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Scanning electron microscopy of stretched elastin fibres
Micron, 1974,5:127-133 with II plates
127
Scanning electron microscopy of stretched elastin fibres R. J. MINNS and F. S. STEVEN Manchester University Connective Tissue Research Group, Department of Medical Biochemistry, University of Manchester, Stopford Building, Manchester M 1 3 9PL, England, U.K.
Manuscript received April 8, 1974
Strips of bovine ligamentum nuchae were (a) stretched to twice their length and then released, (b) stretched until they ruptured, while a third series (c) were not subjected to either procedure and were used as controls. All three series were then treated with buffered guanidine hydrochloride and bacterial collagenase in order to obtain pure preparations of elastin fibres free of their collagenous sheath and matrix. Examination of the controls by scanning electron microscopy revealed a network of elastin fibres with some cross-weaving and cross-branching. However, in strips which had been stretched and released the fibres were orientated in the direction of the strain suggesting that in the absence of the collagen they were unable to return to their original pattern. No broken fbres were observed after stretching. Examination of fibres in ruptured strips revealed some with a smooth fracture face associated with a slight decrease in fibre diameter, an appearance consistent with a brittle fracture. Des bandes de tissus ligamentum nuchae furent respectivement : (a) dtirdesjusqu'it une longueur double de leurs dimensions initiales, puis reldchges; (b) ~tir~esjusqu'it leurs points de rupture; (c) un certain nombre ne fut sujet it aucune des contraintes dgcrites ci-dessus et furent utilisdes comme ~lgments de rdfdrences. Chacune des trois sgries rut ensuite traitde avec une solution tampon d'hydrochlorure de guanidine et de collaggnase bacterial afi~i d'obtenir une prdparation de fibres d¥1astine pures d~pourvues de leur gaines et matrices de collagkne. L' examen des specimens de rdf~rence par microscopie ~lectronique it balq~,age rgv~la un rdseau de fibres d¥lastine primaires et comportant des branches secondaires entrem~lges. Toutefois dans le cas des bandes soumises it une tension puis reldchdes lesfibres gtaient orientges paral#lement au sens de la dgformation, ce qui conduit ~ penser qu'elles sont incapables de revenir it leur situation primitive en l'absence de collag~ne. Aucune fibre rompue ne fut observ~e apr~s dtirage. Quant aux bandesfracturdes l' examen desfibres r~vglaparfois une surface lisse associ~e ~ une diminution du diam~tre de la fibre, c'est-&dire une apparence compatible avec une fracture fragile. Streifen von Ligamentum nuchae des Rindes wurden (a) auf ihre zweifache Liinge gestreckt und dann losgelassen oder (b) bis zum Zerreiflen gestreckt, wiihrend eine dritte Gruppe (c) keiner Behandlung unterzogen wurde und als Kontrollmaterial diente. Alle drei Gruppen wurden dann mit Guanidinhydrochlorid und bakterieller Ifollagenase behandelt, um reine Elastinfaser ohne Kollagenhiille und Bindegewebe zu erhalten. Das mit R E M untersuchte Kontrollmaterial zeigte ein Geflecht aus gewebten Elastinfasern und solche mit Querkluften. In Streifen, die gestreckt wurden, waren die Faser in der Streckrichtung orientiert, was daraufhin deutet, daft sie zu ihrer Originalform nicht mehr zuriick konnten. In diesen Proben gab es keine Anzeichen yon abgerissenen Fasern. In den zerrissenen Streifen sah man einige feink6rnige Bruchfliichen zusammen mit einer Verringerung des Faserdurchmessers, die t"iir einen Spr6dbruch charakteristisch sind.
INTRODUCTION Adult bovine ligamentum nuchae is known to contain a highly organized fibrous network of elastin, each fibre being encircled by a knitted sheath of collagenous material and embedded in a matrix containing a continuous collagen network interconnecting the fibres (Gotte, Mammi and Pezzin, 1972; Finlay and Steven, 1973).
i2t{
NIINNS : \ N I ) S I'EVEN
Which of" tl{ese networks contribute to the mechanical strength and which to> the elastic behaviour is not too well understood. The effect of removing the collagcm~us component from connective tissue by enzymic digestion and then testing the tissue mechanically has been examined by several workers (Hoffman, Grande, Gibson, Park, Daly, Bornstein and Ros;, 1972; Steven, Minns and Thomas, 1974) who found that the elastic fibres were only able to extend to approximately half their original length before rupture occurred. This suggested that at strains above 80-100',a the elastin fibres from adult bovine ligamentum nuchae ruptured while the collagenous network was still intact. This paper reports on the appearance in the scanning electron microscope of elastin fibres from adult bovine ligamentum nuchae that were stretched to 100°o strain and released and other strips which were extended to rupture. The results are compared with those for unstretched elastin fibres from the same tissue and the effect of loading on the structural architecture is discussed. M A T E R I A L S AND M E T H O D S Strips of elastic tissue approximately 3mm thick and 10mm long were cut parallel to the main axis from the surface of the ligamentum nuchae of an adult cow. The internal faces of the strips were clearly marked by knife cuts so that the surface fibres were easily identified after treatment. One series of strips were extended mechanically to 100% of their original length and released. In a second series the strips were extended to rupture (Minns, Soden and Jackson, 1973). A further series were left untouched as controls. All the strips were then stored at --4°C prior to enzymic digestion in order to visualize pure elastin fibres in the scanning microscope. To prepare chemically pure elastin (Steven et al., 1974) all the strips were treated with 6M guanidine hydrochloride buffered to p H 7.0 with 1N N a O H for 18hr at 16°C followed by repeated washing in distilled water. The tissue was then digested with 0.01% bacterial collagenase (Worthington, CLSPA 380 unit/mg) in 0.05M Tris-HC1 buffer pH 7.,5 containing 0.005M CaC12 at 37°C for 18hr followed by repeated washing with distilled water. The tissue was then treated again with the buffered guanidine hydrochloride. Dehydration of the strips was carried out with increasing concentrations of ethanol followed by drying in air in a desiccator for lhr. The specimens were glued to marked metallic stubs and coated under vacuum on a rotating stage with gold-palladium to produce a uniform coating of approximately 20nm. The coated specimens were then examined in a Stereoscan $4 scanning electron microscope (Cambridge Instrument Co.) operated at an accelerating voltage of 30kV. The results were recorded with an Exakta VX500 camera on Ilford FP4 135 film. RESULTS Following treatment with the guanidine hydrochloride and collagenase, the elastin fibres were clearly revealed. In ligaments which had not been subjected to stretching the fibres weaved over and under each other to form an elaborate mesh or net (Figure 1). At high magnification it was observed that while many of the fibres tended to be orientated in the direction of the ligament, there was a considerable degree of branching across the main axis (Figure 2). None of the fibres appeared to be broken.
SEM OF STRETCHED ELASTIN FIBRES
129
A distinct difference in the onela[tttNti 01~ [}[e tlbres was observed in strips which had first been stretched and then allowed to return to their original length• Almost all of the fibres were orientated in the direction of the strain and there was little evidence of the cross-weaving and cross-branching seen previously (Figure 3). Again, however, none of the fibres appeared to be broken. In the strips which had been loaded to rupture and then released, the arrangement of the fibres was again similar to that seen after stretching• This time, however, some of the fibres were broken. As shown in Figure 4, the fracture surface was smooth and there was a slight reduction in the diameter of the fibre at the broken tip, presumably due to 'necking' that occurred just before rupture. The diameter of the fibres ranged from 7-9~. DISCUSSION In studies on horse extensor tendon that had been strained and then allowed to relax, Abrahams (1967) reported that the collagen fibres appeared to uncoil and align themselves parallel to the long axis. In the present work, the elastin fibres appeared to undergo a similar transition during loading, a result shown quite clearly by scanning electron microscopy. Since these fibres are normally covered by a fine framework of collagen fibres (Finlay and Steven, 1973), it seems most likely that during stretching the collagen framework itself becomes considerably elongated. O f particular interest is the fact that in the absence of the collagen, the elastin fibres appear to be unable to return to their normal configuration after stretching. The collagen, therefore, appears to be essential to maintain the cross-weaving pattern seen in the relaxed state and which presumably is of functional significance, for example, by allowing and compensating for lateral stress. In ligaments that were stretched to twice their length there was no evidence that any of the elastin fibres were broken. This suggests that the tensile strength of the ligamentum nuchae depends on the collagen which in turn would obviously afford a measure of protection to the elastin fibres at high load. Finally, it may be mentioned that the appearance of ruptured elastin fibres is characteristic of a brittle failure, with a smooth fracture face associated with a slight reduction in diameter of the fibre at the break. ACKNOWLEDGEMENTS We should like to thank the Department of Biology, Liverpool Polytechnic for the use of the scanning electron microscope and Miss Susan Browne for the technical assistance and in its operation. REFERENCES
ABRAHAMS, M., 1967. Mechanical behaviour of tendon in vitro. A preliminary report. Med. Biol. Engng, 5: 433-443. FINLAY,J. B. and STEW~, F. S., 1973. The fibrous components of bovine ligamentum nuchae observed in the scanning electron microscope. 07. Microscopy, 99: 57-63. GOTTE, L., MAMMI, M. and P~.zzxN, G., 1972. Scanning electron microscope observations on elastin. Connect. Tissue Res., 1: 61-68.
l?,~
MINNS \NI) STEVEN
HOFFMAN, A. S., GRANDE, [,..\., GIBSON, P.. PARK, J. P., I)A1,Y, (i. H., 13ORNs'rvJN. I'. ;l~ld Ross, R., 1972. Preliminary studies on mechanochemical-structure relationships in connective tissues using enzymolysis techniques. In : Perspectives in Biomedical Ertgim'erm4,, Kenedi, R. (ed.), Macmillan, London, 173 176. Mixxs, R. J., SOOEN, P. D. and,JAcKSON, D. S., 1973. The role of the fibrous components and ground substance in the mechanical behaviour of biological tissues: A preliminar}' investigation. J. Biomech., 6 : 1 5 3 165. STEVF,N, F. S., MINNS, R. J. and THOMAS, H., 1974. The isolation of chemically pure elastins in a form suitable for mechanical testing. Connect. Tissue Res., 2: 85-90.
F I G U R E S 1 and 2
Elastin fibres of bovine ligamentum nuchae Figure 1. Scanning electron micrograph of the surface of adult bovine ligamentum nuchae showing the elastin fibre network. Many of the fibres are orientated in the direction of the long axis of the ligament. × 150.
Figure 2. Scanning electron micrograph showing the same network at higher magnification with surface fibres crossing the main fibre axis. x 1,500.
SEM OF STRETCHED ELASTIN FIBRES
131
13_'
MINN,% \Nl) SII:I\EN
F I G U R E S 3 and 4
Elastin fibres of bovine ligamentum nuchae from tissue which has been stretched or ruptured and allowed to relax Figure 3. Scanning electron micrograph of elastin fibres in a ligament that was stretched and then allowed to relax. All the fibres are orientated in the direction of the original strain (cf. Figure 2). Note the bifurcation of elastin fibres. × 3,500. Figure 4. Scanning electron micrograph of the end of a ruptured elastin fibre showing the smooth appearance of the fracture surface typical of a brittle fracture. × 8,250.
SEM OF STRETCHED ELASTIN FIBRES
133
127
Scanning electron microscopy of stretched elastin fibres R. J. MINNS and F. S. STEVEN Manchester University Connective Tissue Research Group, Department of Medical Biochemistry, University of Manchester, Stopford Building, Manchester M 1 3 9PL, England, U.K.
Manuscript received April 8, 1974
Strips of bovine ligamentum nuchae were (a) stretched to twice their length and then released, (b) stretched until they ruptured, while a third series (c) were not subjected to either procedure and were used as controls. All three series were then treated with buffered guanidine hydrochloride and bacterial collagenase in order to obtain pure preparations of elastin fibres free of their collagenous sheath and matrix. Examination of the controls by scanning electron microscopy revealed a network of elastin fibres with some cross-weaving and cross-branching. However, in strips which had been stretched and released the fibres were orientated in the direction of the strain suggesting that in the absence of the collagen they were unable to return to their original pattern. No broken fbres were observed after stretching. Examination of fibres in ruptured strips revealed some with a smooth fracture face associated with a slight decrease in fibre diameter, an appearance consistent with a brittle fracture. Des bandes de tissus ligamentum nuchae furent respectivement : (a) dtirdesjusqu'it une longueur double de leurs dimensions initiales, puis reldchges; (b) ~tir~esjusqu'it leurs points de rupture; (c) un certain nombre ne fut sujet it aucune des contraintes dgcrites ci-dessus et furent utilisdes comme ~lgments de rdfdrences. Chacune des trois sgries rut ensuite traitde avec une solution tampon d'hydrochlorure de guanidine et de collaggnase bacterial afi~i d'obtenir une prdparation de fibres d¥1astine pures d~pourvues de leur gaines et matrices de collagkne. L' examen des specimens de rdf~rence par microscopie ~lectronique it balq~,age rgv~la un rdseau de fibres d¥lastine primaires et comportant des branches secondaires entrem~lges. Toutefois dans le cas des bandes soumises it une tension puis reldchdes lesfibres gtaient orientges paral#lement au sens de la dgformation, ce qui conduit ~ penser qu'elles sont incapables de revenir it leur situation primitive en l'absence de collag~ne. Aucune fibre rompue ne fut observ~e apr~s dtirage. Quant aux bandesfracturdes l' examen desfibres r~vglaparfois une surface lisse associ~e ~ une diminution du diam~tre de la fibre, c'est-&dire une apparence compatible avec une fracture fragile. Streifen von Ligamentum nuchae des Rindes wurden (a) auf ihre zweifache Liinge gestreckt und dann losgelassen oder (b) bis zum Zerreiflen gestreckt, wiihrend eine dritte Gruppe (c) keiner Behandlung unterzogen wurde und als Kontrollmaterial diente. Alle drei Gruppen wurden dann mit Guanidinhydrochlorid und bakterieller Ifollagenase behandelt, um reine Elastinfaser ohne Kollagenhiille und Bindegewebe zu erhalten. Das mit R E M untersuchte Kontrollmaterial zeigte ein Geflecht aus gewebten Elastinfasern und solche mit Querkluften. In Streifen, die gestreckt wurden, waren die Faser in der Streckrichtung orientiert, was daraufhin deutet, daft sie zu ihrer Originalform nicht mehr zuriick konnten. In diesen Proben gab es keine Anzeichen yon abgerissenen Fasern. In den zerrissenen Streifen sah man einige feink6rnige Bruchfliichen zusammen mit einer Verringerung des Faserdurchmessers, die t"iir einen Spr6dbruch charakteristisch sind.
INTRODUCTION Adult bovine ligamentum nuchae is known to contain a highly organized fibrous network of elastin, each fibre being encircled by a knitted sheath of collagenous material and embedded in a matrix containing a continuous collagen network interconnecting the fibres (Gotte, Mammi and Pezzin, 1972; Finlay and Steven, 1973).
i2t{
NIINNS : \ N I ) S I'EVEN
Which of" tl{ese networks contribute to the mechanical strength and which to> the elastic behaviour is not too well understood. The effect of removing the collagcm~us component from connective tissue by enzymic digestion and then testing the tissue mechanically has been examined by several workers (Hoffman, Grande, Gibson, Park, Daly, Bornstein and Ros;, 1972; Steven, Minns and Thomas, 1974) who found that the elastic fibres were only able to extend to approximately half their original length before rupture occurred. This suggested that at strains above 80-100',a the elastin fibres from adult bovine ligamentum nuchae ruptured while the collagenous network was still intact. This paper reports on the appearance in the scanning electron microscope of elastin fibres from adult bovine ligamentum nuchae that were stretched to 100°o strain and released and other strips which were extended to rupture. The results are compared with those for unstretched elastin fibres from the same tissue and the effect of loading on the structural architecture is discussed. M A T E R I A L S AND M E T H O D S Strips of elastic tissue approximately 3mm thick and 10mm long were cut parallel to the main axis from the surface of the ligamentum nuchae of an adult cow. The internal faces of the strips were clearly marked by knife cuts so that the surface fibres were easily identified after treatment. One series of strips were extended mechanically to 100% of their original length and released. In a second series the strips were extended to rupture (Minns, Soden and Jackson, 1973). A further series were left untouched as controls. All the strips were then stored at --4°C prior to enzymic digestion in order to visualize pure elastin fibres in the scanning microscope. To prepare chemically pure elastin (Steven et al., 1974) all the strips were treated with 6M guanidine hydrochloride buffered to p H 7.0 with 1N N a O H for 18hr at 16°C followed by repeated washing in distilled water. The tissue was then digested with 0.01% bacterial collagenase (Worthington, CLSPA 380 unit/mg) in 0.05M Tris-HC1 buffer pH 7.,5 containing 0.005M CaC12 at 37°C for 18hr followed by repeated washing with distilled water. The tissue was then treated again with the buffered guanidine hydrochloride. Dehydration of the strips was carried out with increasing concentrations of ethanol followed by drying in air in a desiccator for lhr. The specimens were glued to marked metallic stubs and coated under vacuum on a rotating stage with gold-palladium to produce a uniform coating of approximately 20nm. The coated specimens were then examined in a Stereoscan $4 scanning electron microscope (Cambridge Instrument Co.) operated at an accelerating voltage of 30kV. The results were recorded with an Exakta VX500 camera on Ilford FP4 135 film. RESULTS Following treatment with the guanidine hydrochloride and collagenase, the elastin fibres were clearly revealed. In ligaments which had not been subjected to stretching the fibres weaved over and under each other to form an elaborate mesh or net (Figure 1). At high magnification it was observed that while many of the fibres tended to be orientated in the direction of the ligament, there was a considerable degree of branching across the main axis (Figure 2). None of the fibres appeared to be broken.
SEM OF STRETCHED ELASTIN FIBRES
129
A distinct difference in the onela[tttNti 01~ [}[e tlbres was observed in strips which had first been stretched and then allowed to return to their original length• Almost all of the fibres were orientated in the direction of the strain and there was little evidence of the cross-weaving and cross-branching seen previously (Figure 3). Again, however, none of the fibres appeared to be broken. In the strips which had been loaded to rupture and then released, the arrangement of the fibres was again similar to that seen after stretching• This time, however, some of the fibres were broken. As shown in Figure 4, the fracture surface was smooth and there was a slight reduction in the diameter of the fibre at the broken tip, presumably due to 'necking' that occurred just before rupture. The diameter of the fibres ranged from 7-9~. DISCUSSION In studies on horse extensor tendon that had been strained and then allowed to relax, Abrahams (1967) reported that the collagen fibres appeared to uncoil and align themselves parallel to the long axis. In the present work, the elastin fibres appeared to undergo a similar transition during loading, a result shown quite clearly by scanning electron microscopy. Since these fibres are normally covered by a fine framework of collagen fibres (Finlay and Steven, 1973), it seems most likely that during stretching the collagen framework itself becomes considerably elongated. O f particular interest is the fact that in the absence of the collagen, the elastin fibres appear to be unable to return to their normal configuration after stretching. The collagen, therefore, appears to be essential to maintain the cross-weaving pattern seen in the relaxed state and which presumably is of functional significance, for example, by allowing and compensating for lateral stress. In ligaments that were stretched to twice their length there was no evidence that any of the elastin fibres were broken. This suggests that the tensile strength of the ligamentum nuchae depends on the collagen which in turn would obviously afford a measure of protection to the elastin fibres at high load. Finally, it may be mentioned that the appearance of ruptured elastin fibres is characteristic of a brittle failure, with a smooth fracture face associated with a slight reduction in diameter of the fibre at the break. ACKNOWLEDGEMENTS We should like to thank the Department of Biology, Liverpool Polytechnic for the use of the scanning electron microscope and Miss Susan Browne for the technical assistance and in its operation. REFERENCES
ABRAHAMS, M., 1967. Mechanical behaviour of tendon in vitro. A preliminary report. Med. Biol. Engng, 5: 433-443. FINLAY,J. B. and STEW~, F. S., 1973. The fibrous components of bovine ligamentum nuchae observed in the scanning electron microscope. 07. Microscopy, 99: 57-63. GOTTE, L., MAMMI, M. and P~.zzxN, G., 1972. Scanning electron microscope observations on elastin. Connect. Tissue Res., 1: 61-68.
l?,~
MINNS \NI) STEVEN
HOFFMAN, A. S., GRANDE, [,..\., GIBSON, P.. PARK, J. P., I)A1,Y, (i. H., 13ORNs'rvJN. I'. ;l~ld Ross, R., 1972. Preliminary studies on mechanochemical-structure relationships in connective tissues using enzymolysis techniques. In : Perspectives in Biomedical Ertgim'erm4,, Kenedi, R. (ed.), Macmillan, London, 173 176. Mixxs, R. J., SOOEN, P. D. and,JAcKSON, D. S., 1973. The role of the fibrous components and ground substance in the mechanical behaviour of biological tissues: A preliminar}' investigation. J. Biomech., 6 : 1 5 3 165. STEVF,N, F. S., MINNS, R. J. and THOMAS, H., 1974. The isolation of chemically pure elastins in a form suitable for mechanical testing. Connect. Tissue Res., 2: 85-90.
F I G U R E S 1 and 2
Elastin fibres of bovine ligamentum nuchae Figure 1. Scanning electron micrograph of the surface of adult bovine ligamentum nuchae showing the elastin fibre network. Many of the fibres are orientated in the direction of the long axis of the ligament. × 150.
Figure 2. Scanning electron micrograph showing the same network at higher magnification with surface fibres crossing the main fibre axis. x 1,500.
SEM OF STRETCHED ELASTIN FIBRES
131
13_'
MINN,% \Nl) SII:I\EN
F I G U R E S 3 and 4
Elastin fibres of bovine ligamentum nuchae from tissue which has been stretched or ruptured and allowed to relax Figure 3. Scanning electron micrograph of elastin fibres in a ligament that was stretched and then allowed to relax. All the fibres are orientated in the direction of the original strain (cf. Figure 2). Note the bifurcation of elastin fibres. × 3,500. Figure 4. Scanning electron micrograph of the end of a ruptured elastin fibre showing the smooth appearance of the fracture surface typical of a brittle fracture. × 8,250.
SEM OF STRETCHED ELASTIN FIBRES
133