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Biochimica et Biophysica Acta, 533 (1978) 147--155 © Elsevier/North-Holland Biomedical Press

BBA 37860

ISOMETRIC TENSION DEVELOPED DURING HEATING OF COLLAGENOUS TISSUES

RELATIONSHIPS WITH COLLAGEN CROSS-LINKING

J.C. ALLAIN, M. LE LOUS, S. BAZIN, A.J. BAILEY a and A. DELAUNAY b

Unit~ de Recherche de Gdn~tique Mddicale,H6pital des Enfants Malades, 149, rue de Sdvres, 75015 Paris (France), a Agricultural Research Council, Meat Research Institute, Langford, Bristol (U.K.), and b Insti tut Pasteur, Paris (France)

(Received July 1st, 1977)

Summary

A sensitive instrument has been constructed to measure the development and subsequent relaxation of the isometric tension during the heating of col- lagenous tissues.

The curves of the tension developed by rat skin plot ted against temperature exhibited different shapes according to the age of the animal. In skin from new born rats the maintenance of the maximum tension up to temperatures around 95°C appeared to be correlated with the presence of the thermally stable " k e t o " cross-link. In adult skin the dramatic relaxation of the tension that occurred above 75°C correlated with the increased proport ion of the heat labile "aldimine" cross-link. With increasing age the extent of this relaxation decreased due to the increasing proport ion of thermally stable cross-links of unknown origin.

The curves obtained with granulation tissues of acute inflammation during the development phase and subsequent resorption phase of the granuloma were similar to those obtained during the growth of young animals from birth to 1 month. On the other hand granulation tissues of chronic inflammation retained an isometric tension curve similar to that of the new born rat skin, even after a long period of inflammation (8 months).

Determination of the isometric tension developed in heated tissues provides a rapid assay useful for obtaining preliminary information on the cross-linking of collagen fibres in normal and pathological situations.

Introduction

Hydrothermal shrinkage is a well known characteristic of collagen fibres, the extent of which increases with the age of the animal [1,2,3]. These findings,

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together with the enhanced shrinkage resulting from tanning [4] indicated a relationship between hydrothermal shrinkage and cross-linking of collagen.

The contraction of the collagen fibres can be measured directly through shortening of the tissue; but a more accurate method is by measurement of the tension developed in the tissue when mechanically restrained at its lineal dimensions. Such isometric tension measurements have been used to demon- strate an increased cross-linking with age [5] and the presence of thermally labile cross-links [6].

We have built a very sensitive instrument in our laboratory capable of mea- suring the small changes in tension developed during heating of samples of rat skin and granulation tissues. In the same tissues, the nature of the reducible cross-links of collagen were chemically determined. In this paper we report a correlation between the shape of the isometric tension curve and the nature of the cross-links that could be useful when studying growth changes or some pathological processes.

Material and Methods

(1) Preparation of tissues (a) Skin. Skin was taken from female Wistar rats from our laboratory breed-

ing stock: animals of increasing age from birth to 18 months were used. Imme- diately after the animals had been killed and bled, the skin was shaved. An area of 4 cm X 3 cm was delineated on the back of the normally extended animal. A piece of skin slightly larger than the delineated surface was then removed and pinned on a cork plaque under sufficient tension to maintain the delineated area with the same dimension as when marked on the whole animal. A speci- men of skin of exactly 1.8 cm X 1.8 cm was then stamped out and kept at --20°C until the isometric tension determinations were undertaken.

Some samples of skin from adult rats (3 months) were reduced in 50 ml sodium borohydride solution (0.025 mg to 5 mg/ml) at 20°C for 5 min before the isometric tension determination.

(b) Granulomas (i) Acute inflammation: Sterile inflammation was induced by subcutaneous injection of 0.25 ml turpentine oil on the back of 2.5 months old female Wistar rats, according to our method described previously [ 7].

The tissue of very recent granulomas was too loose to be used in assays of isometric tension. However, 5 to 10 day granuloma were sufficiently devel- oped, and were stamped out as previously described for the skin samples. After 12 days, the granuloma had partially resorbed and had become too small for isometric tension determinations.

Another type of granuloma resulting from acute inflammation was obtained from burns following a 10 s contact of a 14 mm diamter metal disc heated at 250 ° C, on the shaved dorsal skin of anesthesized rats. The granulomas were removed, together with surrounding normal tissue at 12 to 21 days after burning.

(ii) Chronic inflammation: Two pieces of sterile polyvinyl sponge 1.8 cm X 1.8 cm X 0.3. cm were implantes subcutaneously on the backs of 2.5 months old female Wistar rats, according to our usual method [7].

The sponges, together with the invading and covering granulation tissue,

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Fig. 1. A, b a l a n c i n g sca le -maxker a r m ; B, t r a n s d u c e r ( L C F B 3 0 t y p e S e n t o s e c ) : C, A d j u s t e m e n t f o r regula - t i o n o f s a m p l e t e n s i o n ; D, s o u n d i n g r o d a n d c o n t a c t t h e r m o m e t e r , b o t h f ixed o n the s a m e h o l d e r ; E, m o t o r c o n t r o l l i n g m a g n e t o f t he c o n t a c t t h e r m o m e t e r ; F , h e a t i n g coi l f r o m g l y c e r o l b a t h ; G , e l ec t r i ca l in. p u t a n d r e g u l a t i o n o f t h e s o u n d i n g r o d a n d t r a n s d u c e r ; H , c o n t a i n e r f i l led w i t h p h y s i o l o g i c a l sa l ine a n d t h e r m a l l y insula ted~ I, g l y c e r o l b a t h h e a t e d b y a n e lec t r ic r e s i s t ance ; J , r e c o r d e r ( G e p e r a c t y p e , S e f r a m ) ; P, s a m p l e h o l d e r w i t h space r e g u l a t e d b y a m e c h a n i c device .

were harvested from 7 days to 8 months after implantation. The whole granulomas were used for isometric tension determinations, using

a piece of the original polyvinyl sponge as the control.

(2) Isometric tension A device was built in our laboratory according to the scheme in Fig. 1.

Before use, the electrical systems (transducer, sounding rod, recorder, heater) were tested and regulated. The specimen of tissue was clamped between two holders (P in Fig. 1), thus making a connection between the scale marker arm (A) and the tightener (C) and then immersed in physiological saline maintained at 37°C.

The scale marker arm was then freed, connected to the transducer and the tissue subjected to a tension of approximately 1/50 of the anticipated maximal tension.

The temperature of the saline bath was progressively increased from 37°C to 100°C at a speed of 1.15°C per min, and the tension developed in the tissue simultaneously recorded. This tension is expressed as 1 mV = 1.2 g weight.

Preliminary assays were performed to demonstrate the accuracy and repro- ducibility of the isometric tension determinations during the increase of tem- perature between 37°C and 100°C. The influence of surface, thickness, storage and freezing of the tissue samples, rate of increase of temperature in the saline bath, sensitivity of the galvanometric recorder, were all examined. The con- ditions described above were chosen as the most reproducible and convenient. All subsequent determinations were made under these standard conditions.

(3) Reducible collagen cross-links A portion of tissue was used to identify the reducible cross-links present.

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This was achived by reaction of the intact tissue with tritiated potassium boro- hydride and subsequent separations of the acid or basic hydrolysate as pre- viously described [ 8].

Results

(1) Isometric tension When a specimen of tissue was heated over the range 37°C to 100°C a ten-

sion started to develo p at a temperature of approximately 60 ° C. At temperatures above 60°C the tension increased rapidly up to a maximum,

then decreased more or less rapidly according to the nature of the tissue spe- cimen. For a given age of specimen and a standard rate of temperature increase, the maximum tension was always registered at the same temperature, within 0.5°C. The maximum tension generated from identical samples was slightly variable, of the order of 15%, similar to that obtained by other workers [1,2,3] . More importantly the change in shape of the tension curves with age was entirely reproducible. As a result, the shape of the curve of the changes in tension, plotted against temperature, appeared to be characteristic of the tissue.

(a) Normal skin. From birth to 1 month, the changes of tension between 37°C and 100°C are shown in Fig. 2, and from 1 month to 18 months in Fig. 3.

It can be seen that the maximum tension was reached at a much higher tem- perature in skin of new born animals (95°C) than in adult animals (67°C). In

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Fig . 3 . M o d i f i c a t i o n o f i s o m e t r i c t e n s i o n b e t w e e n 3 7 ° C a n d 1 0 0 ° C in rat s k i n f r o m 2 t o 1 8 m o n t h s .

, 2 m o n t h s ; . . . . . . , 5 m o n t h s : . . . . , 1 8 m o n t h s .

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new born rat skin, the tension was also much more heat stable than in adult skin. However when the temperature was increased above 67°C significant differ-

ences between the tissues were observed: in the new-born skin the tension did not reach a maximum until 95 ° C; in the 1 week skin the maximum was reached at 73 ° C, whilst in the 1 month skin and the skins of adults up to 1 year the maximum tension developed at the sharp shrinkage temperature around 67°C. Although no change in the temperature at the onset of shrinkage occurred with age, there was a significant increase in the temperature at which the maximum tension was generated; 67°C for 1--3 months, 71°C around 6 months and 78°C at 18 months.

Further, the rate of relaxation of the tension when the temperature was further increased after the maximum tension had been developed varied considerably with the age of tissue. The most rapid decreases in tension occured between 1 and 5 months, whilst in contrast, the 18 month old and par- ticularly the new-born skins, the tension was found to be maintained to a con- siderable extent up to 100 ° C.

(b) Pathological tissues. In granulation tissues of recently developed acute inflammations (5 days after turpentine injection or 14 days after burns} and in those of chronic inflammation of short or prolonged duration, the maximum tension was reached at the higher temperature (95 ° C): Figs. 4 and 5.

In tissues with acute inflammation and undergoing remodelling during the healing process the maximum tension attained occurred at a decreasing temper-

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Fig. 5. M o d i f i c a t i o n o f i som e tr i c t ens ion b e t w e e n 3 7 ° C and 1 0 0 ° C in granulat ion t issue o f chronic i n f l a m m a t i o n : . . . . , 2 1 days granuloma; - - - , 8 m o n t h s granuloma.

152

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h y d r i d e / m l ; - - - , 0 . 5 m g s o d i u m b o r o h y d r i d e / m l ; - - - , c o n t r o l s k i n .

F i g . 7 . R e d u c e d c o l l a g e n c r o s s - l i n k s in rat s k i n : I - - n e w b o r n r a t , I I - - 3 w e e k s o l d rat ; I I I - - 3 m o n t h s o l d ra t . L y s ( O H ) 2 N l e , d i h y d r o x y l s i n o n o r l e u c i n e , L y s ( O H ) - N l e , h y d r o x y l y s i n o n o r l e u c i n e ; H i s - M e t ( O H ) ,

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ature and gradually the isometric tension curve returned to one with a com- parable heat lability to that of the normal adult skin {Fig. 4).

(c) Reduced normal skin. When the normal skin of 3 month old rats was reduced with sodium borohydride for 5 min only prior to heating, the tension reached its maximum at a higher temperature and also became more heat stable in that the tension was maintained at higher temperatures. The extent of the tension developed was shown to be dependent on the sodium borohydride con- centration in the reduction medium (Fig. 6). At the lower concentration, the reduction is incomplete after 5 min and demonstrates the progressive effect of stabilization of the reducible aldimine cross-links.

(2) Collagen cross-links Elution patterns of the radioactive components of acid hydrolysates

obtained from boro[3H]hydride reduced skin showed that the major cross- link in collagen of new born rat skin was almost exclusively dihydroxylysinon- orleucine. In adult rat skin, the major radioactive peaks are those of hydroxyly- sinonorleucine and histidinohydroxymerodesmosine. During growth, from 1 week to 1 month, there is a decrease of the dihydroxylated cross-links and a progressive increase of the monohydroxylated cross-links (Fig. 7).

When the skins from new born rats or from 3 week old rats were heated to 73°C, rapidly cooled and then reduced with borohydride, the monohydroxyl-

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ated cross-links progressively disappeared but, the dihydroxylated cross-link was unmodified (Fig. 8).

In the granulation tissues, as previously described [ 9,10], the major reducible cross-link is of the dihydroxylated type but when the tissue is remodelling to finally produce a normal composition and structure, the cross-link pattern gradually reverts to one comparable to that of the control skin.

During chronic inflammation (sponge implants), the major cross-links are of the mono and dihydroxylated type and very little change occurs with time of development the cross-link elution pattern being practically the same from 1 to 8 months development of the granulation tissue [9].

Discussion

The instrument was found to give extremely reproducible results and to be sufficiently sensitive to detect small changes in tension.

Considering first the tension at the shrinkage temperature (65--67°C) the results obtained with normal skin showed that the maximum tension developed during heating increased with the age of the animal from 3--5 mV at 1--3 weeks to 192 mV at 18 months. This observation is in agreement with the results described by other authors on tendon [ 1,2] and human skin [3 ].

We suggest that the changing shapes of these curves can be explained in terms of the intermolecular cross-links stabilizing the fibre. In new-born to 3 week old skin there is a decreasing proportion of the cross-link hydroxyly- sino-5-keto-norleucine (revealed as dihydroxylysinonorleucine following boro-

154

hydride reduction) (Fig. 7). This cross-link is thermally stable (Fig. 8) and would therefore account for the absence of any relaxation of the tension fol- lowing the collapse of the collagen molecule at the shrinkage temperature. From 1 to 5 months the major cross-link stabilizing the native fibre is dehydro- hydroxylysinonorleucine (revaled as hydroxylysinonorleucine following boro- hydride reduction). The thermal lability of this cross-link (Fig. 8) could readily account for the rapid relaxation of the tension above the shrinkage tempera- ture.

With further increases in age of the tissue the labile aldimine bond disap- pears and is replaced by a non-reducible thermally stable bond believed to be derived from the original aldimine [8]. The increasing proport ion of this ther- mally stable bond, the chemical nature of which is as ye t unknown, would therefore account for the maintenance of a high tension above the shrinkage temperature in old skin. This is supported by the negligible relaxation at 100°C of tissue in which the aldimine bond has been artificially stabilized in vitro by borohydride reduction (Fig. 6). It is also notewor thy that the temperature at which the maximum tension was generated increases following a t reatment that stabilizes existing aldimine cross-links and this is consistent with the higher temperature maximum observed with increasing age (Fig. 3). A number of stable cross-link structures have been proposed [9,10,11] but their increase with age has not been demonstrated and their relative importance is unknown since it is not possible at the present time to quantify them.

These changes in the tension curves with age correlated so well with the nature of cross-links that we feel they must be the major determining factor. The presence of Type III in skin is an unknown factor but we feel this compo- nent is unlikely to affect the shrinkage characteristic dramatically since only 10--15% is present and its cross-links are similar to Type I [12].

Analysis of the isometric tension curves produced by granulation tissues when compared with the previous studies on the nature of the cross-links [13--14] also gave, as anticipated, a good correlation. The progressive replace- ment of the thermally stable "ke to" cross-link by the labile aldimine bond during the healing process is clearly reflected in the progressively more rapid relaxation at higher temperatures (Fig. 4). This sequence of events in wound healing is analogous to the changes from embryonic to adult skin.

In contrast, the granulomas from chronic inflammation have previously been shown to fail to mature in that they maintained their reducible cross-links over a considerable period [13]. This retention of stable reducible "ke to" cross- link appears to be reflected in the high shrinkage temperature and the negli- gible relaxation at higher temperatures.

Although the nature of cross-links can account to a considerable degree for the changes in shape of the isometric tension curves, the high temperatures at which the maximum tension is developed in embryonic skin, 5 day turpentine granuloma, 14 day burns and in chronic granuloma, could also be influenced by other factors. Whether these involve the type of collagens, the proport ion of Type III collagen being higher in all these tissues [14], or interaction with other tissue components remains to be elucidated.

In conclusion it is clear that the total shape of the curves demonstrating the changes in the isometric tension developed between 37°C and 100°C appears to

155

be more sensitive in distinguishing the heat stability of collagen during growth and ageing than determination of the absolute value of tension at a predeter- mined temperature as studied by other workers.

Our results show that when accurately measured, the determination of the isometric tension developed in small specimens of tissues is able to rapidly sup- ply preliminary indications about the nature and degree of cross-linking of col- lagen without resorting to time consuming and sophisticated techniques. Such determination can be used to fol low the growth of young individuals and to detect possible abnormal cross-linking of collagen in pathological tissues.

Preliminary assays with a miniaturized device are now in progress and will supply, in the near future, a method of carrying out the determinations on specimens of tissues as small as biopsies.

References

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Press, N e w York 5 Verzar, F. (1964) Intern. Rev. Connect . Tissue Res. 2 ,243- -300 6 Bailey, A.J. and Lister, D. (1968) Nature 220 ,280- -281 7 Bazin, S. and Delaunay, A. (1964) Ann. Inst. Pasteur, 107, 163--172. 8 Robins, S.P., Shimokomaki , M. and Bailey, A.J. (1973) Biochem. J. 131 ,771- -780 9 Davis, N.R., Risen, O.N. and Pringle, S.A. (1975) Biochemistry 14, 2031--2035

10 FIousley, T., Tanzer, M.L., Henson, E. and Gallop, P.M. (1975) Biochem. Biophys. Res. Commun. 67, 824--830

11 Fuj imoto, D., Akiba, K. and Nakamura, N. (1977) Biochem. Biophys . Res. Commun. 76, 1124--1129 12 Bailey, A.J. and Sims, T.J. (1976) Biochem. J. 153, 211--215 13 Bailey, A.J., Bazin, S. and Delaunay, A. (1973) Biochim. Biophys. Acta 328 ,383- -390 14 Bailey, A.J., Bazin, 8., Sims, T.J., Le Lous, M., Nicoletis, C. and Delaunay, A. (1975) Biochim. Bio-

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