Molecular Biomimetics of Non-Mineralized Hard tissues

Minerals impart strength, stiffness, and wear resistance functionalities to biomaterials that assist in performing their mechanically active or supportive roles. In vertebrates, bones and teeth, composed of 70-90% of hydroxyapatite, are two classic examples. Recently, it has been recognized that in addition to using minerals, Nature also applies a contrasting strategy to make tissues with grinding or biting functionalities. For instance, studies highlight that in invertebrates’ jaws, strength and wear-resistance are achieved by metal ion cross-linking within a proteinaceous network. This involves dense protein cross-linking, and/or the formation of polysaccharide or protein complexes.

Such findings offer a novel paradigm for the biomimetic synthesis of robust and biocompatible structural polymers with little or no mineral content. These biomacromolecular networks feature remarkable mechanical performance twice in magnitude to that of the strongest synthetic polymers. In conjunction with that, their mechanical gradients can be controlled by a biomolecular gradient, which minimize interfacial stresses and confers the material with unrivaled abrasion resistance. In joint implants, wear and abrasion-resistance is a long-standing problem. Successful mimics of these organic networks can potentially be applied as restorative materials. Further, in the field of wear-resistant materials, the absence of minerals offers a new paradigm of research. Protein-based polymers also hold great potential to solve the growing sustainability issues associated with the production of polymers from fossil fuels (plastics) and their enormous accumulation in oceans and landfills (learn more).

To synthesize such biopolymers, it is essential to understand their natural processing and the incorporation of building blocks (proteins, polysaccharides) into the final assembled tissue. Exploring these elusive features is necessary to duplicate Nature’s benign processing conditions/materials and constitutes a core aspect of our research.

Squid Sucker Ring Teeth and Suckerin Proteins

Sucker ring teeth (SRT) from cephalopods such as jumbo squids (Dosidicus gigas) or cuttlefish are a fascinating model system of interest that we have thoroughly investigated in recent years. These extra-cellular hard tissues, embedded on the squid’s arms and tentacles, assist in firmly grasping and handling the prey; a process that is mechanically demanding and requires high resistance against compressive, bending, and shearing load regimes.

Our research efforts concluded that the SRTs are made of proteins only and exhibit mechanical properties comparable to the best structural synthetic polymers (Advanced Materials, 2009). Unusually, such high mechanical performance is achieved in the absence of inter-chain chemical cross-linking. It is noted that a dense network of hydrogen bonds stabilizes SRTs. By combining RNA-sequencing (first report in the field of natural biopolymers) with classical and high-throughput proteomic techniques, we have shown that in SRT, modular “suckerin” proteins assemble into a supramolecular network reinforced by nano-confined β-sheets (Nature Biotechnology, 2013, ACS Nano, 2014)

Owing to the supra-molecular assembly observed, we illustrated that SRTs exhibit thermoplastic properties, which is an exceptional feature for a protein-based material. Therefore, SRTs can be re-processed, and the proteins can be molded into complex shapes by simple lithographic techniques. These characteristics make SRT a promising material as “bio-ink” for 3D bio printing (Nature Communications, 2015).

Revealing the molecular-scale structure-property relationships in SRTs and suckerins, identifying their full-length protein sequences, we established recombinant expression systems to express suckerins in large quantities. Utilizing the distinct molecular architecture of suckerins, we have designed biomaterials that span 7-orders of magnitude in elasticity. The soft gels match the elasticity of the liver, while the stiff films have similar Young’s modulus to that of the bone (Biomacromolecules, 2014).

The ease of processability and redox activity of suckerins can induce the growth of metallic nanoparticles without reducing agents, including redox-active nano-structured solid substrates (Macromol. Rapid Commun., 2015).

We have also engineered suckerins for nanomedicine applications. Suckerins can be prepared into nanoparticles with a controlled particle size ranging between 100-200 nm. It is successfully shown that these nanoparticles can be used for efficient drug delivery, gene transfection (in vitro & in vivo), and can effectively inhibit tumor growth in vivo. Furthermore, hydrophobic drugs can be encapsulated in these nanoparticles for pH-dependent release in vitro. Suckerins can also complex with and stabilize plasmid DNA. The hydrophobic interactions in the β-sheet domains stabilize complexes, as opposed to electrostatic interactions commonly achieved with cationic polymers. Hence, the chances of cytotoxicity are lowered, which is traditionally associated with such polymers (ACS Nano, 2017).

Biomolecular gradients: Squid Beak proteins

Another model biological structure we have investigated, that also originates from cephalopods are squid beaks. The beak exhibits a remarkable 200-fold mechanical gradient - from its soft base embedded within its buccal mass to its hard tip. This hard tip is used to tear apart preys and is one of the stiffest, wholly organic materials. The beak is solely made of organic components, namely chitin and proteins, the latter being the dominant phase at the tip. The research findings were published in Science (2008).

Due to the extremely high cross-link density made of covalent cross-links between histidine side chains and catechols (JBC, 2010), the extraction and sequencing of squid beak proteins were virtually intractable for years. However, with next-generation RNA-sequencing technology, the transcriptome of the precursor cells that secrete the beak (“becublasts”) in combination with proteomics of short protein extracts was constructed. This enabled the full-length sequencing of squid beak proteins (Chemical Biology, 2015).

Squid beak proteins were divided into two protein families. One family consists of chitin-binding proteins (DgCBPs), predicted to physically join chitin chains to form the beak nano-porous scaffold. The other family comprises of highly modular histidine-rich proteins (DgHBPs) that play key roles during beak bioprocessing. We have demonstrated that DgHBPs can form concentrated protein “coacervates” that can spontaneously spread and infiltrate the nanoporous chitin scaffold, comparable to classical processing methods used to fabricate polymer matrix composites. Furthermore, DgHBPs contain the Glycine-Histidine-Glycine (GHG) sequence motif in abundance. This motif is responsible for final cross-linking and curing via a pH-triggered mechanism. These processes generate spatially controlled desolvation, resulting in the impressive biomechanical gradient of the beak.

Our objective is to utilize the knowledge to design functionally gradient materials from biomolecular building blocks such as recombinant proteins or shorter peptides. We also aim to create eco-friendly polymer-matrix composites using aqueous-based chemistry.