Studies on stabilization of collagen using Cr-doped polydopamine complex.

The stabilization of collagen using different small molecules have been explored for the development of biomaterials. One of the most studied molecules in biomaterials research is polydopamine (PDA) due to its ability to bind to different substrates that ranges from metal surface to collagen. Similarly, in leather tanning, chromium has been an extensively used metal ion as it binds strongly with collagen and enhances its stability. However, as per regulatory authority, the presence of chromium in leather has been restricted to minimum level. Here, we studied the application of chromium doped polydopamine (Cr-PDA) complex as collagen stabilizing agent. The preparation and characterization of Cr-PDA were confirmed using FE-SEM, DLS and FT-IR techniques. Cr-PDA did not alter the triple helical structure of collagen as evidenced from the CD spectral data. Cr-PDA delays the fibrillation in collagen compared to collagen or PDA alone. Calorimetric data shows the enhanced stability of collagen when treated with Cr-PDA compared to collagen control but lesser than PDA alone. Viscometry studies have shown that Cr-PDA reduces the viscosity of collagen compared to PDA or collagen alone. Contact angle studies showed that PDA and Cr-PDA imparts more hydrophobicity to collagen compared to control. Tensile strength studies showed that addition of Cr-PDA or PDA increases the tensile strength of the collagen fiber. The present study on stabilization of collagen using Cr-PDA might be helpful in development of crosslinking agents with eco-friendly approach.

Physico-chemical characterization studies of collagen labelled with Ru(II) polypyridyl complex.

The rich luminescence behaviour exerted by transition metal complexes has found significant role in the development of biomolecular and cellular probes. The conjugation of fluorophore to a protein has its own advantage over the label-free system due to its high sensitivity. While numerous proteins have been labelled with either organic or inorganic fluorophores, the conjugation of luminescent transition metal complexes with collagen has not yet been attempted. Here, in this study, the conjugation of a Ru(II) polypyridyl complex with collagen was carried out and its physico-chemical characterization was studied. The conjugation of Ru(II) to collagen was characterized by UV-Visible, fluorescence and ATR-FT-IR spectroscopy. The conjugation of Ru(II) did not alter the triple helical structure of the collagen as evidenced from CD spectral data. The luminescence behaviour of the Ru-tagged collagen was found to be similar to that of the commercially available fluorescein isothiocyanate (FITC) tagged collagen with increase in luminescence upon addition of collagenase. Gel-based collagenase assay showed that the digestion of collagen can be vizualized using UV light due to intrinsic fluorophore tag without carrying out the staining-destaining processes. Energy dispersive X-Ray analysis (EDAX) confirms the presence of Ru in Ru-collagen fibrils. To the best of our knowledge, this is the first report on the conjugation of a Ru(II) complex with the fibrous protein collagen that exhibits similar property as of FITC-collagen and can be used as an alternative.

Fibrous protein composite scaffolds (3D) for tissue regeneration: An in vitro study on skeletal muscle regeneration.

The present study explores the differentiation of myoblasts in bioengineered 3D composite scaffolds containing keratin and gelatin. Based on the composition and rheological properties three different scaffolds namely HM1, HM2 and HM3 were prepared, characterized and employed for the present study. The scaffolds were then subjected to C2C12 myoblasts differentiation under in vitro conditions as per the standard protocols. Results reveal a wide variation in the elastic modulus, water uptake and degradation rate of the scaffolds significantly impact the myogenesis processes. Composite scaffolds HM2 and HM3 ease the myogenesis compared to HM1, wherein, results in nil myogenesis. Among HM2 and HM3, accelerated myogenesis and the significant expression of myogenin mRNA levels along with extensive myotube development were observed in the HM3 scaffold. In conclusion, scaffolds modulus play a vital role in myogenesis and the observations of the present study provide a possible strategy for better skeletal muscle regeneration using composite scaffolds.

Physical Properties of Extrudates with Fibrous Structures Made of Faba Bean Protein Ingredients Using High Moisture Extrusion.

Faba bean is a potential ingredient due to its high protein yield and its possible cultivation in colder climate regions. In this study, meat analogues made from faba bean protein isolate (FPI) and concentrate (FPC) blends were produced using high moisture extrusion. The aim of this study was to investigate the effect of the FPI content (FPIc), feed water content (FWC), and temperature of the long cooling die (LT) during extrusion on the mechanical and physicochemical properties as well as on the structure of the meat analogues. Increased FPIc resulted in higher values in hardness, gumminess, chewiness, and cutting strengths as well as in darker colour and decreased water absorption capacity. The effect of increased FWC on these properties was weaker and the opposite. Images from microtomography revealed that higher FPIc led to a less organised fibrous structure. In conclusion, fibrous structures can be achieved by utilising a mixture of faba bean protein ingredients, and a higher FPC content seemed to promote fibre formation in the meat analogue.

Natural Fibrous Protein for Advanced Tissue Engineering Applications: Focusing on Silk Fibroin and Keratin.

As one of the important branches of natural biopolymer, natural fibrous protein has a lot of advantages including good mechanical properties, excellent biocompatibility, controllable biodegradability, renewability, abundant sources, and so on. Moreover, natural fibrous protein is also a protein that could only be used for structure supporting without any bioactivities, which attracts a lot of attentions in the field of tissue engineering scaffold. This chapter is taking silk fibroin and keratin as model materials of natural fibrous protein, focusing on their protein structure, chemical compositions, processing and extraction methods, chemical modification methods, and their applications in tissue engineering through advanced manufacturing.

The predominant roles of the sequence periodicity in the self-assembly of collagen-mimetic mini-fibrils.

Collagen fibrils represent a unique case of protein folding and self-association. We have recently successfully developed triple-helical peptides that can further self-assemble into collagen-mimetic mini-fibrils. The 35 nm axially repeating structure of the mini-fibrils, which is designated the d-period, is highly reminiscent of the well-known 67 nm D-period of native collagens when examined using TEM and atomic force spectroscopy. We postulate that it is the pseudo-identical repeating sequence units in the primary structure of the designed peptides that give rise to the d-period of the quaternary structure of the mini-fibrils. In this work, we characterize the self-assembly of two additional designed peptides: peptide Col877 and peptide Col108rr. The triple-helix domain of Col877 consists of three pseudo-identical amino acid sequence units arranged in tandem, whereas that of Col108rr consists of three sequence units identical in amino acid composition but different in sequence. Both peptides form stable collagen triple helices, but only triple helices Col877 self-associate laterally under fibril forming conditions to form mini-fibrils having the predicted d-period. The Co108rr triple helices, however, only form nonspecific aggregates having no identifiable structural features. These results further accentuate the critical involvement of the repeating sequence units in the self-assembly of collagen mini-fibrils; the actual amino acid sequence of each unit has only secondary effects. Collagen is essential for tissue development and function. This novel approach to creating collagen-mimetic fibrils can potentially impact fundamental research and have a wide range of biomedical and industrial applications.

Review on Electromechanical Coupling Properties of Biomaterials.

Electromechanical coupling properties of biological materials, especially cellulose from plant cell walls and proteins from animals, are of great interest for applications in biocompatible sensors and actuators and ecofriendly energy harvesters. On the basis of their anisotropic nanostructures, cellulose and fibrous proteins such as collagen, silk, keratin, etc. are expected to be piezoelectric; however, this property does not necessarily translate to cellulose- or protein-containing bulk materials. In fact, the values of piezoelectric coefficients reported for cellulose and proteins in the literature vary over several orders of magnitude, which raises the question of whether these are truly intrinsic piezoelectric properties of biological materials or whether they are obscured with other electromechanical coupling processes such as electrostriction, flexoelectricity, electrochemical transport, or electrostatic deformation. This critical question about intrinsic and extrinsic electromechanical coupling mechanisms is reviewed in this article. The origin of piezoelectricity of cellulose and collagen (the most widely studied protein for piezoelectricity) is discussed based on their molecular structures. Key requirements to construct macroscopic piezoelectric biocomposites are addressed in terms of packing orders or arrangements of polar domains in composites. On the basis of this structural argument, truly piezoelectric responses of macroscopic materials fabricated with or containing cellulose and collagen are found to be extremely difficult to observe or quantify; most values reported in the literature as piezoelectric coefficients of such materials appear to originate from other electromechanical coupling mechanisms. Clarifying these mechanisms is important to properly design electromechanical devices using biobased materials.

Crystal Structure of the Carboxy-Terminal Region of the Bacteriophage T4 Proximal Long Tail Fiber Protein Gp34.

Long tail fibers of bacteriophage T4 are formed by proteins gp34, gp35, gp36, and gp37, with gp34 located at the phage-proximal end and gp37 at the phage-distal, receptor-binding end. We have solved the structure of the carboxy-terminal region of gp34, consisting of amino acids 894-1289, by single-wavelength anomalous diffraction and extended the structure to amino acids 744-1289 using data collected from crystals containing longer gp34-fragments. The structure reveals three repeats of a mixed α-ß fibrous domain in residues 744 to 877. A triple-helical neck connects to an extended triple ß-helix domain (amino acids 900-1127) punctuated by two ß-prism domains. Next, a ß-prism domain decorated with short helices and extended ß-helices is present (residues 1146-1238), while the C-terminal end is capped with another short ß-helical region and three ß-hairpins. The structure provides insight into the stability of the fibrous gp34 protein.

Induced oxidative stress management in wounds through phenolic acids engineered fibrous protein: An in vitro assessment using polymorphonuclear (PMN) cells.

The present study explores the preparation, characterization and the role of phenolic acid tethered fibrous protein in the management of induced oxidative stress studied under in vitro conditions. In brief, the biomaterial is prepared by engineering the fibrous protein with dihydroxy and trihydroxy phenolic acid moieties and subjected to characterization to ensure the tethering. The resultant biomaterial studied for its efficacy as a free radical scavenger using polymorphonuclear (PMN) cells with induced oxidative stress and also as an agent for cell migration using fibroblasts cells. Results revealed that induced oxidative stress in PMN cells after exposure to UVB radiation managed well with the prepared biomaterial by reducing the levels of superoxide anion, oxygen and hydroxyl radicals. Further, the protein and the phenolic acid interaction supports the cell migration as evidenced from the scratch assay. In conclusion, though phenolic acids are well known for their antimicrobial and antioxidant potential, indenting these acids directly to the wounds is not sensible, but tethering to protein explored the scavenging activity as expected. The present study infers that phenolic acid engineered protein has a significant role in managing the imbalance in the redox state prevailing in wounds and supports the healing at appreciable level.

Recent advances in nanoscale bioinspired materials.

Natural materials have been a fundamental part of human life since the dawn of civilization. However, due to exploitation of natural resources and cost issues, synthetic materials replaced bio-derived materials in the last century. Recent advances in bio- and nano-technologies pave the way for developing eco-friendly materials that could be produced easily from renewable resources at reduced cost and in a broad array of useful applications. This feature article highlights structural and functional characteristics of bio-derived materials, which will expedite the design fabrication and synthesis of eco-friendly and recyclable advanced nano-materials and devices.

Circular permutation directs orthogonal assembly in complex collagen peptide mixtures.

Multiple types of natural collagens specifically assemble and co-exist in the extracellular matrix. Although noncollagenous trimerization domains facilitate the folding of triple-helical regions, it is intriguing to ask whether collagen sequences are also capable of controlling heterospecific association. In this study, we designed a model system mimicking simultaneous specific assembly of two collagen heterotrimers using a genetically inspired operation, circular permutation. Previously, surface charge-pair interactions were optimized on three collagen peptides to promote the formation of an abc-type heterotrimer. Circular permutation of these sequences retained networks of stabilizing interactions, preserving both triple-helical structure and heterospecificity of assembly. Combining original peptides A, B, and C and permuted peptides D, E, and F resulted primarily in formation of A:B:C and D:E:F, a heterospecificity of 2 of 56 possible stoichiometries. This degree of specificity in collagen molecular recognition is unprecedented in natural or synthetic collagens. Analysis of natural collagen sequences indicates low similarity between the neighboring exons. Combining the synthetic collagen model and bioinformatic analysis provides insight on how fibrillar collagens might have arisen from the duplication of smaller domains.