Vineetha Jayawarna

Introducing chemical functionality in Fmoc-peptide gels for cell culture

Aromatic short peptide derivatives, i.e. peptides modified with aromatic groups such as 9-fluorenylmethoxycarbonyl (Fmoc), can self-assemble into self-supporting hydrogels. These hydrogels have some similarities to extracellular matrices due to their high hydration, relative stiffness and nanofibrous architecture. We previously demonstrated that Fmoc-diphenylalanine (Fmoc-F(2)) provides a suitable matrix for two-dimensional (2D) or three-dimensional (3D) culture of primary bovine chondrocytes. In this paper we investigate whether the introduction of chemical functionality, such as NH(2), COOH or OH, enhances compatibility with different cell types. A series of hydrogel compositions consisting of combinations of Fmoc-F(2) and n-protected Fmoc amino acids, lysine (K, with side chain R=(CH(2))(4)NH(2)), glutamic acid (D, with side chain R=CH(2)COOH), and serine (S, with side chain R=CH(2)OH) were studied. All compositions produced fibrous scaffolds with fibre diameters in the range of 32-65 nm as assessed by cryo-scanning electron microscopy and atomic force microscopy. Fourier transform infrared spectroscopy analysis suggested that peptide segments adopt a predominantly antiparallel beta-sheet conformation. Oscillatory rheology results show that all four hydrogels have mechanical profiles of soft viscoelastic materials with elastic moduli dependent on the chemical composition, ranging from 502 Pa (Fmoc-F(2)/D) to 21.2 KPa (Fmoc-F(2)). All gels supported the viability of bovine chondrocytes as assessed by a live-dead staining assay. Fmoc-F(2)/S and Fmoc-F(2)/D hydrogels in addition supported viability for human dermal fibroblasts (HDF) while Fmoc-F(2)/S hydrogel was the only gel type that supported viability for all three cell types tested. Fmoc-F(2)/S was therefore investigated further by studying cell proliferation, cytoskeletal organization and histological analysis in 2D culture. In addition, the Fmoc-F(2)/S gel was shown to support retention of cell morphology in 3D culture of bovine chondrocytes. These results demonstrate that introduction of chemical functionality into Fmoc-peptide scaffolds may provide gels with tunable chemical and mechanical properties for in vitro cell culture.

Three-dimensional cell culture of chondrocytes on modified di-phenylalanine scaffolds

The design of self-assembled peptide-based structures for three-dimensional cell culture and tissue repair has been a key objective in biomaterials science for decades. In search of the simplest possible peptide system that can self-assemble, we discovered that combinations of di-peptides that are modified with aromatic stacking ligands could form nanometre-sized fibres when exposed to physiological conditions. For example, we demonstrated that a number of Fmoc (fluoren-9-ylmethyloxycarbonyl) modified di- and tri-peptides form highly ordered hydrogels via hydrogen-bonding and pi-pi interactions from the fluorenyl rings. These highly hydrated gels allowed for cell proliferation of chondrocytes in three dimensions [Jayawarna, Ali, Jowitt, Miller, Saiani, Gough and Ulijn (2006) Adv. Mater. 18, 611-614]. We demonstrated that fibrous architecture and physical properties of the resulting materials were dictated by the nature of the amino acid building blocks. Here, we report the self-assembly process of three di-phenylalanine analogues, Fmoc-Phe-Phe-OH, Nap (naphthalene)-Phe-Phe-OH and Cbz (benzyloxycarbonyl)-Phe-Phe-OH, to compare and contrast the self-assembly properties and cell culture conditions attributable to their protecting group difference. Fibre morphology analysis of the three structures using cryo-SEM (scanning electron microscopy) and TEM (transmission electron microscopy) suggested fibrous structures with dramatically varying fibril dimensions, depending on the aromatic ligand used. CD and FTIR (Fourier-transform IR) data confirmed beta-sheet arrangements in all three samples in the gel state. The ability of these three new hydrogels to support cell proliferation of chondrocytes was confirmed for all three materials.

Working together: the combined application of a magnetic field and penetratin for the delivery of magnetic nanoparticles to cells in 3D

Nanoparticles (NPs) are currently being developed as vehicles for in vivo drug delivery. Two of the biggest barriers facing this therapy are the site-specific targeting and consequent cellular uptake of drug-loaded NPs(1). In vitro studies in 2D cell cultures have shown that an external magnetic field (MF) and functionalization with cell-penetrating peptides (CPPs) have the capacity to overcome these barriers. This study aimed to investigate if the potential of these techniques, which has been reported in 2D, can be successfully applied to cells growing in a 3D environment. As such, this study provides a more realistic assessment of how these techniques might perform in future clinical settings. The effect of a MF and/or penetratin attachment on the uptake of 100 and 200 nm fluorescent iron oxide magnetic NPs (mNPs) into a fibroblast-seeded 3D collagen gel was quantified by inductively coupled plasma mass spectrometry. The most suitable mNP species was further investigated by fluorescence microscopy, histology, confocal microscopy, and TEM. Results show that gel mNP uptake occurred on average twice as fast in the presence of a MF and up to three times faster with penetratin attachment. In addition, a MF increased the distance of mNP travel through the gel, while penetratin increased mNP cell localization. This work is one of the first to demonstrate that MFs and CPPs can be effectively translated for use in 3D systems and, if applied together, will make excellent partners to achieve therapeutic drug delivery in vivo.

Exploiting CH-π interactions in supramolecular hydrogels of aromatic carbohydrate amphiphiles

A novel class of supramolecular hydrogels derived from amino sugars is reported, where the self-assembly of aromatic carbohydrate amphiphiles is driven by CH-π interactions, rather than π–π stacking and H-bonding associated with gelators based on aromatic peptide amphiphiles. Spectroscopic data is provided as evidence for this mode of self-assembly and in silico studies revealed that a combination of CH-π and T-stacking of the fluorenyl groups contribute to the formation of the aggregated structures.

Biogelx: Cell Culture on Self-Assembling Peptide Gels

Aromatic peptide amphiphiles can form self-supporting nanostructured hydrogels with tunable mechanical properties and chemical compositions. These hydrogels are increasingly applied in two-dimensional (2D) and three-dimensional (3D) cell culture, where there is a rapidly growing need to store, grow, proliferate, and manipulate naturally derived cells within a hydrated, 3D matrix. Biogelx Limited is a biomaterials company, created to commercialize these bio-inspired hydrogels to cell biologists for a range of cell culture applications. This chapter describes methods of various characterization and cell culture techniques specifically optimized for compatibility with Biogelx products.

Self-assembling peptide hydrogels : directing cell behaviour by chemical composition

For tissue engineering of small-diameter blood vessels, biodegradable, flexible and elastic porous tubular structures are most suited. In this study, we prepared crosslinked porous tubular structures from poly(trimethylene carbonate) (PTMC), in which smooth muscle cells (SMCs) were seeded and cultured in a pulsatile bioreactor mimicking the physiological conditions. PTMC was synthesized and porous tubular structures were prepared by dipping coating, cross-linking by g-irradiation, and leaching. SMCs were seeded into the porous structures by perfusion and then the constructs were cultured in a pulsatile bioreactor system. The morphologies, mechnical properties were analyzed and SMCs attachment and proliferation were evaluated by histology studies and CyQuant. Flexible tubular structures were obtained by dip coating with 3mm inner diameter and 1mm wall thickness. The porosity of the structures in wet state reached 85 vol% and the pore sizes were 60-150 mm. PTMC tubular structures showed comparable tensile strength and higher elongation compared with natural blood vessels. A pulsatile bioreactor system mimicking the conditions in vivo (dynamic pressure 70 mmHg, 75 beats/min) was successfully built. Experiements showed 7-day dilation was <10% and variation of diameter at each pulse was <1%. SMCs were homogeneously seeded in the porous scaffolds by perfusion. SMCs proliferate well to form confluent cell layer during a time period of up to 14 days, leading to constructs with even better mechanical performance. PTMC Porous tubular structures were prepared with good microstructures, elasticity and biocompatibility. SMCs were seeded and proliferated well in pulsatile bioreactor system and significant improvement of mechnical strength was observed.

Development of biomaterials for cellular differentiation using a metabolomics approach

Adequate cellular in-growth into biomaterials is one of the fundamental requirements in regenerative medicine. Type-I-collagen is the most commonly used material for soft tissue engineering, because it is nonimmunogenic and a highly porous network for cellular support. However, adequate cell in-growth and cell seeding has been suboptimal. Different densities of collagen scaffolds (0.3% to 0.8% (w/v)) with/without polymer knitting (poly-caprolactone (PCL)) were prepared. The structure of collagen scaffolds was characterized using scanning electronic microscopy (SEM) and HE staining. The mechanical strength of hybrid scaffolds was determined using tensile strength analysis. Cellular penetration and interconnectivity were evaluated using fluorescent bead distribution and human bladder smooth muscle cells and urothelium seeding. SEM and HE analysis showed the honeycomb structure and the hybrid scaffolds were adequately connected. The hybrid scaffolds were much stronger than collagen alone. The distribution of the beads and cells were highly dependent on the collagen density: at lower densities the beads and cells were more evenly distributed and penetrated deeper into the scaffold. The lower density collagen scaffolds showed remarkably deeper cellular penetration and by combining it with PCL knitting the tensile strength was enhanced. This study indicated that a 0.4% hybrid scaffold strengthened with knitting achieved the best cellular distribution.Human adult heart harbors a population of resident progenitor cells that can be isolated by Sca-1 antibody and expanded in culture. These cells can differentiate into cardiomyocytes and vascular cells in vitro and contribute to cardiac regeneration in vivo. However, when directly injected as single cell suspension, the survival rate and retention is really poor, less than 1% of injected cells being detectable in the hosttissue within few weeks. The present study aimed at investigating the possibility to produce scaffoldless, thick cardiac progenitor cell-derived cardiac patches by thermo-responsive technology. Human cardiac progenitors obtained from the auricles of patients were cultured as scaffoldless engineered tissues fabricated using temperature-responsive surfaces obtained by poly-N-isopropylacrylamide (PNIPAAm) surface immobilization. In the engineered tissue, progenitor cells established proper three-dimensional intercellular relationships and produced abundant extracellular matrix, while preserving their phenotype and plasticity. Cell phenotype and viability within the 3D construct were followed for 1 week, showing that no significant differentiation or apoptotic events occurred within the construct. After engineered tissues were leant on visceral pericardium, a number of cells migrated into the myocardium and in the vascular walls, where they integrated in the respective textures. The study demonstrates the suitability of such approach to deliver stem cells.Spinal cord injury and repair is one of the important focus areas in tissue regeneration. Mechanical trauma caused due to factors such as contusion, compression or involuntary stretching induce post-traumatic secondary tissue damage in many Spinal Cord Injury (SCI) patients. Therefore, there is a need for scaffolds that provide a conducive threedimensionsal (3D) environment for injured cells to attach and grow. In this study we propose to synthesize 3D polymeric scaffolds in order to study the mechanical and adhesive properties & the nature of the interactions between hyaluronan-based (HY) biomaterials and cells and tissues both in vitroandin vivo. Here we have synthesized 3D HY-based hydrogels with robust mechanical and adhesive properties and demonstrate the use of this material for neuronal-related applications such as the treatment of SCI. Cell culture and survivability studies were done with NSC-34 cells. Live/Dead assay performed on the cells revealed significant differences in the staining of live cells and showed increased viability and proliferation. The number of live cells in the HY-based hydrogels with 0.1% collagen showed higher cell numbers compared with the other hydrogels. In this study we show that Injectable HYbased hydrogels with high elasticity, comparable to the mechanical properties of nervous tissue have been used in this study to study their biocompatibility and neuroprotective properties and they show better affinity for neuronal cells.Calcium phosphates (CaP) obtained by biomineralisation in Simulated Boby Fluid have been used for decades to assess the mineralisation capability of biomaterials. Recently, they have been envisioned as potential agents to promote bone formation. In this study, we have fabricated and coated with calcium phosphate melt electrospun scaffolds whereby macropores permit adequate cell migration and nutrient transfer. We have systematically investigated the effect of coating and osteoinduction onto the response of ovine osteoblasts and we observed that the coating up-regulated alkaline phosphatase activity regardless of the in vitro culture conditions. Micro Computed Tomography revealed that only scaffolds cultured in an osteoinductive cocktail were capable of depositing mineralised matrix, and that CaP coated scaffolds were more efficient at promoting mineralisation. Theses scaffolds were subcutaneously implanted in athymic rats and this demonstrated that the osteoinduction was a pre-requisite for bone formation in this ectopic model. It showed that although the bone formation was not significantly different after 8 weeks, the CaP coated scaffolds were superior at inducing bone formation as evidenced by higher levels of mineralisation at earlier time points. This work demonstrated that CaP coating is not sufficient to induce bone formation; however the combination of osteoinduction and CaP coating resulted in earlier bone formation in an ectopic model.Introduction: Bladder regeneration using minced bladder mucosa is an alternative to costly and time-consuming conventional in vitro culturing of urothelial cells. In this method, the uroepithelium ...

Fmoc hydrogels from aromatic carbohydrate amphiphiles

Purpose: To model and interpret drug distribution in the dermis and underlying tissues after topical application which is relevant to the treatment of local conditions. Methods: We created a new physiological pharmacokinetic model to describe the effect of blood flow, blood protein binding and dermal binding on the rate and depth of penetration of topical drugs into the underlying skin. We used this model to interpret literature in vivo human biopsy data on dermal drug concentration at various depths in the dermis after topical application of 6 substances. This interpretation was facilitated by our in vitro human dermal penetration studies in which dermal diffusion coefficient and binding were estimated. Results: The model shows that dermal diffusion alone cannot explain the in vivo data and blood and/or lymphatic transport to deep tissues must be present for almost all of the drugs tested. Conclusion: Topical drug delivery systems for deeper tissue delivery should recognise that blood/ lymphatic transport may dominate over dermal diffusion for certain compounds.