Biotechnological Potential of MCT

Consideration of the biotechnological potential of MCT must of necessity be highly speculative in view of our currently incomplete knowledge of the molecular organisation of MCT and the molecular mechanism underpinning its variable tensility. Theoretically MCT could be a source of, or an inspiration for, (1) new pharmacological agents or strategies and (2) new composite materials.

Pharmacological Agents or Strategies

It hardly needs to be reiterated that the outstanding property of MCT is its capacity for reversible changes in stiffness. There are certain clinical conditions that would benefit from the therapeutic manipulation of connective tissue mechanical properties, perhaps as an alternative to surgery or other interventions. Most of these conditions would require the temporary or permanent plasticisation or weakening of the connective tissue, rather than its strengthening. This applies to problems like joint contractures due to immobilisation, burn scar contractures, breast capsule contractures following enhancement procedures, Dupuytren's contracture and peritendinous lesions following tendon surgery. Previous suggestions for the pharmacological treatment of some of these conditions have focused on the suppression of collagen synthesis and deposition by, for example, the topical application of lathyrogens such as b-aminopropionitrile, which inhibits an enzyme - lysyl oxidase - involved in intermolecular cross-link formation (Chvapil 1988). On the other hand, structures that need to be strengthened include ligaments and tendons weakened by immobilisation (Nordin and Frankel 1980) and repair sites in trau-matically or surgically transected tendons, which rarely regain full tensile strength (Koob 2002).

Is it possible that MCT contains molecules that could affect the mechanical properties of mammalian connective tissue? As noted above, holothurian chondroitin sulphate relieves joint pain, though there is at the moment no reason to believe that this is due to anything more than the anti-inflammatory or anti-oxidant effect exerted by other chondroitin sulphates and other gly-cosaminoglycans (GAGs) (Delehedde et al. 2002; Campo et al. 2003). It is intriguing, however, that GAGs contribute significantly to interfibrillar force transfer, and therefore the overall mechanical properties, of mammalian connective tissue (Redaelli et al. 2003), and that stiffness changes in the uterine cervix are accompanied by significant shifts in the expression of certain GAGs (Westergren-Thorsson et al. 1998). Whilst this implies that the best way to treat fibrotic lesions might be to engineer in them a GAG composition mimicking that of the compliant cervix, it is feasible that, since holothurian GAGs are components of much more mutable connective tissue, they possess features that might facilitate the 'loosening' of fibrotic tissue and that could be incorporated pharmacologically or genetically into the latter. This illustrates the need to determine both the chemical structure and the precise role in MCT mutability of GAGs, the proteoglycans of which they are constituents, and other, as yet incompletely characterised, interfibrillar components. The benefits that would accrue from a therapeutic strategy that treats successfully the fibrotic conditions referred to above, as well as other common pathophys-iological processes such as pulmonary fibrosis and connective tissue-related stiffening of the walls of hypertensive blood vessels, cannot be exaggerated.

Regarding a completely separate feature of MCT, Szulgit and Shadwick (1998) discovered that the mutable dermis of the holothurian Parastichopus parvimensis has remarkable self-adhesive properties. Dermal autografts or allografts adhered to their implantation site without external pressure or assistance of any sort and shear stresses of 200-500 Pa were required to separate isolated samples after they had been in contact with each other for only 2 h. This property is not due to the entangling of collagen fibres, capillary adhesion or viscous shear forces, but seems to be based on weak chemical bonds. It is independent of the mechanical state of the tissue and is not cell-dependent, and so it seems to be unrelated to mutability, although Szulgit and Shadwick (1998) speculated that a collagen fibril-aggregating factor, such as stiparin, might be involved. This phenomenon merits further investigation, since its elucidation might lead to the identification of chemical factors or mechanisms that could be exploited to promote the adhesion of tissue grafts or artificial skin to wound areas or could be incorporated into MCT-derived artificial tissue (see below).

New Composite Materials

In a recent review, Langer and Tirrell (2004) have drawn attention to the outstanding impact that biomaterials have had on health care, particularly in the context of prosthetic and drug delivery devices, and they commented that the extracellular matrix "provides an important model for biomaterial design". With regard to the development of new structural materials that have medical applications, connective tissue has been employed in three different ways: 1. An entire connective tissue, either living (i.e. with cellular elements left in situ) or with cellular elements removed, may be used as a graft or prosthesis. Examples of this include the use of Achilles tendon allografts for the reconstruction of cruciate ligaments (DeFrate et al. 2004) and skin repair using dermis from different species prepared by various methods (Ramos-e-Silva and Ribeiro de Castro 2002). Obviously, it would be ideal if the mechanical properties of each implant could be adjusted precisely to match the needs of the respective implantation site. For example, skin replace ments need to be more extensible and elastic over the extension sides of joints, such as those of the finger, than over the flexion sides. It has to be admitted, however, that there is unlikely to be much scope for using whole MCT in this way, due to the immunological challenge it would present and the low probability that any echinoderm structure would have a microarchitecture more suitable than that of a mammalian alternative. Techniques would also have to be developed to fix the MCT xenograft in the optimal mechanical state. Furthermore, because of the labile nature of the bonds upon which the integrity of MCT depends, many mutable collage-nous structures become unmanageably friable in the softened state. It is possible, though, that some of those that demonstrate a limited range of tensile changes and never become compliant to the point of disintegration could be exploited. The echinoid compass depressor ligament and peristo-mial membrane are examples of such structures (Wilkie et al. 1992,1993).

2. Components may be isolated from the connective tissue and then reassembled with other biological or synthetic elements to form a novel composite. Examples of biomaterials produced in this way are Integra a combination of bovine collagen, shark chondroitin-6-sulphate and a silicone sheet (the last acting as an artificial epidermis), which is used as a skin substitute (Ramos-e-Silva and Ribeiro de Castro 2002), and acellular blood vessel grafts that consist of intestinal submucosa and bovine collagen (Huynh et al. 1999). It is as a source of such components that MCT could make the most direct contribution to biomaterial design, largely by virtue of the extractability of its collagen fibrils. It is notoriously difficult to extract intact collagen fibrils from the post-fetal connective tissue of vertebrates (Trotter et al. 1997). For this reason, the collagen used in existing reassembled biomaterials is in the form of disaggregated molecules. These have the advantageous property of aggregating spontaneously to form fibrils, but, due to the absence of covalent intermolecular bonds, the fibrils have a low tensile strength. Koob (2002) has developed a method for chemically crosslinking such reconstituted fibrils to make a product that could be used to bridge gaps in damaged tendons. In stark contrast to the vertebrate situation, intact collagen fibrils can be isolated easily from MCT by mild non-denaturing techniques. A solution containing 0.5 M NaCl, 0.05 M EDTA, 0.2 M b-mercaptoethanol and 0.1 M TRIS buffer (pH 8.0) disaggregates holothurian dermis, asteroid body wall and echinoid spine ligaments (Mat-sumura 1973; Matsumura et al. 1973; Trotter and Koob 1989). Even more remarkably, Trotter et al. (1996) discovered that fibrils could be isolated from holothurian dermis using sequential 24-h extractions in artificial sea-water alone. This is evidently a consequence of the weak nature of the interactions that maintain interfibrillar cohesion in MCT, and it means that holothurian dermis and other mutable collagenous structures represent a cheap and easily accessible source of intact collagen fibrils that retain their tensile strength and stiffness and could be used for the manufacture of artificial tissues.

However, a more exciting way in which MCT could contribute to the design of new biomaterials, whether these incorporate MCT-derived components or not, is through the mimicking of the mechanisms responsible for its variable tensility and contractility. There is a clinical need for artificial tissues with site-specific micro-architectures and mechanical properties that are either pre-set or continuously adjustable and responsive to changing physiological parameters or to therapeutic manipulation (Langer and Tir-rell 2004). Amongst possible applications for such 'smart' materials (i.e. which combine the functions of sensors and actuators) would be vascular implants or cuffs that controlled blood pressure or local blood flow, and whose stiffness and/or contractile state were directly sensitive to blood biochemistry or to precisely targeted pharmacological agents, thereby offering an alternative to systemic antihypertensive drugs and their spectrum of unwanted side effects.

Trotter et al. (2000b) have already examined the possibility of developing a simple 'hybrid' biomaterial assembled from collagen fibrils extracted from holothurian dermis and a synthetic interfibrillar matrix. The interaction between the fibrils and the matrix, and therefore the tensile properties of the whole system, would depend on a pair of synthetic molecules that have been shown to selectively and reversibly associate with each other in physiological conditions. These are a catechol and a phenylboronic acid, which complex to form a boronic ester. This interaction can be reversed by oxidizing the catechol to orthoquinone which does not bind to boronic acid (Fig. 6). The synthetic material would consist of collagen fibrils, to which catechol groups had been attached chemically, linked by a soluble poly-acrylamide polymer complexed with phenylboronic acid groups. Trotter et al. envisaged that the association between fibrils and matrix could be repeatedly switched on and off by sequential oxidations and reductions controlled perhaps by optical or electrical signals that change the redox potential of the matrix (Fig. 7). The only MCT-derived elements in this device are collagen fibrils. It is possible that further investigation of MCT will reveal other components that could be built into new controllable biomaterials.


Fig. 6. Interaction between catechol and phenylboronic acid, which provides the reversible cross-links in proposed hybrid biomaterial. (Adapted from Trotter, unpubl.)


Fig. 6. Interaction between catechol and phenylboronic acid, which provides the reversible cross-links in proposed hybrid biomaterial. (Adapted from Trotter, unpubl.)

Fig. 7A, B. Model of proposed hybrid biomaterial. Collagen fibrils (horizontally striated; only one shown) with attached catechol groups are embedded in polyacrylamide polymer (stippled) complexed with phenylboronic acid. A Stiff condition, in which cross-links are formed by interaction of catechol and phenylboronic acid. B Compliant condition, in which cross-links are reversed by oxidation of catechol to orthoquinone. (Adapted from Trotter, unpubl.)

Matrix Model Trotter

3. Connective tissue may provide inspiration for entirely synthetic materials. A simple example resulting from this approach is an artificial tendon which is manufactured from poly(ethylene terephthalate) fibres embedded in a swollen hydrogel matrix and can be designed to have mechanical properties that suit specific implantation sites (Kolarik 1995). Trotter (unpubl.) has speculated that MCT could serve as a model system for the construction of dynamically controllable ligaments with adjustable stiffness and adjustable damping. Trotter has also suggested that these might be incorporated into energy-efficient robots, and it is therefore relevant that there has been interest in the design of'compliant' robots for specialist purposes such as pipeline inspection (Suzumori 1996). As for reassembled biomaterials (see above), both the variable passive mechanical properties and the contractility of MCT may yield design principles applicable to the development of fully synthetic devices.

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