Biomaterials (v.28, #14)

Unconstrained recovery characterization of shape-memory polymer networks for cardiovascular applications by Christopher Michael Yakacki; Robin Shandas; Craig Lanning; Bryan Rech; Alex Eckstein; Ken Gall (2255-2263).
Shape-memory materials have been proposed in biomedical device design due to their ability to facilitate minimally invasive surgery and recover to a predetermined shape in vivo. Use of the shape-memory effect in polymers is proposed for cardiovascular stent interventions to reduce the catheter size for delivery and offer highly controlled and tailored deployment at body temperature. Shape-memory polymer networks were synthesized via photopolymerization of tert-butyl acrylate and poly(ethylene glycol) dimethacrylate to provide precise control over the thermomechanical response of the system. The free recovery response of the polymer stents at body temperature was studied as a function of glass transition temperature (T g), crosslink density, geometrical perforation, and deformation temperature, all of which can be independently controlled. Room temperature storage of the stents was shown to be highly dependent on T g and crosslink density. The pressurized response of the stents is also demonstrated to depend on crosslink density. This polymer system exhibits a wide range of shape-memory and thermomechanical responses to adapt and meet specific needs of minimally invasive cardiovascular devices.
Keywords: Shape memory; Polymer; Stent; Vascular graft; Photopolymerization; Crosslinking;

Influence of the physical properties of two-dimensional polyester substrates on the growth of normal human urothelial and urinary smooth muscle cells in vitro by Géraldine Rohman; Jennifer J. Pettit; Françoise Isaure; Neil R. Cameron; Jennifer Southgate (2264-2274).
Although synthetic biomaterials have a wide range of promising applications in regenerative medicine and tissue engineering, there is limited insight into the basic materials properties that influence cellularisation events. The aim of this study was to investigate the influence of the physical properties of polyester films on the adherence and growth of normal human urothelial and urinary smooth muscle (SM) cells, as part of a programme for the development of potential biomaterials for bladder tissue engineering. Films of different thickness were produced by spin coating from solution. Cell attachment and proliferation were analysed and revealed a reproducible and significant growth advantage over the initial 7 days for both cell types on poly(lactide-co-glycolide) (PLGA) versus poly(ε-caprolactone) (PCL), and on thick versus thin films. In order to understand the basis of the variation in cell growth, the surface morphology, degradation behaviour and mechanical properties of the films were investigated. The pattern of cell attachment and growth was found to be unrelated to surface topography and no distinction in film degradation behaviour was found to account for differences in cell growth, except at late time points (14 days), where degradation of thin PLGA films became significant. By contrast, the flexural loss and storage moduli were found to be reduced in films composed of PLGA versus PCL, and also as film thickness increased, indicating that mechanical properties of biomaterials can influence cell growth. We conclude that elastic modulus is relevant to biology at the cellular scale and may also be influential at the tissue/organ level, and is a critical parameter to be considered during development of synthetic biomaterials for tissue engineering.
Keywords: Cell proliferation; Urinary tract; Mechanical properties; Poly(lactide-co-glycolide); Poly(ε-caprolactone);

Self-organization of hydroxyapatite nanorods through oriented attachment by Jing Di Chen; Ying Jun Wang; Kun Wei; Shu Hua Zhang; Xue Tao Shi (2275-2280).
The oriented attachment of hydroxyapatite (HAP) nanorods was achieved through a hydrothermal treatment. The nanorods were arranged in order along the c-axis of the HAP. These formed ca. 3.5 slip-shaped pores between the rods. High-resolution transmission electron (HRTEM) also revealed that single crystals of HAP may grow in the [0 0 2] direction of the HAP structure. This work extends the understanding of the potential for using biomimetic principles to synthesize bone-like composite materials.
Keywords: Hydroxyapatite; Self-organization; Oriented attachment;

Microfabricated tapered pits in vivo can stimulate connective tissue and bone attachment to percutaneous devices, secondarily preventing epithelial migration, and promoting long-term implant survival. Epithelial cells, which form a seal with a dental implant, acting as a barrier, and osteoblasts, which form bone, can come into contact with the same implant topography. To investigate whether the phenotypic characteristics of each cell type influenced cell response to micro-topography, we compared the response of the two cell types to the same dimensions of tapered pits, in vitro, and in vivo. Increased spreading, mature FAs, and restricted migration characterized individual PLE cell response to tapered pits. In contrast, osteoblasts were highly migratory, formed smaller, punctate adhesions and mineralized. Epithelial sheets formed from high-density PLE cultures demonstrated that tapered pits did not inhibit migration of the PLE sheets in vitro, similar to in vivo observations. In vitro, PLE sheet migration correlated with increases in vinculin, tyrosine phosphorylation, cytokeratin and ERK 1/2 phosphorylation. The findings of this study show that tapered pits stimulate osteoblast mineral deposition in vitro and in vivo, but do not prevent epithelial sheet migration. In vitro results suggest that epithelial sheet migration could involve altered FA mediated signal transduction.
Keywords: Substratum topography; Dental implant; Osteoblasts; Epithelium; Osseointegration; Epithelial downgrowth;

Composite chondroitin-6-sulfate/dermatan sulfate/chitosan scaffolds for cartilage tissue engineering by Yen-Lin Chen; Hsiao-Ping Lee; Hing-Yuen Chan; Li-Yu Sung; Huang-Chi Chen; Yu-Chen Hu (2294-2305).
Conjugating a single glycosaminoglycan (GAG) species such as chondroitin-6-sulfate (CSC) to chitosan is beneficial to chondrocyte culture and extracellular matrix (ECM) production, but whether fabrication of 3D chitosan scaffolds with additional minor GAG species such as dermatan sulfate (DS) further improves the ECM production is unknown. In this study, Response Surface Methodology (RSM) was employed to design CSC/DS/chitosan scaffolds of various formulations for cartilage engineering and to investigate the roles of individual GAG species in cartilage formation. The CSC/DS formulation affected neither the physical properties of scaffolds nor cell adhesion, but influenced cell morphology, GAGs and collagen production and chondrocytic gene expression. The linear effects elucidated by RSM analysis suggested that within the level range higher CSC levels favored GAGs and collagen production, whereas lower DS levels were desired for these responses. Nonetheless, the quadratic effects of DS and two-way interactions between CSC and DS also contributed to the GAGs and collagen production. Accordingly, the optimal formulation, as predicted by RSM and validated by experiments, comprised 2.8 mg CSC and 0.01 mg DS per scaffold. This study confirmed the importance of DS in cartilage tissue engineering and implicated the feasibility of rational CSC/DS/chitosan scaffold design with the aid of RSM.
Keywords: Chondrocyte; Chitosan; Chondroitin sulfate; Dermatan sulfate; Response surface methodology; Glycosaminoglycan;

Cytocompatibility and blood compatibility of multifunctional fibroin/collagen/heparin scaffolds by Qiang Lu; Shenjia Zhang; Kun Hu; Qingling Feng; Chuanbao Cao; Fuzhai Cui (2306-2313).
An applicable matrix used in tissue engineering should not only have suitable mechanical properties, porous structures and biocompatibility that facilitate the adhesion, growth and proliferation of tissue cells, but also have the ability to release bioactive factors to provide a more conducive and inductive environment for tissue growth. Because of the harsh preparation conditions and deficiency of mechanical properties, it is still difficult for fibroin and collagen matrices to possess these multifunctional properties. In this research, we successfully prepared fibroin/collagen hybrid scaffolds containing heparin that possess multifunctional properties under mild conditions. These scaffolds maintain outstanding mechanical properties and porous structures of fibroin-based scaffolds. Furthermore, the scaffolds keep the bioactivity of collagen, becoming delivering systems that release heparin slowly to make the scaffolds blood compatible. Compared with fibroin/collagen scaffolds, the scaffolds containing heparin further facilitate the growth of HepG2 cells since a more complex, dynamic environment was formed to promote the cell growth. Considering the mild aqueous preparation environment without crosslinking reaction, besides promoting the progress in blood contacting tissue engineering, our research has also opened a door to prepare various multifunctional fibroin/collagen hybrid matrices that combine the advantages of fibroin and collagen.
Keywords: Fibroin/collagen; Blood compatibility; Cytocompatibility; Scaffolds;

The phenomenon of infection with abdominal wall reconstruction by Anton F. Engelsman; Henny C. van der Mei; Rutger J. Ploeg; Henk J. Busscher (2314-2327).
This review presents the clinical background to abdominal wall reconstruction, the different types of surgical meshes employed and known mechanisms of infection. It is shown that there are major physico-chemical differences between available meshes, which, in combination with the location of the mesh, the surgical technique applied and hernia type involved influence the infection potential.
Keywords: Abdomen; Bacterial adhesion; Biofilm; Infection; Polytetrafluorethylene; Polypropylene;

Cell micropatterning has important applications in the development of biosensors and lab-on-a-chip devices, tissue engineering and fundamental cell biology studies. The conventional micropatterning techniques involve patterning of cells over a planar substrate. In this paper, we propose the introduction of topographical features on cell adhesive regions to enhance cell adhesion and function. The textured surface is created by assembly of polystyrene microspheres and the topographical parameters can be varied systematically by changing the size and density of the particles. A technique of generating spatial arrangement of microspheres on a nonfouling background is developed. This is achieved by using a bi-functional template which has a patterned hydrophobic parylene film to facilitate self-assembly of particles; after assembly, the film is liftoff, revealing a cell resistance background which is compatible with cell micropatterning. Particles were assembled by selective wetting of the hydrophobic–hydrophilic template. A fluidic chamber was designed to control the movement of the particle suspension across the template so as to attain uniform particle array over large area. This method of cell micropatterning can improve the efficiency and functionality of cell-based devices. It can also be used for examining the effects of topographical cues on cell–substrate adhesion which can provide valuable insights into cell biology and design of biomaterials.
Keywords: Microsphere; Patterning; Cell; Self-assembly;

In this study, we compared two systems which can be applied for transfection in vitro and in vivo: polyplexes based on the polymer dextran–spermine (D–SPM) and lipoplexes based on 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/cholesterol. The carriers differ in (1) solubility in aqueous media, (2) source of the positive charges (quaternary amines for DOTAP and primary plus secondary amines for D–SPM), (3) electrostatics, i.e., for lipid and polymer, respectively: zeta-potential (81.0 and 48.1 mV), surface potential (180 and 92 mV), and surface pH (10.47 and 8.97), and (4) charge distribution (ordered versus non-ordered). The stability of the complex upon interaction with serum proteins was studied by means of fluorescence resonance energy transfer (FRET) between rhodamine-labeled cationic carriers and fluorescein-labeled DNA. Addition of serum increases the lipid–DNA average distance and decreases the polymer–DNA distance. However, FRET efficiency indicates that serum proteins do not induce a major DNA dissociation for either polyplexes or lipoplexes. Comparing the biodistribution of rhodamine-labeled complexes and the transgene expression after intravenous (i.v.), intramuscular (i.m.), and intranasal (i.n.) administration, we found that local administration of lipoplexes resulted in the lipoplexes remaining at the site of injection, whereas the polyplexes showed systemic distribution, accompanied by transgene expression in lungs and liver. It is suggested that the high water-solubility of the polymer combined with its lower positive charge (compared to DOTAP), which makes its association with the cells at the site of injection weaker, enables the polymer to reach and transfect distant organs through the blood stream. Using chemically modified D–SPM, we demonstrated the importance of high density of positive charges and a sufficient level of secondary amines for achieving efficient transgene expression in vivo.
Keywords: Gene delivery; Charge-distribution; PEGylation; Biodistribution; Transgene expression;

A major challenge in the use of gene transfer vectors as therapeutic tools is controlling vector administration at a desired tissue site. One potential solution is implanting tissue-engineering constructs loaded with gene transfer vectors such as viruses for localized transgene delivery. In this work, we conjugated recombinant adeno-associated virus serotype 2 (rAAV2) to a heparinized small intestinal submucosa (H-SIS) matrix, which resulted in vector transduction upon cellular adhesion. H-SIS was prepared by incorporating heparin, the rAAV2 receptor, into SIS through N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) mediated crosslinking. Incorporated heparin adsorbed rAAV2 onto the H-SIS matrix for conjugation. Using green fluorescent protein and β-galactosidase as reporters, we showed that conjugated rAAV2 was active and capable of mediating transgene delivery in cell culture. Additionally, we applied H-SIS to adsorb unpurified rAAV2 from the crude lysate of packaging cells for conjugation, avoiding the use of ultracentrifugation or chromatography in preparation of infectious rAAV2 for transduction. Our work provides a unique, modified tissue substrate H-SIS for rAAV2 binding and transduction, which can be a useful tool in developing localized gene transfer.
Keywords: Adsorption; Fibroblast; Gene expression; Gene transfer; Heparin; SIS;

Spider silks exhibit remarkable mechanical properties while dentin matrix protein 1 provides controlled nucleation and hydroxyapatite growth. In the present work, these two attributes were combined via genetic engineering to form a chimera, a clone encoding consensus repeats from the major protein in the spider dragline silk of Nephila clavipes fused to the carboxyl terminal domain of dentin matrix protein 1 (CDMP1). The objective was to exploit the self-assembly and material properties of silk proteins with controlled hydroxyapatite (HA) formation from CDMP1, for novel biomaterial composites. The purified recombinant protein retained native-silk like self-assembly properties and β-sheet structure when formed into films and treated with methanol. When the chimeric protein in solution was incubated with CaCl2, the secondary structure shifted from random coil to α-helix and β-sheet, due to the interactions between the CDMP1 domain and Ca2+. The control protein without the CDMP1 domain did not undergo a similar transition. Films formed from the recombinant protein were mineralized using simulated body fluids and induced the formation of calcium-deficient carbonated HA, Ca10(PO4)6(OH)2 based on SEM, EDS, FTIR and TEM analysis. This mineral phase was not formed on the films formed from the control spider silk protein without the CDMP1 domain. Considering the osteoconductivity of HA and the novel material features of spider silks, these new hybrid systems offer potential as biomaterials for a number of potential applications.
Keywords: Silk; Hydroxyapatite; Apatite; Composites; Dentin matrix protein 1; Mineralization;

Protein mapping of calcium carbonate biominerals by immunogold by Frédéric Marin; Boaz Pokroy; Gilles Luquet; Pierre Layrolle; Klaas De Groot (2368-2377).
The construction of metazoan calcium carbonate skeletons is finely regulated by a proteinaceous extracellular matrix, which remains embedded within the exoskeleton. In spite of numerous biochemical studies, the precise localization of skeletal proteins has remained for a long time as an elusive goal. In this paper, we describe a technique for visualizing shell matrix proteins on the surface of calcium carbonate crystals or within the biominerals. The technique is as follows: freshly broken pieces of biominerals or NaOCl then EDTA-etched polished surfaces are incubated with an antibody elicited against one matrix protein, then with a secondary gold-coupled antibody. After silver enhancement, the samples are subsequently observed with scanning electron microscopy by using back-scattered electron mode. In the present case, the technique is applied to a particular example, the calcitic prisms that compose the outer shell layer of the mediterranean fan mussel Pinna nobilis. One major soluble protein, caspartin, which was identified recently, was partly de novo sequenced after enzymatic digestions. A polyclonal antibody raised against caspartin was used for its localization within and on the prisms. The immunogold localization indicated that caspartin surrounds the calcitic prisms, but is also dispersed within the biominerals. This example illustrates the deep impact of the technique on the definition of intracrystalline versus intercrystalline matrix proteins. Furthermore, it is an important tool for assigning a putative function to a matrix protein of interest.
Keywords: Calcium carbonate; Immunogold; SEM; Caspartin; Back-scattered electrons; Surface treatment;