Mechanical Adaptability of MCT

Passive Mechanical Properties

Each mutable collagenous structure exhibits one of three patterns of change in its passive tensile properties: (1) only reversible changes (e.g. in viscosity or stiffness); (2) irreversible destabilisation (always associated with autotomy) as well as reversible changes; or (3) only irreversible destabilisation (Wilkie 2002).

The most extreme manifestation of MCT mechanical adaptability is the rapid and irreversible loss of tensile strength undergone by collagenous structures that cross echinoderm autotomy planes. For example, when an arm of the ophiuroid Ophiocomina nigra is autotomised, the ultimate tensile

Fig. 1A—I. Passive mechanical behaviour of MCT. A Creep test (in which extension of sample under constant load is recorded): in response to 100 mM K+ (K) syzygial ligament of crinoid Antedon mediterranea shows sudden decrease in viscosity culminating in rupture (star). Horizontal scale bar 1 min; vertical scale bar 1 mm (adapted from Wilkie et al. 1999). B, C Creep tests: viscosity of dental ligament of echinoid Diadema setosum is increased reversibly by 1 mM acetylcholine (ACh) and 100 mM K+ (K). ASW Artificial seawater (adapted from Birenheide et al. 1996). D Stress relaxation tests (in which samples are subjected to constant deformation and force is recorded): average force relaxation curves of 20 dental ligaments of echinoid Dendraster excentricus treated with artificial seawater (ASW) and 15 ligaments treated with divalent cation-free seawater (DCF) (adapted from Ellers and Telford 1996). E, F Stress-strain tests (in which force is recorded while samples are stretched at fixed extension rate) conducted on spine ligament of echinoid Anthocidaris crassispina: Examples of stress-strain curves produced by samples treated with E 0.1 mM acetylcholine

(ACh) and F 0.1 mM adrenaline (Adr). Horizontal scale bar Strain of 10% in both cases; vertical scale bar 10 MPa in E and 1 MPa in F (adapted from Hidaka and Takahashi 1983). G-I Dynamic stress-strain tests (in which force is recorded while samples are subjected to oscillating strain): hysteresis loops produced by repetitive testing of dermis of holothurian Actinopyga mauritiana in three mechanical states. During these tests, maximum strain (indicated by number above each curve) was increased incrementally; note that sample in 'soft' state showed strain-induced softening. (Adapted from Motokawa and Tsuchi 2003)

strength of the intervertebral ligament at the autotomising joint drops to less than 0.1 % of the normal value in a timescale of 0.4-5.4 s (Wilkie 1988). These drastic changes in mechanical properties can also be demonstrated experimentally using isolated tissue preparations undergoing creep tests (in which their extension under constant load is recorded). Treatment with neuro-active agents such as elevated [K+] or appropriate neurotransmitter chemicals causes an abrupt decrease in viscosity (stress-strain rate) culminating in tissue rupture (Fig. 1A).

Reversible changes in mechanical properties have been quantified and analysed by means of various testing methods. Reversible changes in viscosity have been demonstrated in creep tests and stress relaxation tests (in which specimens are stretched to a particular length, which is fixed whilst force decay is recorded) (Fig. 1B-D). Reversible changes in tensile strength, tensile stiffness, dynamic shear stiffness, relative damping, etc. have been investigated by means of standard stress-strain tests (Fig. 1E,F) and by dynamic testing methods in which samples are subjected to oscillating strain (Fig. 1G-I). Evidence of mutability is often apparent in the wide variability in the mechanical properties of untreated tissues. Motokawa (1983), for example, found a 200-fold difference in the viscosity of untreated central spine ligaments of Diadema setosum taken from different joints of the same animal. In order to overcome this problem and compare tissues in predictable mechanical states, a common strategy has been to subject them to different treatments that induce either maximal or minimal values of stiffness, viscosity, etc. (see Fig. 1G-I). Although biologically relevant stimuli, such as mechanical compression, have been used in these investigations, the artificial nature of some treatments, especially those employed to bring about a compliant state, engender some uncertainty about the physiological relevance of the results thus obtained. Nevertheless, these methods no doubt give an indication of the magnitude of the reversible changes that MCT can accommodate in vivo. Hidaka and Takahashi (1983), for instance, found that at a low strain rate the ultimate tensile strength and elastic modulus of an echinoid spine ligament in the compliant condition (induced by 0.1 mM adrenaline) were around 1 % of the values measured in the stiffened condition (induced by 0.1 mM acetyl-choline), the latter falling within the range reported for mammalian tendon (Redaelli et al. 2003; Fig. 1E,F).

Data from these biomechanical studies have been used to generate models with the ultimate intention of specifying the contribution of different extracellular components to net mechanical properties and determining which of them contribute to variable tensility. Due to the diversity of testing regimes employed, the conclusions from different investigations have been incompatible (see, e.g., Szulgit and Shadwick 2000; Motokawa and Tsuchi 2003) and are difficult to interpret in the light of the increasingly complex picture of the MCT extracellular matrix that is emerging (see Sect. 2.3 below). It seems likely that the full potential of this methodology will not be realised until it can be applied to tissues from which specific components have been eliminated chemically, enzymatically or, ideally, by genetic knockout (see Bornstein et al. 2000 and Chakravarti 2002 for examples of this last approach applied to mammalian connective tissues).

Active Contractility

Certain mutable collagenous structures can generate tensile force. In the case of the capsular ligament, or 'catch apparatus', of the echinoid spine-test joint, this is attributable to the presence of fine (diameter 0.1-1.0 |m) muscle fibres that are distributed between the bundles of collagen fibrils and constitute 1-3 % of the total cross-sectional area of the ligament. The neurotransmitter acetylcholine increases the tensile strength and stiffness of this ligament (Hidaka and Takahashi 1983; Morales et al. 1989), but also causes isolated preparations of it to shorten and develop mechanical force, which relaxes as soon as the acetylcholine is removed. Both the contraction and relaxation phases of this response can be very fast, the former in some cases reaching 90 % of the maximum in under 1 s (Fig. 2A; Vidal et al. 1993). The functional significance of the muscle fibres is at present unknown. Del Castillo et al. (1995), ignoring incontrovertible evidence that the spine ligament consists of MCT (see, e.g., Hidaka and Takahashi 1983; Szulgit and Shadwick 1994), hypothesised that they are responsible for varying its passive stiffness, which they achieve by adjusting the frictional forces between the ligament fibres and the skeletal ossicles into which they are inserted. Having provoked a frank exchange of views (see Del Castillo and Smith 1996; Wilkie 1996,2002; Pérez-Acevedo et al. 1998; Elphick and Melarange 2001), this hypothesis was tested and disproved by Takemae and Motokawa (2002). The muscle fibres also do not seem to be involved in straightening out the wrinkles that form transiently in regions of the ligament that are compressed by contraction of the spine muscle (Pérez-Acevedo et al. 1998). This leaves the possibilities that they assist the reshortening of stretched ligament fibres or that they operate syner-gistically with the spine muscle, perhaps during specific manoeuvres such as re-erection of the spine.

The reputation of echinoderms for being an inexhaustible mine of biological novelty has been enhanced by the discovery that, as well as varying their passive mechanical properties, ligaments in the cirri and arms of crinoids have the capacity for active contractility, though they lack myocytes. Cirri are finger-like appendages supported by a single series of interarticulating ossicles. The only mechanically significant structures connecting adjacent ossicles are myocyte-free collagenous ligaments. Cirri attached to the stalk of sea lilies bend upwards, against gravity, when the stalk or the cirri themselves are stimulated mechanically. In stress relaxation tests, isolated cirri display slow force production in response to the cholinergic agonists muscarine and methacholine at concentrations as low as 0.1 |M (Fig. 2B). Since force produc-

Fig. 2A-F. Force generation by MCT. A Contraction of spine ligament of echinoid Eucidaris tribuloides induced by 0.1 mM acetylcholine (arrow) which was removed as soon as force peaked (adapted from Vidal et al. 1993). B Contraction of cirral ligaments of crinoid Metacrinus rotundus induced by 0.1 |m methacholine (M) (adapted from Birenheide et al. 2000). C-F Responses to 100 mM K+ (K) of arm ligaments of M. rotundus. In each case, upper trace shows upward displacement of arm tip (caused by shortening of ligaments) and lower trace shows stiffness changes in ligament. Horizontal scale bar 3 min in all cases. C Contraction associated with stiffening. D Contraction without stiffness change in 100 mM K+ and contraction with stiffening when excess K+ was removed. E No contraction, but with marked stiffening. F No contraction, but with destiffening. (Adapted from Motokawa et al. 2004)

Fig. 2A-F. Force generation by MCT. A Contraction of spine ligament of echinoid Eucidaris tribuloides induced by 0.1 mM acetylcholine (arrow) which was removed as soon as force peaked (adapted from Vidal et al. 1993). B Contraction of cirral ligaments of crinoid Metacrinus rotundus induced by 0.1 |m methacholine (M) (adapted from Birenheide et al. 2000). C-F Responses to 100 mM K+ (K) of arm ligaments of M. rotundus. In each case, upper trace shows upward displacement of arm tip (caused by shortening of ligaments) and lower trace shows stiffness changes in ligament. Horizontal scale bar 3 min in all cases. C Contraction associated with stiffening. D Contraction without stiffness change in 100 mM K+ and contraction with stiffening when excess K+ was removed. E No contraction, but with marked stiffening. F No contraction, but with destiffening. (Adapted from Motokawa et al. 2004)

tion can be induced in preparations that have undergone some stress relaxation prior to stimulation, it cannot be explained in terms of the passive recoil of a previously stretched elastic element (Birenheide and Motokawa 1995, 1998; Birenheide et al. 2000).

The mobility of crinoid arms depends on the presence of muscular articulations between adjacent arm ossicles. Below the fulcral ridge of each articulation is a single aboral ligament and above it are paired oral ligaments and paired muscles. Contraction of the muscles bends the arm orally (upwards),yet the power stroke for locomotion by swimming, crawling or climbing is generated by the aboral (downward) flexion of the arm and must be effected by the ligaments. It had long been assumed that this involved the purely passive elastic recoil of stretched aboral ligaments, and perhaps of compressed oral ligaments, these functioning like expanded and compressed springs respectively (see, e.g.,Young and Emson 1995). However, Birenheide and Motokawa (1996, 1998) established that the aboral ligament can shorten slowly and generate a tension of up to ca. 5.6 kPa. Because this ligament is a mutable collagenous structure, it is feasible that its apparent contractility results from its becoming stiff whilst it is stretched (by muscle-mediated oral flexion); it would then store strain energy until appropriate stimulation induced its destiffening and thereby allowed it to recoil elastically and reshorten. This 'spring-with-a-lock' hypothesis was shown to be untenable by Motokawa et al. (2004) who, by recording simultaneously stiffness and shortening, demonstrated the independence of passive mechanical properties and contraction: contraction, for example, does not require the ligament to be in a destiffened condition (Fig. 2C-F). The fact that contracting ligaments are usually destiffened, however, indicates that there is coordination of the passive and active mechanical properties,both of which appear to be under cholinergic control.

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