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A synchrotron radiation X-ray diffraction study of the crystallinity of tendon fibres
A synchrotron radiation X-ray d action study of the crystallinity of tendon fibres Keid H. Svendsen* Physics Laboratory, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Copenhagen, Denmark
Michel H. J. Kocht European Molecular Biology Laboratory, Hambur9 Outstation, c/o DESY, Notkestrasse 85, D-2000 HamburO 52, FRG
C. Boulin European Molecular Biology Laboratory, Meyerhofstrasse 1, D-6900 Heidelberg, FRG
and A. Gabriel European Molecular Biology Laboratory, Grenoble Outstation, BP 156X, F-38041 Grenoble C~dex, France
(Received 30 April 1984; revised 16 July 1984) The equatorial diffraction pattern of tendon collagen fibres was measured during short successive exposures at different lengths using a double focusing X-ray synchrotron radiation camera with film and with an area detector. Similarly, patterns from thin fibres from premature rats were recorded. The patterns unambiguously illustrate the relationship between fibre crystallinity and the age of the animal. Further, the results indicate that in the initial part of the linear reoion of the stiffness-versus-length curve, the collagen fibres are characterized by a quasihexaoonal arrangement of collagen molecules, whereas at the end of this region, the molecular arrangement becomes hexagonal. Keywords: Collagenfibres;X-raysynchrotronradiation;crystallinity;ageing
Introduction The mechanical function of connective tissue relies on the fact that the collagen fibres are nearly inextensible, i.e. have high values of stiffness and strength resulting from the packing and crosslinking of the collagen molecules. The aggregation of collagen molecules into fibrils has been studied by electron microscopy, X-ray diffraction and mechanical techniques. Electron microscopic studies on stained and dehydrated fibres have failed to give unambiguous evidence of the molecular arrangement 1. Parry and Craig 2 have observed that the total content of collagen molecules in the fibrils remains constant throughout life, but that the content of aggregated molecules increases with age. These observations are in accordance with results from biomechanical studies a'4 and also correspond to recent X-ray diffraction results obtained by Svendsen et al. s, who observed that the features of diffraction patterns from tendon fibres are related to the age of the animal. The crystallinity indices relative to fibres from 240 day old animals range from 0.55 to 0.80 for fibres from 40 and 90 day old animals. With conventional X-ray generators, exposure times for film patterns are of the order of half a day even with thick fibres from old animals which have a large diffraction mass. These fibres have to be treated to * Present address: Sandoegade6, DK 8200 Aarhus,Denmark. t To whomcorrespondenceshould be addressed. 0141-8130/84/060298-05503.00 © 1984 Butterworth& Co. (Publishers) Ltd 298 Int. J. Biol. Macromol., 1984, Vol 6, December
minimize denaturation°'7, so that studies on ageing phenomena are precluded. With X-ray cameras using synchrotron radiation from storage rings, exposure times of 2-5 min can easily be achieved, even with thin fibres. This is adequate for exposures of untreated rat-tail tendon fibres in isometric states and allows re-exposures at different lengths on the same fibre. Low angle X-ray diffraction patterns are characterized by meridional layer-lines and a series of equatorial reflections. The layer-line pattern has been analysed by several groups s and it is interpreted as resulting from the axial staggering of the collagen molecules. Changes in this pattern have been studied under dynamic conditions by Nemetschek et al. 9 and the length versus tension behaviour of the fibres was related to the position of the layer-line. Although the equatorial pattern has been difficult to interpret, a detailed analysis by Fraser et al. 1o strongly indicates that straight segments of collagen molecules are packed in quasihexagonal lattices. The diffuse scatter between the reflections is caused by noncrystalline regions in the collagen molecular arrangement and/or contributions from less ordered segments. The position of the reflections appears not to depend on the animals' age, whereas their intensity, relative to that of the diffuse scatter does. Detailed analysis shows that this intensity ratio can be used to monitor the increase of fibrillar crystallinity with age s. X-ray diffraction studies have also been performed on rat-tail fibres at different pH
Crystallinity of tendonfibres: K. H. Svendsen et al. values and ionic strengths' '. Svendsen and Koch 12 have demonstrated that the acid effect decreases with the age of the animal and that aldimine crosslinks are a controlling component of the lateral packing of collagen molecules. Low pH values had no effect on the crosslinks when the fibres were pretreated with sodium borohydride. These observations are in accordance with the results of biochemical studies 13. The purpose of the present investigations was to study the equatorial reflections in order to relate crystallinity of the fibres to their relative extension and to the age of the animal.
Experimental The X-ray patterns were recorded on the double focusing camera X33 of the EMBL, in HASYLAB 14 using the synchrotron light provided by the storage ring DORIS at the Deutsches Elektronen Synchrotron (DESY). The procedures for fibre preparation and the sample holder have been described elsewhere4'1s. The films (Osray T4 Agfa-Gevaert) were densitometered and digitized using an Optronics film scanner with a raster of 100/art. The area detector 16 operated with argon (70%), CO2 (29.5%) and R13B1 freon (0.5%) at atmospheric pressure had an active surface of 100 x 100 mm 2. Its data acquisition system was similar to the one described earlier 17. A lead mask selecting the relevant part of the pattern was placed in front of the detector window to eliminate unwanted background. Exposure times varied between 3 and 5 rain and the total count rate over the whole detector
(c)
did not exceed 2 x l0 s counts/s. The detector patterns were normalized to the intensity of the primary X-ray beam using an ion chamber placed in front of the sample. Finally, the image corresponding to the background due to the empty cell was subtracted. All manipulations and evaluations of the film and detector data were performed using the standard data evaluation programs of the EMBL Outstation' s.
Results Relative length The fibre rest length, Io, is defined as the length where the tension response, at slow stretching rate, exceeds 10 mN. The actual extensions for the X-ray exposure were reached by interval stretching of 0.01 lo4'' s. It should be noted that extrapolation of the linear segment of the length-versus-tension relation intersect the abscissa around 1.02 lo, for all ages. The physiological working range is on the linear segment having an upper limit at 1.08 lo, with respect to stiffness, and 1.13 Io, with respect to strength and final rupture. It has been observed that cycling extensions produce reversible tension relations up to 1.03 lo19 and that such conditioning of the fibres produces sharper diffraction patterns 9.
Film-exposures at different lenoths Diffraction patterns of fibres from 90 day old rats were exposed at 1.01 and 1.03 lo, as illustrated in Fioures la and lb, respectively. The fibres were then recycled five times within the region from 1.005 ~
(d)
(e)
Figure 1 Isointensity contour plot of film patterns from a 90 day old rat-tail tendon fibre at differentfibre lengths. In (a) 1.01 lo, a series of equatorial reflections including the triplet at 1.3 nm is observed, (b) pattern for 1.03 !o. After recycling the fibre five times between tensions corresponding to the above exposures, new patterns were obtained at 1.01 Io (c), 1.06 !o (d) and 1.091o(e). Note that the triplet evolves towards a single peak, and that the other equatorial reflections disappear. The plots correspond to an area of 115 x 50 mm 2
Int. J. Biol. Macromol., 1984, Vol 6, December
299
Crystallinity of tendon fibres: K. H. Svendsen et al.
(a)
(b)
(c)
(d)
Figure 2 Isointensity contour plot of equatorial patterns from a recycled 200 day old fibre. Patterns (a) and (b) correspond to the initial tension limits of the recycling procedure, i.e. 1.01 and 1.03 10, whereas (c) and (d) are at relative lengths of 1.06 and 1.09 lo respectively. The 1.26 nm reflection enters the region of the 1.35 nm reflection before the fanning decreases
were performed at tension values corresponding to 1.01 lo in the previous runs and after additional 5 and 8% extensions (1.06 and 1.09 10). The isointensity contour plots of these films are presented in Figures lc, ld and le, respectively. A series of equatorial reflections were obtained at relative fibre length of 1.01 and 1.03 I0, prior to the recycling procedure, i.e. intensity maxima corresponding to a spacing of 1.26 nm and the pair of offequatorial maxima corresponding to a spacing of 1.35 nm (unresolved 1.33 and 1.37 nm row-lines). Recycling and re-exposure at 1.01 10 result in decreased fanning of the equatorial intensity profile and all equatorial reflections, except those of the triplets, become very weak. At 1.06 lo all reflections but the triplets have disappeared while at 1.09 1o only a circular Bragg reflection is left. For 40 day fibres the patterns at 1.0110 show larger fanning after recycling than observed for 90 day fibres. The situation is reversed for 200 day fibres. Approximately 12 fibres of each age group were exposed. It was further attempted to monitor the convergence of the 1.3 nm triplet towards a single reflection in greater detail to observe how the corresponding 1 . 2 5 and 1.35nm spacings fuse. Representative patterns are illustrated in Figure 2. The contour plots are from a 200 day recycled fibre exposed at tension values corresponding to initial stretches of 1.01 and 1.03 l o and at further 3 and 6~o extensions. It can be observed that the 1.26 nm reflection first converges towards the 1.35 nm reflection before the fanning characteristic of changes in the molecular tilt takes over beyond 1.06 lo. For 40 day fibres the experimental conditions were not ideal, partly due to a relatively large tension relaxation during the 5 min of exposure and partly because the thin fibres produced weak patterns. The experiments were therefore repeated using a multiwire proportional area detector.
300
Int. J. Biol. Macromol., 1984, Vol 6, December
Fibres from animals of different aye The number of age groups was increased from three to four to obtain a better covering of the events prior to maturity. Animals of 30, 60, 120 and 200 days were killed immediately before the X-ray measurements. After exposure of a film pattern at 1.01 lo, detector patterns were recorded at 1.01, 1.03 and 1.07 lo and this was followed by a final film exposure. The relative length values of 1.01 and 1.03 lo were chosen in accordance with earlier studies by Svendsen et al. 5, showing that the region of reversible stretching around 1.02 lo corresponds to optimal crystallinity. Although the tiny fibres from 30 day old animals are difficult to mount, the precontrol films show wellresolved triplet reflections, but with a relatively high diffuse scatter. Comparison with the post controls indicates convergence of the triplet towards a single peak on the equator. The detector data illustrated in Figure 3 for the four age groups clearly indicate that the crystallinity of the collagen fibrils increases drastically, especially during the first 60 days and saturates at maturity.
Discussion Patterns of stretched fibres Low-angle diffraction patterns of collagen fibres can, at present, not be used to deduce the packing arrangement of the molecules. Instead one has to resort to trial-and-error methods in which computed patterns, based on different packing models are compared with the experimental ones. The conventional equatorial patterns of collagen fibres are basically congruent with that of Fioure la. They are interpreted as resulting from quasihexagonal lattices with two types of principal planes, having spacings of 1.26 and 1.35 nm, respectively. The observations of off-equatorial
Crystallinity of tendon fibres: K. H. Svendsen et al.
30 days
60 days
120 days
200 days
Figure 3 Multiwireproportional area detector patterns ofthe equatorial intensity distribution from fibres of 30, 60, 120 and 200 day old animals.The distributions are obtained after recyclingthe fibresand the exposurescorrespond to the lower end of the linear tension relation, i.e. 1.03 lo. The patterns were not corrected for inhomogeneitiesin the detector response to illustrate the quality of the latter splitting on the 1.35 nm reflection makes it necessary to incorporate a straight-tilt within the 1.26 nm plane of the collagen or of a part of it, relative to the fibril axis 2°. The patterns in Figures la to le show that the 1.26 nm spacing approaches the 1.35 spacing when the fibres are stretched to 1.06 1o. Figure 2 shows that the 1.26 nm reflection enters the region of the 1.35 nm reflection and that this onset of the hexagonal phase Occurs before the loss of molecular tilt monitored by the decrease of the equatorial fanning. It is further observed that stretches around 1.09 10cause the molecular tilt to vanish. This stage is reached just at the end of the linear segment of the fibre stiffness, an ultimate physiological limit, only about 5% from rupture. The lower limit of the linear segment is at 1.02 l0 where the triplet pattern appears. Part of the stretching behaviour might be accounted for by decrimping of the collagen molecules. Increased strain on the fibres converts the series of equatorial reflections to a single reflection. This change can be interpreted in two ways. One explanation is that the strain causes shearing of the molecules and decreases lateral order as the molecules pack onto a pseudohexagonal lattice where only short range order is left. Another explanation is that the fibre crystallinity remains nearly constant until the molecular tilt vanishes and that the quasihexagonal lattice is converted to a hexagonal lattice with equally spaced principal planes giving rise to one strong reflection. These two mechanisms might combine to give the observed effect.
Crystallinity and age It is well established that the fibre stiffness and strength increase until maturity and then saturate 4. Combined with electron microscopy observations z, as well as earlier X-ray diffraction studies, these results strongly suggest increased fibril formation until maturity. A quantitative assessment of the percentage of crystallinity as a function of age was reported by Svendsen et al. s. It has also been shown that the aldimine crosslinks are a controlling
component of the packing scheme 12 and that this crosslink is stabilized after maturity 13. The results illustrated in Figure 3 also provide evidence for increased fibril formation until maturity, after which the actual rate of increase might be even lower than indicated by the change from Figure 3c to 3d. Thus stabilizing crosslinks alone might have improved the orientation of already crosslinked molecules, thereby giving an additional contribution and enhancing the effect of further incorporation of collagen molecules into lattice regions.
Conclusions The detector patterns unambiguously illustrate the relationship between fibre crystallinity and the age of the animal. Further, the results indicate that the start of the linear region of the stiffness-versus-length relation of collagen fibres corresponds to a quasihexagonal lateral arrangement of collagen molecules and that this region ends where the molecular arrangement becomes hexagonal. Evaluations of the biological age of collagen fibres on the basis of crystallinity is most satisfactorily performed at the lower end of the linear tension relations. Further, the study confirms the usefulness of area detectors for this kind of study despite their present count rate limitations due to the data acquisition systems. In future these devices should enable studies not only on isometric but also on isotonic states of the fibres.
References 1 2 3 4 5
Huimes, D. J. S., Jesior, J.-C., Miller, A., Berthet-Colominas, C. and Wolff, E. Proc. Natl Acad. Sci. USA 1981, 78, 6, 3567 Parry, D. A. D. and Craig, A. S. Biopolymers 1978, 17, 843 Torp, S., Arridge, P. G. R., Armeniades, C. D. and Baer, E. in 'Colston Res. Soc. 26, Structure of Fibrous Biopolyrners', Butterworths, London, 1975, p. 197 Svendsen, K. H., Thomson, G. and Wismer-Pcdersen, J. Mol. Physiol. 1983, 3, 237 Svendsen, K. H., Thomson, G. and Barrels, K. Int. J. Biol. Macromol. 1983, 5, 204
Int. J. Biol. Macromol., 1984, Vol 6, December
301
Crystallinity o f tendon fibres: K . H. Svendsen et al. 6 7
12
Miller, A. and Wray, J. Nature 1971, 230, 437 Nemetsehek, T., Riedl, H. and Jonak, R. J. Mol. Biol. 1979, 133, 67 Hulmes, D. J. S., Miller, A., White, S. W. and Doyle, B. J. Mol. Biol. 1977, I10, 643 Nemetsehek, T., Riedl, H., Jonak, R., Nemetsehek-Gansler, H., Bordas, J., Koch, M. H. J. and Schilling, V. Virchows Arch. (A) 1980, 386, 125 Fraser, R. D. B., MaeRae, T. P., Miller, A. and Su~aki, E. J. Mol. Biol. 1983, 167, 497 Ripamonti, A., Roveri, N., Brage, D., Hulmes, D. J. S., Miller, A. and Timmins, P. A. Biopolymers 1980, 19, 965 Svendsen, K. H. and Koch, M. H. J. EMBO J. 1982, 1, 6, 669
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8 9 10 11
13 14 15 16 17 18 19 20
Light, N. D. and Bailey, A. J. in 'Biology of Collagen' (Eds A. Viidik and J. Vuust), Academic Press, New York, 1980, pp. 15-38 Koch, M. H. J. and Bordas, J. Nucl. lnstrum. Meth. 1983,208, 461 Svendsen, K. H. and Thomson, G,-J. Biomech. 1984, 17, 225 Gabriel, A. and Dauvergne, F. Nucl. lnstrmn. Mahods 1982, 201, 223 Boulin, C., Dainton, D., Dorrington, E., Eisner, G., Gabriel, A., Bordas, J. and Koch, M. H. J. Nucl. lnstrum. Methods 1982, 201, 209 Koch, M. H. J. and Bendail, P. Proc. Digital Equipment Computer Users Society (DECUS) UK 1981, 13 Rigby, B. J. Nature 1964, 202, 1072 Trus, B. L. and Piez, K. A. J. Mol. Biol. 1976, 108, 705
Michel H. J. Kocht European Molecular Biology Laboratory, Hambur9 Outstation, c/o DESY, Notkestrasse 85, D-2000 HamburO 52, FRG
C. Boulin European Molecular Biology Laboratory, Meyerhofstrasse 1, D-6900 Heidelberg, FRG
and A. Gabriel European Molecular Biology Laboratory, Grenoble Outstation, BP 156X, F-38041 Grenoble C~dex, France
(Received 30 April 1984; revised 16 July 1984) The equatorial diffraction pattern of tendon collagen fibres was measured during short successive exposures at different lengths using a double focusing X-ray synchrotron radiation camera with film and with an area detector. Similarly, patterns from thin fibres from premature rats were recorded. The patterns unambiguously illustrate the relationship between fibre crystallinity and the age of the animal. Further, the results indicate that in the initial part of the linear reoion of the stiffness-versus-length curve, the collagen fibres are characterized by a quasihexaoonal arrangement of collagen molecules, whereas at the end of this region, the molecular arrangement becomes hexagonal. Keywords: Collagenfibres;X-raysynchrotronradiation;crystallinity;ageing
Introduction The mechanical function of connective tissue relies on the fact that the collagen fibres are nearly inextensible, i.e. have high values of stiffness and strength resulting from the packing and crosslinking of the collagen molecules. The aggregation of collagen molecules into fibrils has been studied by electron microscopy, X-ray diffraction and mechanical techniques. Electron microscopic studies on stained and dehydrated fibres have failed to give unambiguous evidence of the molecular arrangement 1. Parry and Craig 2 have observed that the total content of collagen molecules in the fibrils remains constant throughout life, but that the content of aggregated molecules increases with age. These observations are in accordance with results from biomechanical studies a'4 and also correspond to recent X-ray diffraction results obtained by Svendsen et al. s, who observed that the features of diffraction patterns from tendon fibres are related to the age of the animal. The crystallinity indices relative to fibres from 240 day old animals range from 0.55 to 0.80 for fibres from 40 and 90 day old animals. With conventional X-ray generators, exposure times for film patterns are of the order of half a day even with thick fibres from old animals which have a large diffraction mass. These fibres have to be treated to * Present address: Sandoegade6, DK 8200 Aarhus,Denmark. t To whomcorrespondenceshould be addressed. 0141-8130/84/060298-05503.00 © 1984 Butterworth& Co. (Publishers) Ltd 298 Int. J. Biol. Macromol., 1984, Vol 6, December
minimize denaturation°'7, so that studies on ageing phenomena are precluded. With X-ray cameras using synchrotron radiation from storage rings, exposure times of 2-5 min can easily be achieved, even with thin fibres. This is adequate for exposures of untreated rat-tail tendon fibres in isometric states and allows re-exposures at different lengths on the same fibre. Low angle X-ray diffraction patterns are characterized by meridional layer-lines and a series of equatorial reflections. The layer-line pattern has been analysed by several groups s and it is interpreted as resulting from the axial staggering of the collagen molecules. Changes in this pattern have been studied under dynamic conditions by Nemetschek et al. 9 and the length versus tension behaviour of the fibres was related to the position of the layer-line. Although the equatorial pattern has been difficult to interpret, a detailed analysis by Fraser et al. 1o strongly indicates that straight segments of collagen molecules are packed in quasihexagonal lattices. The diffuse scatter between the reflections is caused by noncrystalline regions in the collagen molecular arrangement and/or contributions from less ordered segments. The position of the reflections appears not to depend on the animals' age, whereas their intensity, relative to that of the diffuse scatter does. Detailed analysis shows that this intensity ratio can be used to monitor the increase of fibrillar crystallinity with age s. X-ray diffraction studies have also been performed on rat-tail fibres at different pH
Crystallinity of tendonfibres: K. H. Svendsen et al. values and ionic strengths' '. Svendsen and Koch 12 have demonstrated that the acid effect decreases with the age of the animal and that aldimine crosslinks are a controlling component of the lateral packing of collagen molecules. Low pH values had no effect on the crosslinks when the fibres were pretreated with sodium borohydride. These observations are in accordance with the results of biochemical studies 13. The purpose of the present investigations was to study the equatorial reflections in order to relate crystallinity of the fibres to their relative extension and to the age of the animal.
Experimental The X-ray patterns were recorded on the double focusing camera X33 of the EMBL, in HASYLAB 14 using the synchrotron light provided by the storage ring DORIS at the Deutsches Elektronen Synchrotron (DESY). The procedures for fibre preparation and the sample holder have been described elsewhere4'1s. The films (Osray T4 Agfa-Gevaert) were densitometered and digitized using an Optronics film scanner with a raster of 100/art. The area detector 16 operated with argon (70%), CO2 (29.5%) and R13B1 freon (0.5%) at atmospheric pressure had an active surface of 100 x 100 mm 2. Its data acquisition system was similar to the one described earlier 17. A lead mask selecting the relevant part of the pattern was placed in front of the detector window to eliminate unwanted background. Exposure times varied between 3 and 5 rain and the total count rate over the whole detector
(c)
did not exceed 2 x l0 s counts/s. The detector patterns were normalized to the intensity of the primary X-ray beam using an ion chamber placed in front of the sample. Finally, the image corresponding to the background due to the empty cell was subtracted. All manipulations and evaluations of the film and detector data were performed using the standard data evaluation programs of the EMBL Outstation' s.
Results Relative length The fibre rest length, Io, is defined as the length where the tension response, at slow stretching rate, exceeds 10 mN. The actual extensions for the X-ray exposure were reached by interval stretching of 0.01 lo4'' s. It should be noted that extrapolation of the linear segment of the length-versus-tension relation intersect the abscissa around 1.02 lo, for all ages. The physiological working range is on the linear segment having an upper limit at 1.08 lo, with respect to stiffness, and 1.13 Io, with respect to strength and final rupture. It has been observed that cycling extensions produce reversible tension relations up to 1.03 lo19 and that such conditioning of the fibres produces sharper diffraction patterns 9.
Film-exposures at different lenoths Diffraction patterns of fibres from 90 day old rats were exposed at 1.01 and 1.03 lo, as illustrated in Fioures la and lb, respectively. The fibres were then recycled five times within the region from 1.005 ~
(d)
(e)
Figure 1 Isointensity contour plot of film patterns from a 90 day old rat-tail tendon fibre at differentfibre lengths. In (a) 1.01 lo, a series of equatorial reflections including the triplet at 1.3 nm is observed, (b) pattern for 1.03 !o. After recycling the fibre five times between tensions corresponding to the above exposures, new patterns were obtained at 1.01 Io (c), 1.06 !o (d) and 1.091o(e). Note that the triplet evolves towards a single peak, and that the other equatorial reflections disappear. The plots correspond to an area of 115 x 50 mm 2
Int. J. Biol. Macromol., 1984, Vol 6, December
299
Crystallinity of tendon fibres: K. H. Svendsen et al.
(a)
(b)
(c)
(d)
Figure 2 Isointensity contour plot of equatorial patterns from a recycled 200 day old fibre. Patterns (a) and (b) correspond to the initial tension limits of the recycling procedure, i.e. 1.01 and 1.03 10, whereas (c) and (d) are at relative lengths of 1.06 and 1.09 lo respectively. The 1.26 nm reflection enters the region of the 1.35 nm reflection before the fanning decreases
were performed at tension values corresponding to 1.01 lo in the previous runs and after additional 5 and 8% extensions (1.06 and 1.09 10). The isointensity contour plots of these films are presented in Figures lc, ld and le, respectively. A series of equatorial reflections were obtained at relative fibre length of 1.01 and 1.03 I0, prior to the recycling procedure, i.e. intensity maxima corresponding to a spacing of 1.26 nm and the pair of offequatorial maxima corresponding to a spacing of 1.35 nm (unresolved 1.33 and 1.37 nm row-lines). Recycling and re-exposure at 1.01 10 result in decreased fanning of the equatorial intensity profile and all equatorial reflections, except those of the triplets, become very weak. At 1.06 lo all reflections but the triplets have disappeared while at 1.09 1o only a circular Bragg reflection is left. For 40 day fibres the patterns at 1.0110 show larger fanning after recycling than observed for 90 day fibres. The situation is reversed for 200 day fibres. Approximately 12 fibres of each age group were exposed. It was further attempted to monitor the convergence of the 1.3 nm triplet towards a single reflection in greater detail to observe how the corresponding 1 . 2 5 and 1.35nm spacings fuse. Representative patterns are illustrated in Figure 2. The contour plots are from a 200 day recycled fibre exposed at tension values corresponding to initial stretches of 1.01 and 1.03 l o and at further 3 and 6~o extensions. It can be observed that the 1.26 nm reflection first converges towards the 1.35 nm reflection before the fanning characteristic of changes in the molecular tilt takes over beyond 1.06 lo. For 40 day fibres the experimental conditions were not ideal, partly due to a relatively large tension relaxation during the 5 min of exposure and partly because the thin fibres produced weak patterns. The experiments were therefore repeated using a multiwire proportional area detector.
300
Int. J. Biol. Macromol., 1984, Vol 6, December
Fibres from animals of different aye The number of age groups was increased from three to four to obtain a better covering of the events prior to maturity. Animals of 30, 60, 120 and 200 days were killed immediately before the X-ray measurements. After exposure of a film pattern at 1.01 lo, detector patterns were recorded at 1.01, 1.03 and 1.07 lo and this was followed by a final film exposure. The relative length values of 1.01 and 1.03 lo were chosen in accordance with earlier studies by Svendsen et al. 5, showing that the region of reversible stretching around 1.02 lo corresponds to optimal crystallinity. Although the tiny fibres from 30 day old animals are difficult to mount, the precontrol films show wellresolved triplet reflections, but with a relatively high diffuse scatter. Comparison with the post controls indicates convergence of the triplet towards a single peak on the equator. The detector data illustrated in Figure 3 for the four age groups clearly indicate that the crystallinity of the collagen fibrils increases drastically, especially during the first 60 days and saturates at maturity.
Discussion Patterns of stretched fibres Low-angle diffraction patterns of collagen fibres can, at present, not be used to deduce the packing arrangement of the molecules. Instead one has to resort to trial-and-error methods in which computed patterns, based on different packing models are compared with the experimental ones. The conventional equatorial patterns of collagen fibres are basically congruent with that of Fioure la. They are interpreted as resulting from quasihexagonal lattices with two types of principal planes, having spacings of 1.26 and 1.35 nm, respectively. The observations of off-equatorial
Crystallinity of tendon fibres: K. H. Svendsen et al.
30 days
60 days
120 days
200 days
Figure 3 Multiwireproportional area detector patterns ofthe equatorial intensity distribution from fibres of 30, 60, 120 and 200 day old animals.The distributions are obtained after recyclingthe fibresand the exposurescorrespond to the lower end of the linear tension relation, i.e. 1.03 lo. The patterns were not corrected for inhomogeneitiesin the detector response to illustrate the quality of the latter splitting on the 1.35 nm reflection makes it necessary to incorporate a straight-tilt within the 1.26 nm plane of the collagen or of a part of it, relative to the fibril axis 2°. The patterns in Figures la to le show that the 1.26 nm spacing approaches the 1.35 spacing when the fibres are stretched to 1.06 1o. Figure 2 shows that the 1.26 nm reflection enters the region of the 1.35 nm reflection and that this onset of the hexagonal phase Occurs before the loss of molecular tilt monitored by the decrease of the equatorial fanning. It is further observed that stretches around 1.09 10cause the molecular tilt to vanish. This stage is reached just at the end of the linear segment of the fibre stiffness, an ultimate physiological limit, only about 5% from rupture. The lower limit of the linear segment is at 1.02 l0 where the triplet pattern appears. Part of the stretching behaviour might be accounted for by decrimping of the collagen molecules. Increased strain on the fibres converts the series of equatorial reflections to a single reflection. This change can be interpreted in two ways. One explanation is that the strain causes shearing of the molecules and decreases lateral order as the molecules pack onto a pseudohexagonal lattice where only short range order is left. Another explanation is that the fibre crystallinity remains nearly constant until the molecular tilt vanishes and that the quasihexagonal lattice is converted to a hexagonal lattice with equally spaced principal planes giving rise to one strong reflection. These two mechanisms might combine to give the observed effect.
Crystallinity and age It is well established that the fibre stiffness and strength increase until maturity and then saturate 4. Combined with electron microscopy observations z, as well as earlier X-ray diffraction studies, these results strongly suggest increased fibril formation until maturity. A quantitative assessment of the percentage of crystallinity as a function of age was reported by Svendsen et al. s. It has also been shown that the aldimine crosslinks are a controlling
component of the packing scheme 12 and that this crosslink is stabilized after maturity 13. The results illustrated in Figure 3 also provide evidence for increased fibril formation until maturity, after which the actual rate of increase might be even lower than indicated by the change from Figure 3c to 3d. Thus stabilizing crosslinks alone might have improved the orientation of already crosslinked molecules, thereby giving an additional contribution and enhancing the effect of further incorporation of collagen molecules into lattice regions.
Conclusions The detector patterns unambiguously illustrate the relationship between fibre crystallinity and the age of the animal. Further, the results indicate that the start of the linear region of the stiffness-versus-length relation of collagen fibres corresponds to a quasihexagonal lateral arrangement of collagen molecules and that this region ends where the molecular arrangement becomes hexagonal. Evaluations of the biological age of collagen fibres on the basis of crystallinity is most satisfactorily performed at the lower end of the linear tension relations. Further, the study confirms the usefulness of area detectors for this kind of study despite their present count rate limitations due to the data acquisition systems. In future these devices should enable studies not only on isometric but also on isotonic states of the fibres.
References 1 2 3 4 5
Huimes, D. J. S., Jesior, J.-C., Miller, A., Berthet-Colominas, C. and Wolff, E. Proc. Natl Acad. Sci. USA 1981, 78, 6, 3567 Parry, D. A. D. and Craig, A. S. Biopolymers 1978, 17, 843 Torp, S., Arridge, P. G. R., Armeniades, C. D. and Baer, E. in 'Colston Res. Soc. 26, Structure of Fibrous Biopolyrners', Butterworths, London, 1975, p. 197 Svendsen, K. H., Thomson, G. and Wismer-Pcdersen, J. Mol. Physiol. 1983, 3, 237 Svendsen, K. H., Thomson, G. and Barrels, K. Int. J. Biol. Macromol. 1983, 5, 204
Int. J. Biol. Macromol., 1984, Vol 6, December
301
Crystallinity o f tendon fibres: K . H. Svendsen et al. 6 7
12
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