Biol Cell (1994) 82, 59--65 © Elsevier, Paris

59

Original article

The unique fibrillar arrangement of the bullfrog pressure- bearing tendon as an indicative of great functional deformability

Hernandes Faustino de Carvalho, Benedicto de Campos Vidal

Department of Cell Biology, CP 6109 Unicamp, 13083970 Campinas SP, Brazil (Received 29 August 1994; accepted 25 November 1994)

Summary - The fiber distribution and ultrastructure in the plantaris Iongus pressure-bearing tendon of the bullfrog were investigated. The tension region of the tendon showed a predominant parallel distribution of collagen fibers, but three main zones with different crimp parameters were identified with the use of the polarizing microscope. The compression region showed collagen fibers with aspects of disaggregation and were composed of disperse and undulating fibrils. These collagen fibers establish a three-dimensional network but showed a preferential distribution in planes disposed perpendicularly to the tendon's main axis. It is assumed that the con- voluted and disaggregated collagen fibers must be distended before exerting any reinforcement on the tissue and that this only occurs after a great deformation of the tendon. Groups of 5--6 fibrils not associated in fibers are also dispersed in the compression region. The tissue is assumed to have a highly viscous fluid nature allowing for the deformation needed for collagen fibrils to reinforce the tendon structure. The convoluted and crimped structure of collagen fibers would be especially useful when the tendon is submitted to the sud- den and strong mechanical loading expected to occur during jumping and to provide the tendon with the capacity of great functional deformability necessary for the high amplitude of feet movements attained on jumping and swimming.

compressive forces ! crimp morphology / extracellular matrix structure / fiber reinforcement ! pressure-bearing tendon

Introduct ion

Tendon fibrocartilages are found in sites of insertion to the bone and in locations where the tendon is subjected to com- pressive forces. The flexor digitorum profundus tendons of rabbits [8], dogs [11, 12], and cows [5] are found in situa- tions of high compressive loading and exhibit fibrocartilage plaques in the area under pressure.

Studies on the attachment zones and compressive areas in two tendons of the rat demonstrated the existence of dis- tinct phenotypical modification when facing different degrees of compressive loading [16-18]. Under low levels of compression the tendon accumulates chondroitin sul- phate in the extracellular matrix and vimentin intermediate filaments in the cell cytoplasm, even though morphologi- cally the tissue is typically fibrous [18]. At the other extreme, there are zones with fibrocartilaginous morpho- logy both with respect to the intense deposition of glyco- saminoglycans amongst thick collagen bundles and the chondrocyte-like cells disposed in lacunae [16-18].

In a previous paper we described a pressure-bearing ten- don in the bullfrog [2]. As in the mammalian tendons the compression region of the bullfrog plantaris longus tendon accumulates a large amount of sulphated glycosaminogly- cans between the collagen fibers, and round cells are embedded in this glycosaminoglycan-rich matrix. However, the tendon of the frog showed a unique distribution of con- voluted and disaggregated collagen bundles (when loom- pared to the typical parallel arrangement and aggregational state of tendons) in an apparently disordered array [2].

The purpose of this work was to characterize structural features and the macromolecular organization of the fibril- lar components of both tension and compression regions of the frog pressure-bearing tendon, aiming at giving insights on the reinforcing mechanism through which they are

involved in the ability of the tendon to resist compressive forces. Fiber direction and structure were determined by the use of polarization optics and both transmission and scan- ning electron microscopy.

Materials and methods

Animals

The bullfrog, Rana catesbeiana, reared for market purposes was employed. Adult males weighing 100-120 g were killed by decapitation after low temperature immobilization. The tendon of M plantaris longus [2, 10] was used throughout.

Polarization microscopy

Polarization optics were employed to investigate fiber directions and crimp morphology. Analyses were developed on unstained 10/am thick sections obtained as before [2]. In some instances, the tendon was fixed in an extended position by holding the foot inflected during the procedures for paraffin embedding. Crimp parameters described by Dale et al [4] were measured on photo- graphic enlargements of negatives. Observations and micro- graphs were carried out with a Zeiss polarizing microscope.

Transmission electron microscopy

The tendon was dissected out and immediately fixed with 2.5% glutaraldeyde in 0.1 M phosphate buffer (pH 7.2), followed by treatment with 1% tannic acid in the same buffer for 1 h and by treatment with 1% osmium tetroxide. Alternatively, fragments of the tendon were fixed with 2.5% glutaraldeyde plus 0.5% tannic acid in Millonig's buffer for 1 h, followed by an additional hour in 1% osmium tetroxide. Dehydration was carried out in graded ethanol and by treatment with acetone. The material was then embedded in Epon 812. Silver sections were obtained with a dia- mond knife and double stained with uranyl acetate and lead

60 HF de Carvalho. B de Campos Vidal

citrate. Micrographs were made in a Phillips 201M electron microscope. Collagen fibril diameters were measured with a Kontron MOP Videoplan system using photographic enlarge- ments of negatives containing cross-sectioned collagen fibrils with a final magnification of 66000 x.

Scanning electron microscopy

The tendons were dissected out and immediately immersed in liquid nitrogen. The material was then fractured sagitally with the help of a stainless steel blade, maintaining the procedure described earlier [9]. In some instances the fractured surfaces were subjected to treatments with papain (16 mg/ml in 0.1 M acetate buffer at pH 6.0 in presence of 20 mmol/l EDTA and 10 mmol/1 cystein) or 1% testicular hyaluronidase in PBS before fixation. The fixative was the same as used for TEM. Alterna- tively the whole tendon was fixed and then fractured as above. The fixed material was then dehydrated with ethanol, subjected to the critical point drying procedure and sputtered with gold. Micrographs were obtained in a Jeol T300 scanning electron microscope, operating with an acceleration voltage of 20 kV.

Results

The organization of collagen fibers in the tension and com- pression region

The tension region of the frog pressure-bearing tendon is typica l ly fibrous and exhibi ted a highly ordered array of paral lel fibers. F igure 1 shows the cr imp morpho logy of collagen fibers in the tension region. The crimp observed in the superficial areas is visual ly larger than in the deeper areas nearer to the compression region. Table I summarizes the cr imp parameters measured in three areas arbi t rar i ly defined and shown in figure 1. It must be stressed that the apparent complexi ty of figure 1 arises because each zone had its cr imp morpho logy best def ined in pos i t ions not coincident to each other and that though the values pre- sented in table I demonstrated differences in crimp parame- ters between zones, they do not show the whole variabil i ty inside each of them.

The compression region possesses collagen fibers with an undulat ing path, establ ishing a three-dimensional net- work (fig I). Figure 2 shows collagen fibers of the compres- sion region and aspects of their crimp, the parameters of the which are also given in table I. Analyses of cross-sections confirmed previous results on the preferred distribution of fibers in planes perpendicular to the tendon's is main axis [2].

The fibers of the tension region lose their cr imp mor- phology and appear aligned to the tendon's main direction, when the tendon was fixed in a stretched condition. In this situation, polarization microscopy revealed the existence of fibers bending towards the compression region from differ- ent levels of the tension region (figs 3, 4), and the existence of two main areas inside the tension region, which differed in respect to the collagen fiber distribution and compactness (fig 4).

The ultrastructure of the tension and compression regions

The tens ion reg ion is t yp ica l ly f ibrous and exh ib i t ed a highly ordered array of parallel fibrils with few thin fila- ments running in different directions, and apparently con- necting different bundles. The ultrastructure of the tension region resembles that of typical tendons.

The compression region exhibited convoluted and sinu- soidal fibers. Fibri ls inside these fibers were also convo-

Fig 1. Photomontage of an unstained tendon section observed between crossed polarizers. The tendon's main axis is positioned horizontally and is coincident with the analyser direction. I, II and III represent three arbitrarily defined zones in the tension region, the crimp parameters of which are expressed in table I. (If crimp is considered as a wave form, 1 o corresponds to the wavelength and ~o is the angle indicating the deviation of local fibers from overall fiber direction.) The visceral paratenon (VP) exhibits dark and bright bands corresponding to collagen layers lying perpendicular and parallel to the tendon main axis. The net- work of fibrillar components of the compression region is seen in the upper part of the figure. 460 x. Bar = 50/lm.

Table 1. Crimp parameters determined for different zones in the tension (I-III) and for fibers in the compression region.

Zones 21o (p.m) (~o

I 43 12-15 ° Tension region I1 36 10-20 °

IIl 12 22-24 ° Compression region 3.6 27 °

luted and showed peculiar arrangements (figs 5, 7, 8). In some instances, fibers were composed of groups of fibrils, each of them seeming to be very independent from the oth- ers (fig 7). The apparently disordered arrangement of con- voluting and kinking collagen fibrils is evident in the thin sections, where the marked edge of fibers can be delineated (fig 5). Spaces between fibers as those seen in figure 5 are actually filled with ruthenium red-posi t ive material (Car- valho and Vidal, in preparation) and showed filaments like

-..0

Q4W

.11

Bullfrog tendon arrangement 61

Figs 2--4. 2. Unstained tendon section observed in the polarizing microscope to show the crimp morphology of the thicker fibers in the compression region. The crimp parameters of these fibers are presented in table I. The fibers indicated by the arrows are

those observed in figure 10, which are not resolved at this lower magnification.

In SEM, the major fibers of the compression region were shown to possess a coarse covering of apparently homoge- nous material which masks their fibrillar structure (fig 6). This covering is almost indistinguishable in thin section after the staining procedures employed in this work, as observed in figure 5. The fiber covering was removed by the treatments with either testicular hyaluronidase (fig 7) or papain (fig 8) and the fibrillar structure o f the collagen fibers was revealed to be quite distinct from the parallel array observed in the tension region, but shown to be very convoluted and apparently disordered (figs 7, 8), according to the images obtained with TEM. Cell processes were identified amongst the collagen fibrils in the compression region.

The compression region has also been shown to possess thin fibers composed of groups of 3 to 6 fibrils dispersed in the matrix and with which filamentous and granular materi- als were associated (figs 9, I0). These fibrils exhibited aspects of kinking or folding and a number of microfila- ments were observed around them (fig 10).

Collagen fibril diameters in the tension and compression regions of the frog tendon

Fibrils of the tension region exhibited a large variation in diameter, reaching relatively high values. The fibrils of the compression region had smaller diameter values. Figure 11 representatively shows the distribution of diameter of colla- gen fibrils f rom both tension and compression regions. According to the statistical parameters (table II), a high dis- pers i ty o f values measured in the tension region was observed, and the diameters of fibrils in the compression region concentrated in the smaller values. The mean cross- sectional area occupied by collagen fibrils is higher in the tension region (table II).

Discussion

The organization and ultrastructure of collagen fibers

The tension region of the frog tendon showed a parallel arrangement of fibers, but it was also possible to identify variations in this distribution, as demonstrated by the differ- ences in crimp parameters and the existence of fibers that bend towards the compression region. The existence of fibers bending towards the compression region appears to be involved with the integration of both regions in a unique

parallel to the polarizer. 219 x. Bar = 50/zrn. 3, 4. Unstained sec- tion of a frog tendon which was processed in an inflected posi- tion. The fibers are mainly aligned to the tendon main axis and lack the crimp morphology observed previously. 3. The main axis of the tension region at 45 ° to the polarizers. In this situation two main areas can be seen in the tension region, an outermost one with strong birefringence indicative of greater compactness and an innermost one, close to the compression region with wea- ker birefringence, it being apparently less compact. Dark fibers are also observed bending towards the compression regions. 4. The material has been rotated slightly and the dark fiber seen in figure 3 is now bright. In this position the innermost area of the tension region is almost completely dark and the outermost area is still birefringent. 3, 4. 87.5 x. Bar = 100/.tm.

62 HF de Carvalho, B de Campos Vidal

, g , d ~ . . ~ - 7 " "" ~ , i , -.-. ' _e- - ~ . . . .

f,;~-~ .. "; ~, .. , ..-.,-,; ---,,>

.... - ~.~:-. . . _.- ........ :

qS

.J" ~~

' +. , t,r,

Figs 5-8. 5. Thin section of adjacent fibers in the compression region showing the convolution and "kinking of their composing fibrils. Each bundle has a marked delimitation of its surface by an almost indistinguishable material (arrowheads). The existence of spaces be- tween fibers can also be seen. 11000 x. Bar = l/.tm. 6. SEM of a convoluted fiber of the compression region. The existence of an appa- rently non-fibrillar material which masks the fibrillar structure of the fiber is evident. 2 100 x. Bar = 5 um. 7. SEM of a testicular hyalu- ronidase-treated fiber of the compression region. The enzymatic treatment removes the covering observed in the previous figure and reveals the three-dimensional structure of the convoluted fibrils inside. Each fiber is composed of a number of groups of collagen fibrils which are extremely convoluted inside the limits of the fiber. 1650 x. Bar = 5/.tm. 8. SEM of a papain-treated fiber of the compression region. The treatment with papain also removes the covering of the fiber and reveals the convolution of the fibrillar elements as well. 1100 x. Bar= l0/.tin.

system able to transmit tension and to resist pressure. The cr imp parameters demonst ra ted that the tens ion region exhibited a microdomain organization, possibly associated with the curled morphology of the tendon. The crimp mor- phology variabi l i ty suggests a functional different iat ion alongside of the tension region and this could be related not on ly to d i f fe rent b iomechan ica l propert ies but also to degree with which the different zones are loaded, depending on the intensity of the mechanical stimulus. According to this assumption, only in strong movements would all of the zones of the tension region be loaded. As assumed for

different systems, the crimp may also be responsible for damping strong and sudden strain [6].

The c o n v o l u t e d co l l agen fibers of the compress ion region were shown to be composed of fibrils not only dis- tant from each other but also disposed in directions not coincident with the long axis of the fiber. This apparently disordered array of fibrils produced aspects of disaggrega- tion and weak birefringence.

Each fiber was delimited by a proteoglycan-rich matrix identified by its enzymatic susceptibility and was related to the metachromatic material observed after staining with

Bullfrog tendon arrangement 63

. t .

i t . "

* .

, - b

-,.:

~1~ ̧

o - O ." • .*

Figs 9, 10. Thin fibers of the compression region as seen by SEM and TEM, respectively. SEM images disclose the existence of a large number of thin fibrillar components in the compression region beside the thick fibers. Fibro-granular material associates with the fibrils and extend into the adjacent matrix. TEM demonstrates that the thin fibers of the compression region are composed of a few kinking collagen fibrils. Many fibrillar com- ponents are associated with these thin fibers and extend into the neighboring matrix. 9. 2600 x. Bar = 5/.tm. 10. 55000 x. Bar = 250 nm.

cationic dyes [2]. The accumulation of this proteoglycan- rich material on the fiber surface may be attained artifactu- ally due to collapsing of the non-fibrillar matrix on the col- lagen fiber surface during dehydration.

The convoluted and undulated morphology of the fibers in the compression region and the arrangement of their fibrils indicate that these fibers are capable of undergoing great distension before exerting resistance (fig 12). Consid- ering the mechanisms of fiber reinforcement [6, 7, 13], it is apparent that the unstretched fibers would contribute only with visco-elastic properties against the applied pressure. This is consistent with the view that this region is able to withstand great deformation. The three-dimensional distri- bution of these fibers suggests that the compression region may accomodate deformations in many directions, but the predominant distribution of fibers in planes disposed per- pendicularly to the tendon's main axis [2] indicates that the compressive forces applied to the tendon are dissipated in a perpendicular direction.

The collagen fibrils of the tension region do not exhibit the bimodal distribution of diameter values commonly found in tendons [13-15] and other pressure-bearing ten- dons [8-12]. From the overall study undertaken, it was observed that the tension region in the curled area of the tendon had macromolecular modifications of its own, and that these characteristics might be responsible for the atypi- cal distribution of fibril diameters. However, the tension region possesses both very thick and very thin collagen fibrils, in such an array that 50% of the sectional area is occupied by fibrillar elements. This is in accordance with the scheme proposed for the increase in the volume fraction of fibrils achieved by the bimodal distribution of diameters, without a disbalanced increase in fibril thickness [7, 13].

Increments in the aggregational state of collagen fibrils have been noted after controlled exercise [21] and ageing [19] and this was assumed to be due to increasing loads and correlated with published results on increasing fibril diame- ter in these different situations [13-15]. Following these assumptions, it is suggested that the thinner and less aggre- gated fibrils of the compression region are individually sub- jected to smaller strains.

Featuring relevant aspects of the bullfrog tendon biomechanics

While in cartilages the proteoglycan content and organiza- tion are assumed to have importance in the resilience and capacity to resist compression, it must be assumed that, at least in the frog tendon, the proteoglycans also work in pro- viding the convoluted collagen fibers with a viscous fluid which allows them to become taut before exerting any rein- forcement on the tendon structure.

It is well known that collagen may establish complex arrangements, distinct from those found in tendons and liga- ments, and exhibit biomechanical properties compatible with the tissue structure and functions [20]. The collagen arrangement in the compression region is not comprehen- sive, unless it is assumed they can act under extreme condi- tions of physical stimulation, which is apparently associated with a high degree of deformation, resulting from the exis- tence of compression and friction during the mechanical loading of the tendon.

The analyses carried out in this work demonstrated that the plantaris longus tendon of the frog had a particular arrangement of collagen fibers and non-fibrous matrix which differs from the mammalian tendons under compres- sion hitherto described [1, 22]. The differences observed

64 I-IF de Carvalho, B de Campos Vidal

2 5

0

: ~ :-'q * i , i , , e - t , ,

" , , ¢ . . J t.= , r ~

' L= " "

2'5 .50 #5 1(30 1~.5 150 175 200 2 ~ . 5 2 5 0

fibril d i a m e t e r (nm)

11 Fig 11. Distribution of the diameter values obtained from fibrils of the tension (dashed line) and compression (full line) regions. It can be noted that the values obtained for the compression region concentrate in the small diameters while the tension region shows a wide distribution Of values, ran.ging from a few up to 250 nanometers.

A

A O Number of fibrils Mean diameter

SD II Median

Sectional area I occupied by

B collagen fibrils

A

12 Fig 12. A highly schematic model for the collagen fibers of the compression region and the possible modifications they undergo under progressive loading. A convoluted fiber (I) is initially stretched to the condition shown in 11. Further loading is neces- sary to distend the composing collagen fibrils to the condition set in III. According to principles of fiber reinforcement [7, 13], only in this latter situation are all the collagen fibrils involved with reinforcing the tendon structure. As demonstrated by the position of the markers A and B, a relatively great deformation of the structure occurs before the fibrils resist to further loading, reinforcing the tissue structure.

may likely be related to the degree of deformation these tendons are able to undergo following mechanical stimula- tion, even though, as in the mammalian tendon fibrocartil- ages, this is associated with the existence o f compressive forces.

In a previous paper [2], it was suggested that the frog tendon was capable of undergoing great deformation before the collagen fibrils become taut and act in the reinforcement

Table 11. Morphometrics of collagen fibrils in tension and com- pression regions of the bullfrog pressure-bearing tendon.

Tension Compression region region

422 538 111 nm 78 nm 56 nm 22 nm

100 nm 80 nm

51% 36%

of the structure. The macromolecu la r organizat ion pre- sented here supports this assumption and confirms that the compression region in the frog plantaris longus tendon has structural elements allowing for a functional deformation. The dissipation of compressive forces in multidirectional components in the compression region must occur through its sinous fibers of convoluted fibrils, associated with elastic and pre-dastic components (Carvalho and Vidal, submitted) and special cellular dements [3], working together to pro- vide the necessary compliance and restoration of the resting morphology.

Acknowledgments

The authors gratefully acknowledge the collaboration of Dr CA Ferraz de Carvalho, Dr Fernando Galembeck, Dr EA Greg6rio and Dr MA Cruz-HOming for providing them with facilities during the realization of this work. Thanks are also due to MA Boleta, MH Moreno, AM Ferreira Lima and AC Sarti-Sprogis for the skillful technical assistence in different steps of specimen preparation and operation of the electron microscopes. The Kontron MOP Videoplan System was a kind donation by the Bavarian Cancer Society. Part of this work appeared as abstracts in the Annals of the XIII Meeting of the Brazilian Society for Electron Microscopy and of the VII Brazilian Congress of Cell Biology. This work was car- ded out with partial financial support of FAEP/UNICAMP.

Bullfrog tendon arrangement 65

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