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
© 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
