Recombinant self-assembling peptides as biomaterials for tissue engineering

Synthetic nanostructures based on self-assembling systems that aim to mimic natural extracellular matrix are now being used as substrates in tissue engineering applications. Peptides are excellent starting materials for the self-assembly process as they can be readily synthesised both chemically and biologically. P11-4 is an 11 amino acid peptide that undergoes triggered self-assembly to form a self-supporting hydrogel. It exists as unimers of random coil conformations in water above pH 7.5 but at low pH adopts an antiparallel β-sheet conformation. It also self-assembles under physiological conditions in a concentration-dependent manner. Here we describe an unimer P11-4 production system and the use of a simple site-directed mutagenesis approach to generate a series of other P11-family peptide expression vectors. We have developed an efficient purification strategy for these peptide biomaterials using a simple procedure involving chemical cleavage with cyanogen bromide then repeated filtration, lyophilisation and wash steps. We report peptide-fusion protein yields of ca. 4.64 g/L and we believe the highest reported recovery of a recombinant self-assembling peptide at 203 mg/L of pure recombinant P11-4. This peptide forms a self-supporting hydrogel under physiological conditions with essentially identical physico-chemical properties to the chemically synthesised peptide. Critically it also displays excellent cytocompatibility when tested with primary human dermal fibroblasts. This study demonstrates that high levels of a series of recombinant self-assembling peptides can be purified using a simple process for applications as scaffolds in tissue engineering

Comparative Studies of Hyaluronic Acid Concentration in Normal and Osteoarthritic Equine Joints

Osteoarthritis (OA) is the most common major disabling disease in humans and horses. Hyaluronic acid (HA), naturally abundantly present in synovial fluid (SF), is thought to have crucial impact on the functional rheological and biochemical features of SF in healthy and osteoarthritic joints. Here we present comparative measurements of HA concentration in SF from 35 normal and osteoarthritic equine joints, between two different approaches. On the one hand, an established biochemical HA-specific Enzyme–Linked Immunosorbent Assay (ELISA) assay was employed, which determined that SF in healthy and osteoarthritic equine joints is characterized by HA concentration of ca 0.3–2 mg/mL and 0.1–0.7 mg/mL respectively. On the other hand the same SF samples were also examined with a new exploratory approach of finding out HA concentration, which is based on SF rheology. This was done following “calibration” using appropriate model HA solutions. Comparative analysis of the results obtained by both the biochemical and the rheological approaches, revealed that in most cases the rheological approach greatly overestimates HA concentration in SF, by ca 3 to 8 times and 6 to 11 times, in healthy and diseased SF respectively. Overall these findings support the notion that, contrary to the established view, HA may not be the major contributor of equine SF rheology. This should be taken into account for the development of new more effective preventive strategies, as well as more effective early-stage interventions in osteoarthritis

Organisation of self-assembling peptide nanostructures into macroscopically ordered lamella-like layers by ice crystallisation

Bio-inspired molecular self-assembly has attracted considerable research interest as a promising route to novel nanostructured materials. Self-assembling peptides have proven particularly popular building blocks for the construction of a variety of well-defined nanostructures. There is a great interest in learning to control not only the types and properties of nanostructures, but also their precise macroscopic organisation. Here we investigate the effect of water crystallisation during freezing as a possible method for directed organisation of preformed β-sheet tapes, ribbons and fibrils and for the production of microporous materials comprising lamella-like layers. We employ a range of short, systematically designed self-assembling peptides and a wide variety of techniques including SEM, TEM, X-ray tomography, X-ray diffraction, FTIR spectroscopy and compression testing. We find that ice growth does not alter the peptide nanostructures but templates the formation of lamella-like layers of mesoscopically aligned peptide ribbons and fibrils into nematic-like domains. The lamella are macroscopically oriented into regularly spaced stacks, giving rise to rather brittle peptide aerogels. This behaviour is contrasted with that of other self-assembling networks such as surfactant rod-like micelles and the polysaccharide agar. The differences in the properties of the self-assembling network seem to prescribe the way it will behave during ice crystallisation, and whether or not it will form ordered lamella structures. This approach may lead to the preparation of well-aligned peptide nanostructures, important for high-resolution structural studies; anisotropic microporous materials comprising lamella-like layers of self-assembling peptide fibrils with incorporated protein-like bioactivity may also be useful in medical applications e.g. tissue engineering, and nanotechnology

A Novel Drastic Peptide Genetically Adapted to Biomimetic Scaffolds “Delivers” Osteogenic Signals to Human Mesenchymal Stem Cells

This work describes the design, preparation, and deep investigation of “intelligent nanobiomaterials” that fulfill the safety rules and aim to serve as “signal deliverers” for osteogenesis, harboring a specific peptide that promotes and enhances osteogenesis at the end of their hydrogel fibers. The de novo synthesized protein fibers, besides their mechanical properties owed to their protein constituents from elastin, silk fibroin and mussel-foot adhesive protein-1 as well as to cell-attachment peptides from extracellular matrix glycoproteins, incorporate the Bone Morphogenetic Protein-2 (BMP2) peptide (AISMLYLDEN) that, according to our studies, serves as “signal deliverer” for osteogenesis. The osteogenetic capacity of the biomaterial has been evidenced by investigating the osteogenic marker genes ALP, RUNX2, Osteocalcin, COL1A1, BMPR1A, and BMPR2, which were increased drastically in cells cultured on scaffold-BMP2 for 21 days, even in the absence of osteogenesis medium. In addition, the induction of phosphorylation of intracellular Smad-1/5 and Erk-1/2 proteins clearly supported the osteogenetic capacity of the biomaterial

Freeze-Drying Process for the Fabrication of Collagen-Based Sponges as Medical Devices in Biomedical Engineering

This paper presents a systematic review of a key sector of the much promising and rapidly evolving field of biomedical engineering, specifically on the fabrication of three-dimensional open, porous collagen-based medical devices, using the prominent freeze-drying process. Collagen and its derivatives are the most popular biopolymers in this field, as they constitute the main components of the extracellular matrix, and therefore exhibit desirable properties, such as biocompatibility and biodegradability, for in vivo applications. For this reason, freeze-dried collagen-based sponges with a wide variety of attributes can be produced and have already led to a wide range of successful commercial medical devices, chiefly for dental, orthopedic, hemostatic, and neuronal applications. However, collagen sponges display some vulnerabilities in other key properties, such as low mechanical strength and poor control of their internal architecture, and therefore many studies focus on the settlement of these defects, either by tampering with the steps of the freeze-drying process or by combining collagen with other additives. Furthermore, freeze drying is still considered a high-cost and time-consuming process that is often used in a non-optimized manner. By applying an interdisciplinary approach and combining advances in other technological fields, such as in statistical analysis, implementing the Design of Experiments, and Artificial Intelligence, the opportunity arises to further evolve this process in a sustainable and strategic manner, and optimize the resulting products as well as create new opportunities in this field