Julie E. Gough

Self-assembled peptide-based hydrogels as scaffolds for anchorage-dependent cells.

We report here the design of a biomimetic nanofibrous hydrogel as a 3D-scaffold for anchorage-dependent cells. The peptide-based bioactive hydrogel is formed through molecular self-assembly and the building blocks are a mixture of two aromatic short peptide derivatives: Fmoc-FF (Fluorenylmethoxycarbonyl-diphenylalanine) and Fmoc-RGD (arginine-glycine-aspartate) as the simplest self-assembling moieties reported so far for the construction of small-molecule-based bioactive hydrogels. This hydrogel provides a highly hydrated, stiff and nanofibrous hydrogel network that uniquely presents bioactive ligands at the fibre surface; therefore it mimics certain essential features of the extracellular matrix. The RGD sequence as part of the Fmoc-RGD building block plays a dual role of a structural component and a biological ligand. Spectroscopic and imaging analysis using CD, FTIR, fluorescence, TEM and AFM confirmed that FF and RGD peptide sequences self-assemble into beta-sheets interlocked by pi-pi stacking of the Fmoc groups. This generates the cylindrical nanofibres interwoven within the hydrogel with the presence of RGDs in tunable densities on the fibre surfaces. This rapid gelling material was observed to promote adhesion of encapsulated dermal fibroblasts through specific RGD-integrin binding, with subsequent cell spreading and proliferation; therefore it may offer an economical model scaffold to 3D-culture other anchorage-dependent cells for in-vitro tissue regeneration.

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.

Novel bioresorbable and bioactive composites based on bioactive glass and polylactide foams for bone tissue engineering

Bioresorbable and bioactive tissue engineering scaffolds based on bioactive glass (45S5 Bioglass®) particles and macroporous poly(DL-lactide) (PDLLA) foams were fabricated. A slurry dipping technique in conjunction with pretreatment in ethanol was used to achieve reproducible and well adhering bioactive glass coatings of uniform thickness on the internal and external surfaces of the foams. In vitro studies in simulated body fluid (SBF) demonstrated rapid hydroxyapatite (HA) formation on the surface of the composites, indicating their bioactivity. For comparison, composite foams containing Bioglass® particles as filler for the polymer matrix (in concentration of up to 40 wt %) were prepared by freeze-drying, enabling homogenous glass particle distribution in the polymer matrix. The formation of HA on the composite surfaces after immersion in phosphate buffer saline (PBS) was investigated to confirm the bioactivity of the composites. Human osteoblasts (HOBs) were seeded onto as-fabricated PDLLA foams and onto PDLLA foams coated with Bioglass® particles to determine early cell attachment and spreading. Cells were observed to attach and spread on all surfaces after the first 90 min in culture. The results of this study indicate that the fabricated composite materials have potential as scaffolds for guided bone regeneration.

Directing the Morphology and Differentiation of Skeletal Muscle Cells Using Oriented Cellulose Nanowhiskers

Radially oriented submonolayer surfaces of 10-15 nm diameter cellulose nanowhiskers (CNWs) were prepared by spin-coating. The response of myoblasts (muscle cells) to the surfaces was assessed using atomic force microscopy (AFM), immunocytochemistry, and image analysis. Despite the small size of the CNWs, the myoblasts oriented along the CNW surfaces. Upon differentiation, the myoblasts produced striking radial patterns of myotubes, following the radial pattern of the CNWs. This facile method of nanopatterning surfaces may be applied where the directed growth of tissue is required and shows for the first time the potential of CNWs for tissue engineering applications.

Bacterial cellulose scaffolds and cellulose nanowhiskers for tissue engineering.

As the principle structural polysaccharide in plants, cellulose has been extensively characterized over many decades. In recent years, however, exciting new cellulosic materials have been developed with nanoscale fibrillar structures that have particularly promising applications in the growing field of tissue engineering. The majority of recent studies on cellulose nanomaterials for tissue engineering have employed bacterial cellulose, a material with a profile of properties unique among biomaterials commonly used in tissue engineering scaffolds. In addition, a number of recent studies have explored the biomedical applications of discrete colloidal nanocellulose fibrils known as cellulose nanowhiskers or cellulose nanocrystals. The literature on bacterial cellulose scaffolds for tissue engineering is reviewed, and studies on the biocompatibility of cellulose nanowhiskers and their potential for tissue engineering are discussed. Challenges for future development of these materials and potential future advances are also considered.

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.

Oriented surfaces of adsorbed cellulose nanowhiskers promote skeletal muscle myogenesis.

Cellulose nanowhiskers (CNWs) are high-aspect-ratio rod-like nanoparticles prepared via partial hydrolysis of cellulose. For the first time, CNWs have been extracted from the marine invertebrate Ascidiella aspersa, yielding animal-derived CNWs with particularly small diameters of only a few nanometres. Oriented surfaces of adsorbed CNWs were prepared using a flexible and facile spin-coating method, allowing the modulation of CNW adsorption and relative orientation. Due to the shape and nanoscale dimensions of the CNWs, C2C12 myoblasts adopted increasingly oriented morphologies in response to more densely adsorbed and oriented CNW surfaces. In addition, the degree of myoblast fusion was greatest on the highly oriented CNW surfaces, and even low-orientation CNW surfaces promoted more extensive fusion than flat control surfaces. Highly oriented multinuclear myotubes formed on the oriented CNW surfaces and fibrillar fibronectin deposited on the surfaces was also modelled in a highly oriented arrangement after only 4 days in culture. With a mean feature height of only 5-6 nm, the CNW surfaces present the smallest features ever reported to induce contact guidance in skeletal muscle myoblasts, highlighting the potential for nanoscale materials for engineering oriented tissues such as skeletal muscle.

Expression of alternatively spliced isoforms of human Sp7 in osteoblast-like cells

BackgroundOsteogenic and chondrocytic differentiation involves a cascade of coordinated transcription factor gene expression that regulates proliferation and matrix protein formation in a defined temporo-spatial manner. Bone morphogenetic protein-2 induces expression of the murine Osterix/Specificity protein-7 (Sp7) transcription factor that is required for osteoblast differentiation and bone formation. Regulation of its expression may prove useful for mediating skeletal repair.ResultsSp7, the human homologue of the mouse Osterix gene, maps to 12q13.13, close to Sp1 and homeobox gene cluster-C. The first two exons of the 3-exon gene are alternatively spliced, encoding a 431-residue long protein isoform and an amino-terminus truncated 413-residue short protein isoform. The human Sp7 protein is a member of the Sp family having 78% identity with Sp1 in the three, Cys2-His2 type, DNA-binding zinc-fingers, but there is little homology elsewhere. The Sp7 mRNA was expressed in human foetal osteoblasts and craniofacial osteoblasts, chondrocytes and the osteosarcoma cell lines HOS and MG63, but was not detected in adult femoral osteoblasts. Generally, the expression of the short (or beta) protein isoform of Sp7 was much higher than the long (or alpha) protein isoform. No expression of either isoform was found in a panel of other cell types. However, in tissues, low levels of Sp7 were detected in testis, heart, brain, placenta, lung, pancreas, ovary and spleen.ConclusionsSp7 expression in humans is largely confined to osteoblasts and chondrocytes, both of which differentiate from the mesenchymal lineage. Of the two protein isoforms, the short isoform is most abundant.

Self-assembled octapeptide scaffolds for in vitro chondrocyte culture

Nature has evolved a variety of creative approaches to many aspects of materials synthesis and microstructural control. Molecular self-assembly is a simple and efficient way to fabricate complex nanostructures such as hydrogels. We have recently investigated the gelation properties of a series of ionic-complementary peptides based on the alternation of non-polar hydrophobic and polar hydrophilic residues. In this work we focus on one specific octapeptide, FEFEFKFK (F, phenylalanine; E, glutamic acid; K, lysine). This peptide was shown to self-assemble in solution and form β-sheet-rich nanofibres which, above a critical gelation concentration, entangle to form a self-supporting hydrogel. The fibre morphology of the hydrogel was analysed using transmission electron microscopy and cryo-scanning electron microscopy illustrating a dense fibrillar network of nanometer size fibres. Oscillatory rheology results show that the hydrogel possesses visco-elastic properties. Bovine chondrocytes were used to assess the biocompatibility of the scaffolds over 21 days under two-dimensional (2-D) and three-dimensional (3-D) cell culture conditions, particularly looking at cell morphology, proliferation and matrix deposition. 2-D culture resulted in cell viability and collagen type I deposition. In 3-D culture the mechanically stable gel was shown to support the viability of cells, the retention of cell morphology and collagen type II deposition. Subsequently the scaffold may serve as a template for cartilage tissue engineering.

Osteoblast cell death on methacrylate polymers involves apoptosis

The success of an implant depends on the implant-tissue interface. There are many causes of implant failure, one of which is tissue necrosis. The aim of this in vitro study was to determine whether cell death of primary human osteoblasts (implant site specific cells) occurred by apoptosis (a form of programmed cell death) on two methacrylate polymers. Cells were cultured on poly(ethyl methacrylate)/tetrahydrofurfuryl methacrylate and poly(methyl methacrylate in the form of 13-mm discs, in conditioned medium containing leachable monomer and in the presence of various concentrations of monomer itself in the culture medium. It was found that monomer and leached monomer caused apoptosis of human osteoblast cells in this system. Tetrahydrofurfuryl methacrylate monomer was found to be more toxic than currently used monomer methylmethacrylate. Preincubation of polymers in serum containing medium was found to increase the biocompatibility of the polymers. High levels of apoptosis occurred on polymer used directly after polymerization. Apoptosis levels were decreased after polymer was incubated at 60 degrees C overnight or for 3 days. Apoptosis therefore may occur in cells at the implant site in vivo.