No abstract

The interactions among biomacromolecules within insect cuticle may offer new motifs for biomimetic material design. CPR27 is an abundant protein in the rigid cuticle of the elytron from Tribolium castaneum. CPR27 contains the Rebers-Riddiford (RR) motif, which is hypothesized to bind chitin. In this study, active magnetic microrheology coupled with microscopy and protein particle analysis techniques were used to correlate alterations in the viscosity of chitosan solutions with changes in solution microstructure. Addition of CPR27 to chitosan solutions led to a 3-fold drop in viscosity. This change was accompanied by the presence of micrometer-sized coacervate particles in solution. Coacervate formation had a strong dependence on chitosan concentration. Analysis showed the existence of a critical CPR27 concentration beyond which a significant increase in particle count was observed. These effects were not observed when a non-RR cuticular protein, CP30, was tested, providing evidence of a structure-function relationship related to the RR motif.

Previous dynamic analyses of the temporomandibular joint (TMJ) disc have not included a true preload, i.e., a step stress or strain beyond the initial tare load. However, due to the highly nonlinear stress-strain response of the TMJ disc, we hypothesized that the dynamic mechanical properties would greatly depend on the preload, which could then, in part, account for the large variation in the tensile stiffnesses reported for the TMJ disc in the literature. This study is the first to report the dynamic mechanical properties as a function of prestress. As hypothesized, the storage modulus (E′) of the disc varied by a factor of 25 in the mediolateral direction and a factor of 200 in the anteroposterior direction, depending on the prestress. Multiple constant strain rate sweeps were extracted and superimposed via strain-rate frequency superposition (SRFS), which demonstrated that the strain rate amplitude and strain rate were both important factors in determining the TMJ disc material properties, which is an effect not typically seen with synthetic materials. The presented analysis demonstrated, for the first time, the applicability of viscoelastic models, previously applied to synthetic polymer materials, to a complex hierarchical biomaterial such as the TMJ disc, providing a uniquely comprehensive way to capture the viscoelastic response of biological materials. Finally, we emphasize that the use of a preload, preferably which falls within the linear region of the stress-strain curve, is critical to provide reproducible results for tensile analysis of musculoskeletal tissues. Therefore, we recommend that future dynamic mechanical analyses of the TMJ disc be performed at a controlled prestress corresponding to a strain range of 5–10%.

Summary: CP30 is a major component protein in the rigid cuticle of the beetle elytron which we recently identified. It has a highly unusual amino acid sequence comprised largely of alternating blocks of 3-5 anionic and cationic amino acid residues (three charged residues, glutamic acid, arginine and histidine, make up 76% of the sequence) which suggest an unusual stuctural role. The secondary structure of CP30 was evaluated at a variety of pH, ionic strength and temperature conditions. Circular dichroism (CD), fluorescence spectroscopy and static and dynamic light scattering showed that the protein had an extended a-helical structure at pH 3, 5 and 7 which unfolded around 60 C and reformed upon cooling back to 10 C. The protein aggregated or adhered to the dialysis membrane at pH 5, low salt, which is close to the protein's isoelectric point (5.82), preventing analysis at that condition. Further studies aimed at solubilizing the protein near that condition may uncover evidence of assembly. While the structural function of CP30 is currently unknown, the sequence of CP30 may offer new design motifs for the development of novel polymer or gel biomaterials.