Biomaterials (v.29, #34)

Differentiation of human embryonic stem (hES) cells into cells for regenerative medicine is often initiated by embryoid body (EB) formation. EBs may be treated with soluble biochemicals such as cytokines, growth factors and vitamins to induce differentiation. A scanning electron microscopy analysis, conducted over 14 days, revealed time-dependent changes in EB structure which led to the formation of a shell that significantly reduced the diffusive transport of a model molecule (374 Da) by >80%. We found that the shell consists of 1) an extracellular matrix (ECM) comprised of collagen type I; 2) a squamous cellular layer with tight cell–cell adhesions associated with E-cadherin; and 3) a collagen type IV lining indicative of a basement membrane. Disruption of the basement membrane, by either inhibiting its formation with noggin or permeabilizing it with collagenase, resulted in recovery of diffusive transport. Increasing the diffusive transport of retinoic acid (RA) and serum in EBs by a 15-min collagenase digestion on days 4, 5, 6 and 7 promoted neuronal differentiation. Flow cytometry and quantitative RT-PCR analysis of collagenase-treated EBs revealed 68% of cells expressing neural cell adhesion molecule (NCAM) relative to 28% for untreated EBs. Our results suggest that limitations in diffusive transport of biochemicals need to be considered when formulating EB differentiation strategies.
Keywords: Extracellular matrix; Collagen; Basement membrane; Human embryonic Stem cells; Embryoid bodies; Cell culture; Diffusion;

Protein adsorption and cell adhesion on nanoscale bioactive coatings formed from poly(ethylene glycol) and albumin microgels by Evan A. Scott; Michael D. Nichols; Lee H. Cordova; Brandon J. George; Young-Shin Jun; Donald L. Elbert (4481-4493).
Late-term thrombosis on drug-eluting stents is an emerging problem that might be addressed using extremely thin, biologically active hydrogel coatings. We report a dip-coating strategy to covalently link poly(ethylene glycol) (PEG) to substrates, producing coatings with ⪅100 nm thickness. Gelation of PEG-octavinylsulfone with amines in either bovine serum albumin (BSA) or PEG-octaamine was monitored by dynamic light scattering (DLS), revealing the presence of microgels before macrogelation. NMR also revealed extremely high end-group conversions prior to macrogelation, consistent with the formation of highly crosslinked microgels and deviation from Flory–Stockmayer theory. Before macrogelation, the reacting solutions were diluted and incubated with nucleophile-functionalized surfaces. Using optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microbalance with dissipation (QCM-D), we identified a highly hydrated, protein-resistant layer with a thickness of approximately 75 nm. Atomic force microscopy in buffered water revealed the presence of coalesced spheres of various sizes but with diameters less than about 100 nm. Microgel-coated glass or poly(ethylene terephthalate) exhibited reduced protein adsorption and cell adhesion. Cellular interactions with the surface could be controlled by using different proteins to cap unreacted vinylsulfone groups within the coating.
Keywords: Polyethylene glycol; Albumin; Microgel; Nanogel; Surface modification; Cell adhesion;

Supercritical CO2-assisted embossing for studying cell behaviour on microtextured surfaces by Satoshi Fujita; Daizaburo Ono; Masahiro Ohshima; Hiroo Iwata (4494-4500).
Recently, cell responses to micro- and nanoscale structures have attracted much attention. Although interesting phenomena have been observed, we have encountered some difficulties in elucidating purely topographical effects on cell behaviour. These problems are partially attributable to the introduction of functional groups and the persistence of chemicals during surface processing. In this study, we introduced supercritical CO2-assisted embossing, which plasticizes a polycarbonate plate by dissolving supercritical CO2 and thus can emboss wide-scale patterns onto the plate at a lower temperature than the polycarbonate glass transition temperature. Uniform micro- and nanopatterned surfaces were observed across the whole area of the polycarbonate plate surfaces. Nickel, fluorine, and nitrogen were not detected on the fabricated surfaces, and the surface carbon-to-oxygen ratios were equivalent to the theoretical ratio (C:O = 84.2:15.8) calculated from the polycarbonate molecular structure. Human mesenchymal stem cells were cultured on the fabricated microlens and nanogroove substrata. Cell-adhered areas became smaller on the microlens than on non-treated polycarbonate. Meanwhile, cells aligned along the ridges of nanogrooves with valleys deeper than 90 nm. This supercritical CO2-assisted embossing can produce fine substrates for studying the effects of surface topography of synthetic materials on cell behaviours.
Keywords: Cell adhesion; Cell morphology; Surface topography; Polycarbonate; Mesenchymal stem cell;

Modification of gelation kinetics in bioactive peptide amphiphiles by Krista L. Niece; Catherine Czeisler; Vibhu Sahni; Vicki Tysseling-Mattiace; Eugene T. Pashuck; John A. Kessler; Samuel I. Stupp (4501-4509).
Peptide amphiphiles (PAs) previously designed in our laboratory are known to self-assemble into nanofibers that exhibit bioactivity both in vitro and in vivo. Self-assembly can be triggered by charge neutralization or salt-mediated screening of charged residues in their peptide sequences, and the resulting nanofibers can form macroscopic gels at concentrations as low as 0.5% by weight. Controlling the kinetics of gelation while retaining the bioactivity of nanofibers could be critical in tailoring these materials for specific clinical applications. We report here on a series of PAs with different rates of gelation resulting from changes in their peptide sequence without changing the bioactive segment. The pre-existence of hydrogen-bonded aggregates in the solution state of more hydrophobic PAs appears to accelerate gelation kinetics. Mutation of the peptide sequence to include more hydrophilic and bulky amino acids suppresses formation of these nuclei and effectively slows down gelation through self-assembly of the nanofiber network. The ability to modify gelation kinetics in self-assembling systems without disrupting bioactivity could be important for injectable therapies in regenerative medicine.
Keywords: Self-assembly; Peptide amphiphile; Gelation kinetics; Bioactive nanofibers;

Vascular smooth muscle cells (VSMCs) isolated from rabbit aorta and immortalised A7r5 cells were cultured on conducting polypyrrole (PPy) substrates and were subjected to a 50 μA sinusoidal electrical stimulation at 0.05, 5 and 500 Hz. These substrates were doped with hyaluronic acid and coated with collagen IV followed by Matrigel® in order to mimic the basement membrane and encourage cell attachment. Increased proliferation and expression of smooth muscle phenotype markers (smooth muscle α-actin and smooth muscle myosin heavy chain) were observed in cultures stimulated at 5 and 500 Hz. This increased proliferation and expression of contractile proteins were found to be significantly decreased when L-type voltage-gated calcium channels (VGCC) were blocked with the drug nifedipine. To the best of our knowledge, this is the first work that demonstrates that VSMCs cultured on a conducting polymer substrate and subject to electrical stimulation not only exhibit enhanced proliferation but can be simultaneously encouraged to increase contractile protein expression. This behaviour is somewhat contrary to the classical definition of smooth muscle contractile and synthetic phenotypes that, in general, requires a modulation in phenotype as a prerequisite for smooth muscle proliferation. This interesting result highlights both the inherent plasticity of vascular smooth muscle cells and the potential of electrical stimulation via conducting polymer substrates to manipulate their behaviour.
Keywords: Smooth muscle cell; Electrical stimulation; Electroactive polymer; Cell activation; Cell proliferation;

Engineered extracellular matrices with cleavable crosslinkers for cell expansion and easy cell recovery by Jianxing Zhang; Aleksander Skardal; Glenn D. Prestwich (4521-4531).
An unmet need for expansion of primary cells and progenitor cells in three dimensions (3-D) is a synthetic mimic of the extracellular matrix (ECM) with user-controllable composition that would permit rapid recovery of viable cells under mild, non-enzymatic conditions. Three block copolymers based on disulfide-containing polyethylene glycol diacrylate crosslinkers were synthesized, and were used to crosslink thiol-modified hyaluronan and gelatin macromonomers in the presence of cells. The triblock PEGSSDA contained a single disulfide-containing block, the pentablock PEG(SS)2DA contained two disulfide blocks, and the heptablock PEG(SS)3DA contained three disulfide blocks. For each hydrogel composition, four cell types were encapsulated in 3-D, and growth and proliferation were evaluated. Murine NIH 3T3 fibroblasts, human HepG2 C3A hepatocytes, human bone marrow-derived mesenchymal stem cells (MSCs), and human umbilical vein endothelial cells (HUVECs) all showed excellent viability and growth during expansion in 3-D in the three disulfide block copolymer crosslinkers. After cell expansion, the hydrogels were dissociated using the thiol–disulfide exchange reaction in the presence of N-acetyl-cysteine or glutathione, which dissolved the hydrogel network. After dissolution, cells were recovered in high yield and with high viability by gentle centrifugation.
Keywords: Disulfide-containing crosslinkers; Thiol–disulfide exchange reaction; Synthetic extracellular matrix; In situ crosslinking; Primary cells; Hyaluronic acid;

Electrospun poly(ɛ-caprolactone)/gelatin nanofibrous scaffolds for nerve tissue engineering by Laleh Ghasemi-Mobarakeh; Molamma P. Prabhakaran; Mohammad Morshed; Mohammad-Hossein Nasr-Esfahani; Seeram Ramakrishna (4532-4539).
Nerve tissue engineering is one of the most promising methods to restore nerve systems in human health care. Scaffold design has pivotal role in nerve tissue engineering. Polymer blending is one of the most effective methods for providing new, desirable biocomposites for tissue-engineering applications. Random and aligned PCL/gelatin biocomposite scaffolds were fabricated by varying the ratios of PCL and gelatin concentrations. Chemical and mechanical properties of PCL/gelatin nanofibrous scaffolds were measured by FTIR, porometry, contact angle and tensile measurements, while the in vitro biodegradability of the different nanofibrous scaffolds were evaluated too. PCL/gelatin 70:30 nanofiber was found to exhibit the most balanced properties to meet all the required specifications for nerve tissue and was used for in vitro culture of nerve stem cells (C17.2 cells). MTS assay and SEM results showed that the biocomposite of PCL/gelatin 70:30 nanofibrous scaffolds enhanced the nerve differentiation and proliferation compared to PCL nanofibrous scaffolds and acted as a positive cue to support neurite outgrowth. It was found that the direction of nerve cell elongation and neurite outgrowth on aligned nanofibrous scaffolds is parallel to the direction of fibers. PCL/gelatin 70:30 nanofibrous scaffolds proved to be a promising biomaterial suitable for nerve regeneration.
Keywords: Nerve tissue engineering; Electrospinning; Nanofiber; PCL; Gelatin;

Characterization of an NbTaWZr alloy designed for magnetic resonance angiography compatible stents by Barry J. O'Brien; Jon S. Stinson; Dennis A. Boismier; William M. Carroll (4540-4545).
The majority of stent materials are not fully compatible with magnetic resonance imaging due to their ferromagnetic or paramagnetic compositions. This leads to image artifact which can obscure clinical data in the vicinity of the stent. An Nb–28Ta–3.5W–1.3Zr alloy has been developed specifically to provide reduced magnetic susceptibility and therefore reduce image artifact. This study reports on initial surface characterization, corrosion behaviour, endothelial cell response and MR image performance. Surface analysis confirms the presence of a niobium oxide with some tantalum oxide also present. Electrochemical corrosion testing demonstrates the oxide to be stable with no evidence of film breakdown. Leaching of metallic ions during a 60-day immersion test shows low levels of release, comparable to cobalt–chromium L605. A short term endothelial cell adhesion study shows that the Nb–28Ta–3.5W–1.3Zr may be similar to stainless steel for supporting cell growth. The MR artifact assessment shows that the material has significantly reduced artifact compared to stainless steel. In summary, results from this initial study show that the Nb–28Ta–3.5W–1.3Zr meets many on the criteria expected of a stent material and that improved MR imaging behaviour is also obtained.
Keywords: Magnetic resonance; Stent; Artifact; Niobium;

Sustained intraspinal delivery of neurotrophic factor encapsulated in biodegradable nanoparticles following contusive spinal cord injury by Yu-Chao Wang; Yi-Ting Wu; Hsin-Ying Huang; Hsin-I. Lin; Leu-Wei Lo; Shun-Fen Tzeng; Chung-Shi Yang (4546-4553).
Glial cell line derived neurotrophic factor (GDNF) induces neuronal survival and tissue repair after spinal cord injury (SCI). A continuous GDNF supply is believed to gain greater efficacy in the neural restoration of the injured spinal cord. Accordingly, nanovehicle formulation for their efficient delivery and sustained release in injured spinal cord was examined. We first used fluorescence-labeled bovine serum albumin (FBSA) loaded in biodegradable poly(lactic acid-co-glycolic acid) (PLGA) for intraspinal administration after SCI and for in vitro study. Our results showed that the preservation of PLGA–FBSA was observed in the injured spinal cord at 24 h, and PLGA–FBSA nanoparticles were well absorbed by neurons and glia, indicating that PLGA as a considerable nanovehicle for the delivery of neuroprotective polypeptide into injured spinal cord. Furthermore, intraspinal injection of GDNF encapsulated in PLGA (PLGA-GDNF) nanoparticles into the injured spinal cord proximal to the lesion center had no effect on gliosis when compared to that observed in SCI rats receiving PLGA injection. However, local administration of PLGA-GDNF effectively preserved neuronal fibers and led to the hindlimb locomotor recovery in rats with SCI, providing a potential strategy for the use of PLGA-GDNF in the treatment of SCI.
Keywords: Poly(lactic acid-co-glycolic acid); Nanoparticles; Size-dependent delivery; Spinal cord injury; Glial cell line derived neurotrophic factor;

The targeting of surface modified silica nanoparticles to inflamed tissue in experimental colitis by Brice Moulari; David Pertuit; Yann Pellequer; Alf Lamprecht (4554-4560).
One aspect in the emerging field of nanomedicine is site specific drug delivery via nanoparticles. The use of nanoparticles allows for increased therapeutic efficiency with a lowered risk for and extent of adverse reactions resulting from systemic drug absorption. 5-Amino salicylic acid (5ASA) loaded silica nanoparticles (SiNP) are proposed here as drug delivery system for specific accumulation in inflamed colonic tissues allowing for selective medication delivery to such inflammation sites. The drug was covalently bound to SiNP by a four-step reaction process. In-vitro toxicity of modified SiNP was tested in appropriate cell culture systems, while targeting index and therapeutic efficiency were evaluated in a pre-existing colitis in mice. Particle diameter was around 140 nm after final surface modification. In-vitro drug release demonstrated significant drug retention inside the NP formulation. Toxicity of the different formulations was evaluated in-vitro cell culture exhibiting a lowered toxicity for 5ASA when bound to SiNP. In-vivo, oral SiNP were found to accumulate selectively in the inflamed tissues allowing for significant amounts of drug load. SiNP demonstrated their therapeutic potential by significantly lowering the therapeutically necessary drug dose when evaluating clinical activity score and myeloperoxidase activity (untreated control: 28.0 ± 5.0 U/mg; 5ASA-solution (100 mg/kg): 8.2 ± 3.4 U/mg 5ASA–SiNP (25 mg/kg): 5.2 ± 2.4 U/mg). SiNP allow to combine advantages from selective drug targeting and prodrugs appearing to be a promising therapeutic approach for clinical testing in the therapy of inflammatory bowel disease.
Keywords: Drug targeting; Nanoparticles; Drug delivery; Colon targeting; Colitis;

Drug delivery from gold and titanium surfaces using self-assembled monolayers by Gopinath Mani; Dave M. Johnson; Denes Marton; Marc D. Feldman; Devang Patel; Arturo A. Ayon; C. Mauli Agrawal (4561-4573).
Currently available drug-eluting stents (DES) use polymers for coating and releasing drugs. Increasing evidence suggests that inflammatory and hypersensitive reactions are caused by such polymer coatings. This study focused on developing new techniques for delivering drugs directly from metal implant surfaces. Hydroxyl-terminated self-assembled monolayers (SAMs) were coated on Au and Ti surfaces. Therapeutic self-assembled monolayers (TSAMs) were prepared by chemically attaching the model drug, flufenamic acid, to SAM coated metal surfaces. Three different methods of esterification (acid chloride esterification, dry heat esterification, and direct esterification) were explored to attach flufenamic acid to SAMs. TSAMs were characterized using X-ray photoelectron spectroscopy, fluorescence microscopy, atomic force microscopy, and contact angle goniometry. These techniques collectively confirmed the attachment of drug onto SAM coated metal surfaces. In vitro drug release was investigated by immersing TSAM coated metal specimens in tris-buffered saline (TBS) at 37 °C for 28 days. TBS was analyzed at 1, 3, 7, 14, 21, and 28 days for the amount of drug eluted using high performance liquid chromatography. Large data scatter was observed for the release profiles of TSAMs prepared by acid chloride esterification. TSAMs prepared by dry heat and direct esterification methods showed an initial burst release of the drug followed by a sustained slow release for up to 2 weeks. Thus, this study suggests the potential for using self-assembled monolayers as an alternate system for delivering drugs from coronary stents and other metal implants.
Keywords: Drug-eluting stents; Drug delivery; Self-assembled monolayers; Surface modification;