(100h) Insect Cuticle as a Motif for Biomimetic Materials

Insect cuticle's superb mechanical characteristics, as determined by a variety of mechanical tests, are speculated to originate in its dual network structure comprised of chitin and protein enzymatically cross-linked by catechol derivatives. This work examines the mechanical properties of both native insect cuticle and biomimetic hydrogels based on a dual network motif. This research has both basic aspects ? understanding the origin of the mechanical properties of insect cuticle in terms of the relationships between its components ? and applied aspects in developing a simplified version of this motif to produce a dual network hydrogel with improved strength and durability, two characteristics important in biomedical and other applications of gels. The interactions between proteins, chitin and catechols were examined via dynamic mechanical testing and ultimate property tests using a Rheometrics RSA III Dynamic Mechanical Analyzer. Native insect cuticle was studied at several different stages throughout its tanning process (also known as sclerotization and believed to be the result of a chemical cross-linking process). As tanning proceeds, the catechols react with the proteins via an enzymatic process catalyzed by a laccase. The catechols are hydrophobic components and hence the water content of the cuticle decreases as tanning proceeds. Dynamic mechanical analysis is a means to examine the changes in mechanical properties as a function of tanning state, and also has the potential to determine the relevant importance of water loss during sclerotization vis-à-vis covalent cross-linking. Elytra (wing covers) were the insect cuticle samples tested; they were obtained from two types of beetles: Tribolium castaneum (red flour beetle) and Tenebrio molitor (yellow mealworm). Tribolium was chosen because it is an economically important agricultural pest, and it was possible to use RNA interference techniques to selectively suppress laccase gene expression during sclerotization; as laccase is the key enzyme in the quinone tanning theory of sclerotization this would provide a means to test the role of laccase in cuticle tanning. Tenebrio was selected because it is a species closely related to Tribolium but with larger yet geometrically similar elytra to the Tribolium specimens, and hence more amenable to accurate mechanical testing. In fact, it was determined that both types of untanned beetle elytra have similar structural properties and thus it is assumed that the tanning process leads to similar fully tanned properties in both elytra as well. The elytra were tested at three different stages of tanning, as well as in the dried state and for Tribolium, a laccase-suppressed sample. These stages were untanned (Tribolium), 24 hrs after eclosion (Tribolium), 24 hrs after eclosion (Tenebrio) and fully tanned. It was determined that Tenebrio tans faster than Tribolium and has completed more of its sclerotization process after 24 hrs. Samples were mounted directly using the instrument's mounting brackets with the exception of the dried and untanned Tribolium elytra. These proved too fragile to grip directly and thus were mounted on a test frame that provided the gripping surface. Transient mechanical testing indicated that an untanned elytron displays a fracture stress of 0.7 ± 0.1 MPa, a fracture strain of 10 &plusmn 6 % and a Young's modulus (YM) of 8 ± 3 MPa. This data are consistent with data collected via dynamic analysis. The elastic modulus (E') via strain sweep at 1 Hz is equal to 20 ( 6 MPa. Strong frequency dependence of E' is observed for these untanned elytra, indicating that the untanned material is not significantly crosslinked. When fully tanned, elytron fracture stress increases dramatically to 45 ± 12 MPA whereas fracture strain decreases to 3 ± 1 %. Stiffness increases dramatically as shown by a YM of 1674 ± 383 MPa and an elastic modulus of 4860 ± 1840 MPa. No frequency dependence is observed when cuticle is fully tanned, indicative of a significantly crosslinked structure [1, 2]. It was also shown that water loss upon tanning has a much lesser role in the stiffness of the cuticle. Laccase silencing accompanied by water loss resulted in cuticle with both low fracture stress and strain, proving quantitatively that laccase plays a major role in tanning as well as showing that water loss alone is not responsible for the superior mechanical properties of fully tanned insect cuticle. The data do not distinguish between the roles of the protein and the chitin in the cuticle properties. Cuticle can be viewed as a composite material composed of independent dual polymer networks (protein and chitin), potentially arranged as either interpenetrating networks (IPNs) or as semi-IPNs. Recent work by Gong, Osada, and coworkers illustrated the potential of such motifs in synthetic polymer systems tested under compression [3, 4]. PAMPS/ PAAm - poly(2-acrylamido-2-methylpropanesulfonic acid)/ poly acrylamide - as well as agarose/PHEMA (poly(2-hydroxyethyl methacrylate)) are two IPN and semi-IPN gels that were investigated as synthetic model analogs to insect cuticle. The interpenetrating networks (IPNs) consist of a secondary network formed within a primary one during a two-step process. The first network is cross-linked with methylene bisacrylamide (MBA) as the polymerization reaction is initiated via UV light irradiation at 306nm using α-ketoglutaric acid as the photoinitiator. The secondary polymer may (IPN) or may not (semi-IPN) be cross-linked. While the primary network consists of a low fracture stress and low fracture strain hydrogel such as PAMPS and agarose, the secondary network is more flexible and although still displaying a low fracture stress, fracture strain is large, up to 333 ± 29% for PHEMA. Testing was performed using dogbone-shaped gels that were mounted directly on the instrument's mounting brackets with fine grit sandpaper glued to the gripping surface; failure occurred in the gage section of the samples. Results show that mechanical properties of PAMPS/ PAAm gels improve most dramatically, yielding a fracture stress of 7 - 8 MPa and a fracture strain of 29 ±1 % at a water content of approximately 86%. Agarose/PHEMA DN network gels yield a maximum fracture stress of 0.65 ± 0.5 MPa with a fracture strain of 201 ± 33 % at a water content of approximately 79%. In both cases the IPN is much stiffer than the respective secondary network alone, yet it is far more flexible than the primary network by itself. A semi-IPN has also been synthesized in the case of the PAMPS/PAAm gels, using 600-1000 kDa linear PAAm simultaneously with PAMPS in a one step synthesis. High mechanical strength gels may also be achieved this way, which may be a useful mimic of the cuticle motif that may have the chitin physically entangled within the cross-linked protein network. The fracture stress of these synthetic IPN's is close to that found in human cartilage; thus application in the biomedical field of this motif may be possible.

Acknowledgements

This project was funded by National Science Foundation Grant MCB0236039.

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