BIOCHIMICA ET BIOPHYSICA ACTA

BBA 35806

INTERMOLECULAR CROSS-LINKING OF COLLAGEN IN HUMAN AND

GUINEA PIG SCAR TISSUE

681

L. F O R R E S T AND D. S. J A C K S O N

Department of Medical Biochemistry, Department of Physiology, University of Manchester Con- nective T, ssue Research Group, Lapworth Laboratories, Burlington Street, Manchester Mx3 9PL (Great Bmtain)

(Received October 8th, 197 o)

SUMMARY

I. An increase in the extent of intermolecular covalent cross-linking over a period from i to 4 weeks has been shown in collagen from the scar tissue of open splinted wounds in guinea pig skin.

2. No significant changes in the extent of cross-linking has been shown in the collagen from linear scars of human skin over a period from 4 to I2 weeks.

3. It is suggested that the long term gain in tensile strength cannot be corre- lated by change in extent of covalent cross-links in wound collagen.

INTRODUCTION

During the first 5 days of its existence, what little strength a primarily closed wound possesses is due to cellular adhesion. In the next phase, collagen fibres are laid down in the extra cellular space. During this phase the tensile strength of the wound rises steadily, but assumes no more than 3o % of its ultimate strength 1. There is no doubt that the steady increase in tensile strength is associated with the laying down of extracellular material and it may be due to: (a) an increase in the number of collagen fibres per unit volume of tissue, i.e. the density of collagen; (b) an increase in the extent of intermolecular covalent cross-linking within a fibril; (c) the establish- ment of collagen-ground substance interaction within the wound2, S. Some continuity must also be established between the fibres of the wound tissue and those of the adjacent tissue.

The productive phase comes to an indefinite end and is followed by a period of maturation. The tissue is markedly less cellular and vascular and within this period remodelling occurs. The tensile strength of the wound increases slowly but steadily. The collagen concentration during this time remains constant or may even fall 4 and thus cannot be invoked as responsible for the tensile strength increase. However, either or both of the factors (b) and (c) above may still be operating and

Biochim. Biophys. Acta, 229 (1971) 681-689

682 L. FORREST, D . S . JACKSON

in addition architectural organisation of the scar collagen fibres may occur. These suggestions have been discussed in detail previously 5.

In the present study the occurrence of intermolecular cross-links in the collagen of healing wounds was studied in two situations. In the first, the collagen of healing wounds was studied in two situations. In the first, the collagen was derived from splinted wounds made in guinea pig skin from I to 4 weeks old. In the second, the collagen was obtained from human skin scar tissue obtained flesh at post-mortem. In each case purified polymeric collagen was obtained and the occurrence of covalent intermolecular cross-links investigated by methods previously used on other tissues ~.

METHODS

Guinea pig tissue Mature female guinea pigs (weights 55o-65 o g) were anaesthetized with ether.

Bilateral circular skin wounds, one inch in diameter, were made in the animal's flanks. The tissue removed included the panniculus carnosus and the base of the wound was the loose areolar tissue which lies between the panniculus and the fascia of the underlying muscles. A small V-shaped niche was made in the perimeter of the wound and a suture passed through its edges. A perspex ring with an outer trough

inch. deep was placed in situ and by tying the suture the skin edges of the wound were firmly apposed to the trough. The suture comes to lie below the upper flange of the ring and is inaccessible to the animal's claws and teeth (Figs. I and 2). The

Fig. I. P h o t o g r a p h of r ing in sit,~. The scab cover ing the g ranu la t i on t i ssue can be seen w i t h m the ring.

Bioch~m. B~ophys. Acta, 2z9 (1971) 681-689

CROSS-LINKING OF COLLAGEN 683

1 2 3

/ i l l I I i / I I I I . ~ ' i 7 1 l / I / , ~ T I ~ T / I / I I 1 ~ 1 " ~ I 4

Fig. 2. Cross-section through the skin, showing the position of the ring i, epidermis; 2, dermis; 3, panniculus carnosus, 4, deep fascia and underlying muscle.

wound is sprayed with polybactrin and dressed with elastoplast. The wound granu- lates from the base upwards and scar tissue fills the cavity within the ring. The tissue so obtained was harvested at I, 2, 3 and 4 weeks. For each preparation tissue was pooled from 12 wounds. Infected wounds were discarded.

Human tissue The scars were obtained fresh at post-mortem from cadaver skin by dissection

and treated as below.

Extraction methods The a-amylase method of obtaining polymeric collagen, successfully applied

to other tissues 7, was found on preliminary experiments to be superior to the other available method s using EDTA as regards the purity of the end-product and was, therefore, the method of choice.

The tissue once obtained was immediately chopped into small pieces, frozen in liquid nitrogen and homogenized in a stainless steel mill. I t was extracted initially with I.O M NaC1 buffered to pH 7.4 with 0.o2 M NaH2PO4-Na,HP04 at 4 ° for 24 h with an extractant/homogenate ratio of approx. 50 :I (v/w). This was repeated three times. The residue was extracted for a further 24 h at 4 ° with o.I M acetic acid. The residue from this was dialysed against several changes of distilled water in the cold and finally subjected to the a-amylase treatment. In this, collagen is suspended in a solution of bacterial a-amylase (pH 5.4) for 9 ° h at 4 °, subsequently washed in saline and distilled water and finally dispersed in acetic acid. Electron microscopy of the fibrils so prepared revealed the characteristic 640 A banding.

Analysis of the polymeric collagen (Table I) Amino acid analysis A sample of the material was hydrolysed in 6 M HC1 under nitrogen in a sealed

tube for 24 h at lO5 °. The HC1 was subsequently removed by rotary evaporation and the amino acids quanti tated on a Technicon 2i-h apparatus.

Hydroxyproline A sample of material was hydrolysed under the same conditions as above and

the hydroxyproline content estimated by the method of WOESSNER 9. Hexosamines A sample of material was hydrolysed in 8 M HC1 for 3 h at 95 ° under nitrogen

in a sealed tube 10. The HC1 was removed by rotary evaporation at 3 °o and the es- timation done by the method of JOHANSEN et al. n.

Methods of degradation of polymeric collagen Treatment with aminothiols The polymeric collagen was treated with 0.5 M cysteamine containing 0.5 M

Bwch,m. Biophys. Acta, 229 (1971) 681-689

6 8 4 L. F O R R E S T , D. S. J A C K S O N

T A B L E I

AMINO ACID AND HEKOSAMINE CONTENT OF THE POLYMERIC COLLAGENS

The amino acid contents of the polymeric collagen from wounds of different maturity had the same distribution. The hexosamine content for the z weeks maturity tissue was the highest found, but still represents a low degree of contamination of the collagen with non-collagenous material.

Residues per ~ooo

Guinea p~g Human scar granulation polymerw t~ssue collagen (2 weeks (7 weeks maturity) maturity)

H y p 99 .0 97. I Asp 53.1 50 .8 Thr 24.2 19.1 Ser 5 o .2 37 .3 G l u 79 .7 76-3 Pro lOO. 4 lO8. 5 G l y 3 1 2 . 4 330 .7 Ala 94 .7 I oo .2 Val 22.1 26. 4 C y s - - - - - M e t - - - - I l e 13 .6 13.6 Leu 3o .o z8. I T y r 2 .8 2.2 Phe 18.9 15. 4 H y l 8. 4 8 i L y s 32 .9 27.4 His 7 .8 7.3 Arg 49 .5 51.3 Hexosamines I. 5 o. 3

NaC1 buffered with phosphate to pH 7 for 3 days at 4 ° (ref. 12). At the end of this time the supernatant was separated by centrifugation and dialysed exhaustively against distilled water to remove the cysteamine and salt. The residue was washed with cold distilled water.

Treatment with pepsin The polymeric collagen was redispersed in o.I M acetic acid and pepsin added

so that the enzyme/substrate ratio was approx. 1:50. Digestion was allowed to proceed for 7 days at room temperature with gentle shaking. At the end of this time the dispersion was neutralized and the insoluble material centrifuged down. The super- natant was kept as the cold soluble fraction and the residue washed.

Treatment with cold NaOH (refs. I3, 6) Polymeric collagen was homogenized quickly in 20 ml of pre-cooled 2 M NaOH

and shaken gently for 6 days at 4 °. The solution was then neutralized and the inso- luble residue centrifuged down. The supernatant was again kept as the cold soluble fraction and the residue washed.

Each of the degradation procedures applied to the guinea pig tissue was carried out as a single stage process. The degradation procedure applied to human scar tissue was carried out sequentially as shown in Fig. 3.

Biochim. Biophys. Acta, 2 2 9 (1971) 6 8 1 - 6 8 9

CROSS-LINKING OF COLLAGEN 685

I r . . . . . . . . Residue

Smal l aliquot i L 65 °, 1 h, 100+{, I pH 7 I

t i

I I ttot Remdue

soluble

C y s t e a m m e (0.5 M) 3 days, 4 °

Main aliquot 100'~ p e p s i n , E/S: 1 0 . 1 M a c e t l c acid 7 days

N e u t r a h z a t l o n

1 Cold soluble 0%

50.

I I Residue Cold soluble l l00-x] +~ x'(

r" . . . . . I Mam ahquot t 2 M NaOH,

Smal l ahquot 6 days, 4 ° 65 °, 1 h, [ lO0-xy(

/ c , [ 1 0 0 - x ] ~

Hot soluble R e s i d u e

N e u t r a l i z a t i o n I I

R e s i d u e Cold soluble

I y % I

65 °, lh , I [100-x-Y|~ [100-x-yl '* pH 7 [ F u r t h e r d e g r a d a t i o n i i

Hot soluble Residue

Fig. 3. Following each chemical or enzymat i c degration, the residue was divided into two un- equal parts . A small a l iquot was taken for thermal denaturat ion and the main al iquot subjected to a further degradation. The percentages g iven in the table are those that would obtain had all the residue from a particular degradat ion been subsequent ly treated either by (a) thermal dena- turat ion or (b) by a further degradat ive process. Reference to the flow diagram will make this clear (Fig. i). The fact that no hydroxyprol ine was found in the cys teamine cold soluble fract ion is included.

Thermal denaturation Each degradation procedure provided a soluble fraction and an insoluble

residue. This residue was subjected to thermal denaturation during which a part of the collagen was gelatinized. In the case of guinea pig collagen the gelatinization was carried out at two temperatures, 60 and IOO °. Human collagen was gelatinized only at 65 °. The quantity of collagen in each fraction was determined by its hydroxyproline content.

RESULTS

Guinea pig collagen The percentage of polymeric collagen solubilized by heating at 60 ° decreases

with increasing maturity of the tissue from 42.9% at I week to 19.1#/0 at 4 weeks. Similarly, the percentage solubilized at IOO ° decreases also but the rate of the de- crease is less, falling from 63.3#/0 at I week to 52.6°/0 at 4 weeks. A similar pattern is seen after the cysteamine treatment. The percentage gelatinized falls from 51.5% at I week to 21.3°/0 at 4 weeks on thermally denaturing at 6o ° and again a slower decrease in solubilization is seen on thermally denaturing at IOO °. In each case the percentage solubilized is greater after cysteamine treatment but the difference falls within the spread of the individual result and need not be significant. After pepsin

Biochim. Biophys. Acre, 229 (1971) 681-689

686 L. F O R R E S T , D, S. J A C K S O N

T A B L E I I

EXTENT OF SOLUBILIZATION OF GUINEA PIG SCAR POLYMERIC COLLAGEN BY VARIOUS TREATMENTS

F i g u r e s (as p e r c e n t a g e s ) a r e t h e m e a n s o f t w o o b s e r v a t i o n s . T h e m a x i m u m d i f f e r e n c e b e t w e e n t w o o b s e r v a t i o n s w a s 5 . 6 % .

Treatment Age (weeks) : z 2 3 4

G e l a t i n i z a t l o n a t 6o °, I h G e l a t i n i z a t i o n a t i o o °, i h G e l a t i n i z a t i o n a t 6o °, I h ; a f t e r c y s t e a m m e G e l a t i n i z a t i o n a t IOO °, I h ; a f t e r c y s t e a m i n e G e l a t i n i z a t i o n a t IOO °, I h ; a f t e r p e p s i n

t r e a t m e n t

42 .9 26 .9 - - I9. I 63 .3 57 .9 - - 52 .6 51.5 33.7 21.3 65 .8 58 5 - - 53 .4

98 .0 96 .o 92 .o 6o .5

treatment, for the first 3 weeks nearly all the polymeric collagen is solubilized by thermal denaturation, but in the 4th week this falls sharply to only 60.5 % (Table II).

Human scar collagen Surprisingly, no changes in degree of solubilization with time are seen. The

scar maturi ty extends from 4 to 12 weeks and the figures in any fraction are con- sistent. None of the collagen is directly solubilized by cysteamine, but after heat treatment 14-15% of the residue goes into solution. Pepsin directly solubilizes roughly IO % of the cysteamine-treated residue and about 35 % of this fraction goes into solution on thermal denaturation. Pepsin treatment followed by the alkali treatment solubilizes nearly 95% in the cold, but even on subsequent thermal denaturation there is a small but persistent residue (Table III).

" F A B L E I l I

EXTENT OF SOLUBILIZATION OF HUMAN SCAR POLYMERIC COLLAGEN BY MULTIPLE STAGE D E P O L Y -

M E R I Z A T I O N

F i g u r e s a r e g i v e n as p e r c e n t a g e s .

Treatment

C y s t e a m i n e C o l d s o l u b l e H o t s o l u b l e R e s i d u e

P e p s i n Co ld s o l u b l e H o t s o l u b l e R e s i d u e

N a O H C o l d s o l u b l e H o t s o l u b l e R e s i d u e

Patzent. A C.K. L.D. B .M. Scar maturity (weeks) : 4 7 ±o z2 S*te of scar: Rzght Thorax Thorax Right

arm ~lmc fossa

o o o .o o .o o .o 14.1 14.3 14.9 15.o 85 .9 85 .7 85.1 85 .o

7 .0 13.2 11-3 9 .4 34 .5 35 .8 37 .o 36"5 58 .5 5I ,O 51 .7 54.1

87.5 79 ,0 68 .5 86.2 2 .6 3 ,0 3.1 2 .0 2.9 4 .8 7.1 2.4

Biochim. Biophys. Acta, 229 (1971) 6 8 1 - 6 8 9

CROSS-LINKIAG OF COLLAGEN 687

DISCUSSION

In soft tissue collagens the process of formation of covalent cross-links seems to follow a particular pattern. Initially a lysine residue in the N-terminal telopeptide is oxidatively deaminated to yield allysine. This aldehyde then forms an aldol condensation product with an adjacent allysine to form an intramolecular cross- link 14. Recently two intermolecular cross-links have been identified through their sodium borohydride reduced derivatives 15.

One of these is a Schiff-base which could be derived from the allysine residue of one chain reacting with a hydroxylysine residue on an adjacent chain. The other is an aldol condensation product of allysine and hydroxyallysine. Evidence has also been provided for the existence of a third type of cross-link 15, and recently the re- duction product of a lysine-derived Schiff-base lysinonorleucine has been isolated TM. The Schiff-base is labile to dilute acids, to aminothiols ~7, such as cysteamine and to heat TM. Heat denaturation destroys the helical structure of the collagen molecule and, if the temperature is sufficiently high, cleaves the Schiff-base bond thus solubilizing a fraction of intact collagen as a high molecular weight gelatin. Unpublished studies in this department suggest that the labile bond is not broken below the shrinkage temperature and hence collagen going into solution at 60 ° was probably not covalently cross-linked originally.

Pepsin hydrolyses a segment of the collagen polypeptide chain in the N-ter- minal telopeptide region. Since the covalent cross-links discussed above occupy this region they are simultaneously lost irrespective of their nature. I f heat denaturation is performed on the residue after pepsin hydrolysis then all the collagen which is exclusively cross-linked by bonds in the N-terminal region will be gelatinized. Alkali- labile links have been describedS, 13 but the nature of these has not been determined.

Using this information, an at tempt has been made to quantitate the type of cross-link in wound tissue and the changes that take place with time. The amount of collagen lost by neutral salt and acetic acid extraction is small and is not considered here.

Guinea pig tissue At I week the explosive development of granulation tissue in a wound of this

type has just begun. Practically all the cross-links of the insoluble collagen are contained in the telo-peptide region and are removed by pepsin. Almost one half (42.9%) of the collagen is not covalently cross-linked. 20.4-23.9% is cross-linked by labile bonds presumably of the Schiff-base type. These are labile to both IOO ° gelatinization and to cysteamine. However, cysteamine appears to be less effective than heat since gelatinization at 6o ° is increased by only 8.6% after cysteamine treatment compared to 20.4% increase due to gelatinization at IOO °. Indeed, the former difference is only slightly greater than the maximum error obtained for any individual result and any interpretation must be tentative.

Between I and 4 weeks the proportion of uncross-linked collagen decreases markedly. However, the proportion of labile covalent cross-links decreases much more slowly since by the 4th week 52.6% is gelatinized after IOO ° compared to 63.3% at the first week. Up to the 3rd week the effect of pepsin decreases only slightly, but by the 4th week only 60.5% is gelatinized after pepsin treatment compared with

Biochim. Biophys. Acta, 229 (197 I) 681-680

688 L. FORREST, D. S. JACKSON

980/0 at week one. This is in marked contrast to the effect of pepsin on mature guinea pig skin collagen where virtually lOO% is solubilized in the cold.

I t appears then that the wound collagen cross-links rapidly and to a marked extent during a period which coincides with the fibro-blastic phase. However, in rat skin, the wound achieves no more than 30% of its ultimate strength during this period is. More recently DOUGLAS et al. 4 working with guinea pig skin found a linear increase in wound strength throughout a period of IOO days and even at 60 days the wound strength was only lO-15% of values for intact skin. The rate of increase of tensile strength cannot, therefore, be correlated solely with the observed increase in cross-linking.

H u m a n tissue For various reasons none of the samples obtained and investigated was less

than 4 weeks old and so no information is available on the early stages of wound healing. However, it is apparent from the table that there are no major changes between the 4th and I2th week of healing as regards the degree of collagen cross- linking, but already by the 4th week only I4-150/0 is solubilized by heating and this percentage is not increased by pre-treating the tissue with cysteamine, suggesting that labile Schiff-bases are not present. Of the remainder of the collagen 41-48% contains cross-links susceptible to pepsin and virtually all the pepsin-treated residue appears to be cross-linked by alkali-labile bonds.

The human scar collagen thus has a similar cross-linking pattern to adult human Achilles tendon as measured by the same technique e but it is obviously considerably weaker. It must be concluded that some factor other than increased cross-linking must be sought for that gain in tensile strength which continues for several months at least, after the initial gain which occurs in the productive phase of the first 3 weeks. On the other hand this latter gain in tensile strength is extremely slow 4 and any increase in cross-linking may be likewise slow and not detectable over the relatively short period from 4 to 12 weeks during which scar connective tissue was investigated in the present work.

Whichever of these two hypotheses is true, it is still evident that considerable cross-linking occurs early on. In the latter stages of healing, as indicated in the introduction, the gain in tensile strength may be related to collagen interaction with other macro-molecules such as proteoglycan or glycoprotein 2° or possibly by a slower process of cross-linking between scar tissue collagen and the mature collagen of the skin adjacent to the wound.

In the latter case the weakest part of the wound would be in this region and tensile strength measurements would not reflect the strength of the scar tissue itself. The fact that marked changes occur in the skin adjacent to the wound has been demonstrated sl.

R E F E R E N C E S

i E. L. HowEs , J. W. Soov AND S. C. HARVEY, J. Am. Died. Assoc., 92 (I929) 42. 2 E. R. PARTINGTON AND G. C. WOOD, Biochim. Biophys. Acta, 69 (1963) 485- 3 M. L. R. HARKNESS AND 1:{. D. HARKNESS, in Structure and Function of Connective and

Skeletal Tissue, But t e rwor th , London , 1964. 4 D. M. DOUGLAS, J. C. FORRESTER AND R. R. OGILVlE, Brzt. J. Surg, 56 (1969) z ig .

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CROSS-LINKING OF COLLAGEN 689

5 D, S. JACKSON, in Repair and Regeneration, 6 F. S. STEVEN, Biochim. Biophys. Acta, 13o (1966) 196 and 202. 7 F. S. STEVEN, Ann. Rheumatic Diseases, 23 (1964) 3 oo. 8 F. S. STEVEN, Ann. Rheumatic Diseases, 14o (1967) 522. 9 J. F. WOgSSNER, Arch. Biochem. Biophys., 93 (1961) 44 o.

io D. A. SCHWANN AND E. A. BALAZS, Biochim. Biophys. Acta, 13o (1966) 112. II P. G. JOHANSEN, R. D. MARSHALL AND A. NEUBERGER, B*ochem. J., in the press. 12 M. NIMNI, Bzochem. B~ophys. Res. Commun,, 25 (1966) 434. 13 C. D. HEY AND G. STAINSBY, Biochim. Biophys. Acta. 97 (1965) 364 • 14 K. A. PIEZ, Ann. Rev. Bzochem., (1968) . 15 A. J. BAILEY, C. M. PEACH AND L. J. FOWLER, Bzochem. J., 117 (197 o) 619. 16 M. L. TANZER AND G. MECHANIC, B*ochem. B,ophys. Res. Commun., 39 (197 o) 183. 17 D. MACKAY, Bzochzm. Bzophys. Acta, 73 (1963) 445. 18 A. J. BAILEY, Nature, 220 (1968) 280. 19 J. E. Du,WPHY AND D. S. JACKSON, Am. J. Surg., lO4 (1962) 273. 20 D. S. JACKSON AND J. P. BENTLEY, in B. S. GOULD, Treat*se on Collagen, Vol. 2, Academic

Press, New York, 1968). 2i R. J. ADAMSON AND I. F. ENQUIST, Surg. Gynecol. Obstetr., 123 (1966) 515.

B~ochim. Biophys. Acta, 229 (1971) 681-689