Plasticization of poly-L-lactide for tissue engineering

Plasticization of medical grade poly-L-lactide (PLLA) by addition of polyethylene glycol (PEG) with various molar masses has been evaluated as means of producing low stiffness matrices for bioresorbable scaffolds for soft-tissue engineering applications. As reported previously, the Tg of injection molded specimens of the PLLA/PEG blends decreased strongly with PEG content, so that at PEG contents of 15 and 25 wt % it became significantly lower than normal human body temperature, implying an essentially rubber-like mechanical response in vivo. The degree of crystallinity of the moldings also increased strongly with PEG content, reaching a maximum of about 60 wt % at 25 wt % PEG. Moreover, after the immersion in phosphate-buffered saline for 5 days in 37°C to simulate conditions in vivo, the moldings with the highest PEG contents showed increased water uptake and, for relatively low molar mass PEG, significant mass loss, associated with phase separation and leaching of the PEG. Blends with relatively low PEG contents also showed large increases in their degree of crystallinity. The implications of these changes for the in vivo performance of the blends and their potential for development as matrices for bioresorbable scaffolds are discussed in the light of results from a series of PLLA/PEG copolymers.

Hybrid Polymer Foams for Osteochondral Tissue Engineering

Matthieu Cuénoud

Articular cartilage is a soft tissue that covers the ends of articulating bones, and is essential for maintaining joint motion, i.e. translation and rotation between the bones. Articular cartilage nevertheless has a limited capacity for self-repair, and cartilage lesions following traumatic injury may consequently initiate an irreversible degenerative process. This ultimately leads to progressive loss of joint function, pain and medical costs. Many different clinical treatments have been investigated, but these have not so far led to satisfactory long-term solutions. Tissue engineering research has therefore focused on the preparation of synthetic bioresorbable scaffolds that provide a template for cells and guide the process of tissue regeneration. In the present study, it is proposed to develop an osteochondral scaffold using novel solvent-free processing methods based on supercritical carbon dioxide (CO2) foaming. The approach is first to tailor the mechanical properties of medical grade polylactides (PLA), using poly(ethylene glycol) (PEG) as a plasticizer, to produce scaffolds with reduced elastic moduli intended for cartilage repair. These polymer scaffolds are then combined with an existing biocomposite scaffold (PLLA/bioceramic), developed for bone repair, to give a bilayered scaffold suitable for osteochondral repair. The investigation thus covers: (I) the preparation of plasticized polylactide and the dependence of its properties on composition; (II) the design of processing windows for the production of porous plasticized polylactide scaffolds by supercritical CO2 foaming; (III) the preparation of hybrid structures with gradients in composition and properties, and tailored interfaces; (IV)the properties and in vitro response of sterilized scaffolds. Plasticization of semicrystalline poly-L-lactide (PLLA) with PEG reduces Tg to below 37 °C, depending on the composition, implying the amorphous regions to be in the rubbery state at body temperature. While immersion in an aqueous medium tends to promote phase separation in blends containing low molar mass PEG, relatively good stability is observed with PEG with a molar mass of 35,000 g/mol. It has thus been possible to prepare interconnected, open-pore foams with porosities of more than 75 %, cell diameters in the range 200 to 700 pm and compression moduli of a few MPa. It was also observed that addition of PEG resulted in foams with increased pore diameters and lower foam densities than the neat polylactide, but also increased pore coalescence at high saturation pressures, effects that were accounted for by the large decrease in melt viscosity on addition of PEG. Furthermore, the compression moduli of the foams were shown to be strongly reduced on addition of PEG, which was assumed to be a direct consequence of the plasticization of the polylactide by PEG and the accompanying decrease in glass transition temperature, implying the amorphous content to be in the rubbery state at in vivo temperature, as required. Thus, foams with moduli ranging from 0.3 to 15 MPa could be prepared from the different PLA/PEG blends and copolymers studied. Bilayered scaffolds were produced by assembling the plasticized foams with a biocomposite foam using an adapted fusion bonding method. This allowed control of interfacial properties such as the bonding strength, and the interface density and permeability. These hybrid scaffolds exhibited well-defined layers with suitable properties for the repair of bone and cartilage, respectively. Finally, X-ray and y irradiation was shown to strongly degrade the polymer matrix. However, it was found that treatment with ethylene oxide gas could be used to sterilize the scaffolds without significantly modifying their properties, or compromising their biocompatibility, as borne out by in vitro tests. The approach proposed in this work was hence concluded to be highly promising for cartilage repair, and the resulting hybrid osteochondral scaffolds are ready for the next stage of the developmental work, which will consist of extended in vivo studies.

EPFL2012

A novel star PEG-derived surface coating for specific cell adhesion

Harm-Anton Klok, Stefano Tugulu

In this study a novel approach for the coating and functionalization of substrates for cell culture and tissue engineering is presented. Glass, silicon, and titanium panes were coated with an ultrathin film (30 ± 5 nm) of reactive star-shaped poly(ethylene glycol) prepolymers (Star PEG). Homogeneity of the films was checked by optical microscopy and scanning force microscopy. These coatings prevent unspecific protein adsorption as monitored by fluorescence microscopy and ellipsometry. In order to allow specific cell adhesion the films were modified with linear RGD peptides (gRGDsc) in different concentrations. After sterilization, fibroblast, SaOS, and human mesenchymal stem cells (hMSC) were seeded on these substrates. Cell adhesion, spreading, and survival was observed for up to 30 days on linear RGD peptide (gRGDsc)-modified coatings, whereas no cell adhesion could be detected on unmodified Star PEG layers. By variation of the RGD concentration within the film the amount of cells that became adhesive could be controlled. When differentiation conditions are used for cultivation of hMSCs the cells show the expression of osteogenic marker genes after 14 days which is comparable to cultivation on cell culture plastic. Thus, the Star PEG/RGD film did not negatively influence the differentiation process. The high flexibility of the system considering the incorporation of biologically active compounds opens a broad field of future experiments. © 2005 Wiley Periodicals, Inc.

2005

Solution behavior of peptide and peptide-hybrid materials

Harald Nuhn

Peptide based and peptide hybrid materials represent two interesting and challenging classes of biomaterials. Both classes consist of and contain, respectively, one or more polypeptide blocks, endowing the resulting polymers with enhanced self-assembly properties, biocompatibility or novel characteristics. Peptide based materials, solely comprised of amino acids, benefit from their advanced self-assembly properties. In the case of peptide hybrid materials, being conjugates of peptides and synthetic polymers, either the polypeptide or the synthetic polymer block can drive the self-assembly process. Furthermore, the latter class benefits from combining the advantages of peptides and synthetic polymers, thus overcoming limitations related to the use of the individual components. The application of these materials is still challenging because it is not possible to predict their final self-assembly behavior based on their design. In order to facilitate a target-oriented design of novel materials for application such as drug delivery, tissue engineering, or self-healing materials, a detailed understanding of the complex interactions within and between these molecules is essential. The work presented in this thesis addresses these fundamental questions. Over the last decades, a plethora of peptide and peptide hybrid materials has been reported. Chapter 1 gives an overview of selected articles describing different synthesis routes for peptide as well as peptide hybrid materials, showing examples of molecules and their self-assembly into superstructures, and highlighting recent applications. Chapter 2 describes the synthesis and characterization of different peptide hybrid diblock oligomers composed of two classes of peptide sequences covalently attached to a poly(ethylene glycol) (PEG) block. The first class of peptide sequences contains the intrinsic propensity to form coiled coils, whereas the second class is composed of "switch" peptides and is able to adopt both amphiphilic α-helical and β-strand conformations. Upon dissolution, the first class yielded superstructures comprised of dimeric and tetrameric coiled coil aggregates, whereas the second class formed fibrillar assemblies. In both cases, the self-assembly properties were driven by the peptide block, and the final superstructure consisted of a peptidic core wrapped by the synthetic polymer block. Chapter 3 presents a detailed study on the aqueous solution self-assembly of two poly(styrene)n-b-poly(L-lysine)m peptide hybrid oligomers. The aim was to clarify whether the observed rod-like micelles, obtained by direct dissolution of the diblock oligomers in aqueous solution, reflect intrinsic self-assembly properties of the hybrid diblock oligomers or whether they present structures that are energetically trapped due to the glassy nature of the polystyrene block. To address this question, the influence of a variety of sample preparation techniques, including the use of organic solvents, dialysis from an organic solvent and several thermal treatments, was investigated. All treatments resulted in solution aggregates that differed in size and shape from samples prepared by direct dissolution in aqueous solution. Thus, it seems likely that direct dissolution of polystyrene-b-poly(L-lysine) diblock oligomers in aqueous solution results in kinetically trapped, rod-like aggregates, which can be transformed into non-spherical micelles by the appropriate treatment. Chapter 4 contains a comprehensive study on peptide-based materials, investigating four different series of short elastin-like peptides (ELPs) based on the GVGVP motif. The objective was to test whether and how defined changes in the primary structure of short ELPs affect the secondary structure and the lower critical solution temperature (LCST) behavior. Therefore, the number of repeats was altered, and within one peptide of constant length, all valines were successively substituted by the more hydrophobic amino acids isoleucine, leucine and phenylalanine, respectively. In parallel, a theoretical approach based on the partition coefficient log P was used to predict the shift of the LCST as function of chain length and composition. For short ELPs, the LCST is strongly affected by changes in the primary structure. This has also been predicted by calculating log P. An increase in the hydrophobicity resulted in a shift of the LCST towards lower temperatures. Furthermore, it was shown for the first time that the sensitivity of short ELPs towards external factors, such as salt concentration, is determined by the intrinsic propensity of the incorporated amino acids to form either α-helices or β-strands. In summary, the obtained results on peptide based and peptide-hybrid materials provides deeper insights into their self-assembly characteristics and highlights the potential of these materials to be used for technical and medical applications.