. C. A l l a i n for their excellent technical assistance. REFERENCES 1 Bazin, S. and Delaunay, A. (1964) Ann. Inst. Pasteur 107, 163-172 2 Bazin, S., Bailey, A. J., Nicoletis, C. and Delaunay, A. (1974) Proc. 1st Int. Syrup. on Wound Healing, Rotterdam, in the press 3 Shetlar, M. R., Shetlar, C. L., Cheen, S., Linares, H. A., Dobrkovsky, M. and Larson, D. L. (1972) Proc. Soc. Exp. Biol. Med. 139, 544-547 4 Sibeleva, K. F., Zenkevich, G. D. and Laufer, A. L. (1965) Vopr. Med. Khim. 11, 55 5 Harris, E. D. and Sjoerdsma, A. (1966) (ii) Lancet 707-711 6 Shetlar, M. R., Dobrkovsky, M., Linares, H., Villarante, R., Shetlar, C. L. and Larson, D. L. (1971) Proc. Soc. Exp. Biol. Med. 138, 298-300 7 Kelman Cohen, I., Keiser, H. R. and Diegelman, R. F. (1974) Proc. 1st Int. Syrup. on Wound Healing, Rotterdam, in the press 8 Milsom, J. P. and Craig, R. D. P. (1973) Br. J. Dermatol. 89, 635-644 9 Berry, H. K. (1974) Proc. 1st Int. Syrup. on Wound Healing, Rotterdam, in the press 10 Forrest, T., Shuttleworth, A., Jackson, D. S. and Mechanic, G. L. (1972) Biochem. Biophys. Res. Commun. 46, 1776-1781 11 Bailey, A. J. and Robins, S. P. (1972) FEBS Lett. 21, 330--334 12 Fitch, S. M., Harkness, M. L. R. and Harkness, R. D. (1955) Nature 176, 163 13 Bailey, A. J., Peach, C. M. and Fowler, L. J. (1970) Biochem. J. 117, 819-831 14 Robins, S. P. and Bailey, A. J. (1975) Biochem. J., in the press 15 Chung, A. and Miller, E. J. (1974) Science 183, 1200-1201 16 Epstein, E. H. (1974) J. Biol. Chem. 244, 3225-3231 17 Sykes, B. C. and Bailey, A. J. (1971) Biochem. Biophys. Res. Commun. 43, 340-345 18 Partridge, S. M. (1973) in Biology of the Fibroblast (Kulonen, E. and Pikkarainen, J., eds), pp. 1340, Academic Press, New York 19 Dunphy, J. E. and Udupa, K. N. (1955) New Engl. J. Med. 253, 847-851 20 Grillo, H. C., Watts, G. T. and Gross, J. (1958) Ann. Surg. 148, 145-152 21 Peacock, E. E. (1962) Ann. Surg. 155, 251-257 22 Bailey, A. J., Bazin, S. and Delaunay, A. (1973) Biochim. Biophys. Acta 328, 383-390 23 Robins, S. P. and Bailey, A. J. (1973) FEBS Lett. 33, 167-171 24 Lindberg, K. A., Gunson, D. E., Sims, T. J. and Bailey, A. J. (1975) Biochem. J., in the press 25 Shuttleworth, C. A., Forrest, L. and Jackson, D. S. (1975) Biochim. Biophys. Acta 379, 207-216 26 Gabbiani, G., Hirschel, B. J., Ryan, G. B., Statkov, P. R. and Majno, G. (1972) J. Exp. Med. 135, 719-734 27 Bazin, S., Nicoletis, C. and Delaunay, A. (1973) in Biology of the Fibroblast (Kulonen, E. and Pikkarainen, J., eds), pp. 571-577, Academic Press, New York 28 Kischer, C. W. and Shetlar, M. R. (1974) Connect. Tissue Res. 2, 205-213