Biomaterials (v.28, #28)

Silicon substitution in the calcium phosphate bioceramics by Alexis M. Pietak; Joel W. Reid; Malcom J. Stott; Michael Sayer (4023-4032).
Silicon (Si) substitution in the crystal structures of calcium phosphate (CaP) ceramics such as hydroxyapatite (HA) and tricalcium phosphate (TCP) generates materials with superior biological performance to stoichiometric counterparts. Si, an essential trace element required for healthy bone and connective tissues, influences the biological activity of CaP materials by modifying material properties and by direct effects on the physiological processes in skeletal tissue. The synthesis of Si substituted HA (Si-HA), Si substituted α-TCP (Si-α-TCP), and multiphase systems are reviewed. The biological performance of these Si substituted CaP materials in comparison to stoichiometric counterparts is discussed. Si substitution promotes biological activity by the transformation of the material surface to a biologically equivalent apatite by increasing the solubility of the material, by generating a more electronegative surface and by creating a finer microstructure. When Si is included in the TCP structure, recrystallization to a carbonated HA is mediated by serum proteins and osteoblast-like cells. Release of Si complexes to the extracellular media and the presence of Si at the material surface may induce additional dose-dependent stimulatory effects on cells of the bone and cartilage tissue systems.
Keywords: Calcium phosphates; Silicon; Bioactivity;

Influence of silicon concentration on the haemocompatibility of amorphous carbon by Soon-Eng Ong; Sam Zhang; Hejun Du; Hui-Chin Too; Kyaw-Naing Aung (4033-4038).
Amorphous carbon (a-C) has good blood compatibility and has been proposed as a coating material for blood contacting devices such as heart pumps and stents. In this study, unhydrogenated a-C films with different silicon concentrations were synthesized by magnetron sputtering, and the corresponding evolution of the surface energy and compatibility with blood were analysed. The incorporation of silicon not only decreased the sp2-hybridized carbon bonding configurations, but the static evaluation of the films incubated in human platelet-rich plasma also showed a decrease in platelet adhesion. Bonding structure and surface energy were determined to be factors contributing to the improved haemocompatibility.
Keywords: Silicon-incorporation; Amorphous carbon; Bonding structure; Surface energy; Human platelet adhesion; Haemocompatibility;

Cell adhesion biomaterial based on mussel adhesive protein fused with RGD peptide by Dong Soo Hwang; Sung Bo Sim; Hyung Joon Cha (4039-4046).
Previously, we designed and constructed a hybrid of the mussel adhesive protein (MAP) fp-151, which is a fusion protein with six type 1 (fp-1) decapeptide repeats at each type 5 (fp-5) terminus. Through various cell-adhesion analyses, we previously demonstrated that fp-151 has the potential to be used as a cell or tissue bioadhesive. In the present study, to improve the cell-adhesion properties of fp-151, we designed a new cell-adhesive protein, fp-151-RGD, which is a fusion with the GRGDSP residues, a RGD peptide sequence that has previously been identified at the cell-attachment site of fibronectin, at the C-terminus of fp-151. Although recombinant fp-151-RGD maintained the advantages associated with fp-151, such as a high production yield in Escherichia coli and simple purification, it showed superior spreading ability, which is important for cell proliferation under serum-free conditions, as well as better cell-adhesion ability compared with other commercially produced cell-adhesion materials such as poly-l-lysine (PLL) and the naturally extracted MAP mixture Cell-Tak. The excellent adhesion and spreading abilities of fp-151-RGD might be due to the fact that it utilizes three types of cell-binding mechanisms: DOPA adhesion of Cell-Tak, cationic binding force of PLL, and RGD sequence-mediated adhesion of fibronectin. Therefore, the new recombinant fp-151-RGD is suitable for use as a cell-adhesion material in cell culture or tissue engineering, and in any other area where efficient cell adhesion is required.
Keywords: Cell adhesion material; Mussel adhesive protein; RGD peptide; Cell recognition motif; Fusion protein;

Multi-functional bilayer polymeric coatings are prepared with both controlled nitric oxide (NO) release and surface-bound active thrombomodulin (TM) alone or in combination with immobilized heparin. The outer-layer is made of CarboSil, a commercially available copolymer of silicone rubber (SR) and polyurethane (PU). The CarboSil is either carboxylated or aminated via an allophanate reaction with a diisocyanate compound followed by a urea-forming reaction between the generated isocyanate group of the polymer and the amine group of an amino acid (glycine), an oligopeptide (triglycine) or a diamine. The carboxylated CarboSil can then be used to immobilize TM through the formation of an amide bond between the surface carboxylic acid groups and the lysine residues of TM. Aminated CarboSil can also be employed to initially couple heparin to the surface, and then the carboxylic acid groups on heparin can be further used to anchor TM. Both surface-bound TM and heparin's activity are evaluated by chromogenic assays and found to be at clinically significant levels. The underlying NO release layer is made with another commercial SR–PU copolymer (PurSil) mixed with a lipophilic NO donor (N-diazeniumdiolated dibutylhexanediamine (DBHD/N2O2)). The NO release rate can be tuned by changing the thickness of top coatings, and the duration of NO release at physiologically relevant levels can be as long as 2 weeks. The combination of controlled NO release as well as immobilized active TM and heparin from/on the same polymeric surface mimics the highly thromboresistant endothelium layer. Hence, such multifunctional polymer coatings should provide more blood-compatible surfaces for biomedical devices.
Keywords: Nitric oxide; Thrombomodulin; Heparin; Hemocompatibility; Platelet activation; Platelet adhesion;

Cartilaginous ECM component-modification of the micro-bead culture system for chondrogenic differentiation of mesenchymal stem cells by Ying-Nan Wu; Zheng Yang; James H.P. Hui; Hong-Wei Ouyang; Eng Hin Lee (4056-4067).
In this study a 3-D alginate microbead platform was coated with cartilaginous extracellular matrix (ECM) components to emulate chondrogenic microenvironment in vivo for the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). BMSCs were seeded onto the microbead surface and the effect of the modified microbead on BMSC adhesion, proliferation and chondrogenic differentiation was studied, and compared to chondrogenesis in conventional pellet culture. Our results indicated that microbead system promoted BMSC proliferation and protein deposition resulting in the formation of bigger aggregates compared to conventional pellet culture. Analysis of the aggregates indicated that chondroitin sulfate (CS)- and Col2-coated microbeads enhanced the chondrogenic differentiation of hBMSCs, with increasing formation of glycosaminoglycan (GAG) and collagen II deposition in histology, immunohistochemistry and real time PCR analysis. In addition, Col2-coated microbeads resulted in hypertrophic maturation of the differentiated chondrocytes, similar to conventional pellet culture, while CS-coated microbeads were able to retain the pre-hypertrophy state of the differentiated cells. Our result suggested that provision of suitable cartilaginous microenvironment in a 3-D system can promote the chondrogenic differentiation of BMSC and influence the phenotype of resulting chondrocytes. Our microbead system provides an easy method of processing a 3-D alginate system that allows the possibility of scaling up chondrogenic pellet production for clinical application, while the modifiable microbeads also provide an adjustable 3-D platform for the study of co-interaction of ECM and differentiation factors during the stem cell differentiation.
Keywords: Alginate; Chitosan; Mesenchcymal stem cells; Extracellular matix; Chondrogenic differentiation;

The effect of actin disrupting agents on contact guidance of human embryonic stem cells by Sharon Gerecht; Christopher J. Bettinger; Zhitong Zhang; Jeffrey T. Borenstein; Gordana Vunjak-Novakovic; Robert Langer (4068-4077).
Mammalian cells respond to their substrates by complex changes in gene expression profiles, morphology, proliferation and migration. We report that substrate nanotopography alters morpohology and proliferation of human embryonic stem cells (hESCs). Fibronectin-coated poly(di-methyl siloxane) substrates with line-grating (600 nm ridges with 600 nm spacing and 600±150 nm feature height) induced hESC alignment and elongation, mediated the organization of cytoskeletal components including actin, vimentin, and α-tubulin, and reduced proliferation. Spatial polarization of gamma-tubulin complexes was also observed in response to nanotopography. Furthermore, the addition of actin disrupting agents attenuated the alignment and proliferative effects of nanotopography. These findings further demonstrate the importance of interplay between cytoskeleton and substrate interactions as a key modulator of morphological and proliferative cellular response in hESCs on nanotopography.
Keywords: Stem cell; Microstructure; Cell morphology; Cell proliferation;

Fabrication of porous ultra-short single-walled carbon nanotube nanocomposite scaffolds for bone tissue engineering by Xinfeng Shi; Balaji Sitharaman; Quynh P. Pham; Feng Liang; Katherine Wu; W. Edward Billups; Lon J. Wilson; Antonios G. Mikos (4078-4090).
We investigated the fabrication of highly porous scaffolds made of three different materials [poly(propylene fumarate) (PPF) polymer, an ultra-short single-walled carbon nanotube (US-tube) nanocomposite, and a dodecylated US-tube (F-US-tube) nanocomposite] in order to evaluate the effects of material composition and porosity on scaffold pore structure, mechanical properties, and marrow stromal cell culture. All scaffolds were produced by a thermal-crosslinking particulate-leaching technique at specific porogen contents of 75, 80, 85, and 90 vol%. Scanning electron microcopy, microcomputed tomography, and mercury intrusion porosimetry were used to analyze the pore structures of scaffolds. The porogen content was found to dictate the porosity of scaffolds. There was no significant difference in porosity, pore size, and interconnectivity among the different materials for the same porogen fraction. Nearly 100% of the pore volume was interconnected through 20 μm or larger connections for all scaffolds. While interconnectivity through larger connections improved with higher porosity, compressive mechanical properties of scaffolds declined at the same time. However, the compressive modulus, offset yield strength, and compressive strength of F-US-tube nanocomposites were higher than or similar to the corresponding properties for the PPF polymer and US-tube nanocomposites for all the porosities examined. As for in vitro osteoconductivity, marrow stromal cells demonstrated equally good cell attachment and proliferation on all scaffolds made of different materials at each porosity. These results indicate that functionalized ultra-short single-walled carbon nanotube nanocomposite scaffolds with tunable porosity and mechanical properties hold great promise for bone tissue engineering applications.
Keywords: Ultra-short single-walled carbon nanotube; Poly(propylene fumarate); Nanocomposite; Scaffold; Porosity; Osteoconduction;

Bioactive glass is used as both a bone filler and as a coating on implants, and has been advocated as a potential osteogenic scaffold for tissue engineering. Rat-derived mesenchymal stem cells (MSCs) show elevated levels of alkaline phosphatase activity when grown on 45S5 bioactive glass as compared to standard tissue culture plastic. Similarly, exposure to the dissolution products of 45S5 elevates alkaline phosphatase activity and other osteogenic markers in these cells. We investigated whether human MSCs grown under the same laboratory conditions as rat MSCs would exhibit similar responses. In general, human MSCs produce markedly less alkaline phosphatase activity than rat MSCs, regardless of cell culture conditions, and do not respond to the growth factor BMP-2 in the same way as rat MSCs. In our experiments there was no difference in alkaline phosphatase activity between human MSCs grown on 45S5 bioactive glass or tissue culture plastic, in samples from five different orthopaedic patients, regardless of culture media composition. Neither was there any consistent effect of 45S5 dissolution products on human MSCs from three different donors. These results suggest that the positive effects of bioactive glass on bone growth in human patients are not mediated by accelerated differentiation of mesenchymal stem cells.
Keywords: Alkaline phosphatase; Bioactive glass; Bone tissue engineering; Cell viability; Mesenchymal stem cell; Simulated body fluids (SBF);

A scaffold cell seeding method driven by surface acoustic waves by Haiyan Li; James R. Friend; Leslie Y. Yeo (4098-4104).
Surface acoustic waves (SAW) have been employed to drive a particle suspension into a porous scaffold as a means for cell seeding. Straight, simple interdigital electrode structures were fabricated on lithium niobate to permit the generation of Rayleigh SAW radiation. Fluorescent microscopy was used to investigate the seeding process; the SAW-driven seeding process occurred in approximately 10 s, much quicker than if the scaffold were to be seeded by gravity-driven diffusional processes alone (>30 min). Analysis of high-speed micrographic images demonstrated that the SAW method could also drive particles deeper into the scaffold, thereby significantly improving the uniformity of the particle distribution. The proposed SAW technique therefore offers a promising technology to dramatically improve the speed and uniformity of cell seeding in scaffolds, which might contribute to rapid and uniform tissue regeneration.
Keywords: Surface acoustic wave; Tissue engineering; Rapid cell seeding; Microfluidic actuation;

The inhibition of the adhesion of clinically isolated bacterial strains on multi-component cross-linked poly(ethylene glycol)-based polymer coatings by Isabel C. Saldarriaga Fernández; Henny C. van der Mei; Michael J. Lochhead; David W. Grainger; Henk J. Busscher (4105-4112).
This study examined bacterial adhesion to a new multi-component cross-linked poly(ethylene glycol)-based polymer coating that can be applied by spin-coating or spraying onto diverse biomaterials. Adhesion of five clinically isolated bacterial strains involved in biomaterial-centered infections were studied in a parallel-plate flow chamber at different shear rates and after exposure of the coating to different physiological fluids. The new chemistry inhibits non-specific biomolecular and cell binding interactions. Relative to glass, the coating reduced adhesion of all strains used in this study by more than 80%, with the exception of Escherichia coli O2K2. Reductions in adhesion of Staphylococcus epidermidis 3399 persisted beyond 168 h exposure of the coatings to phosphate buffered saline or urine, but not after exposure to protein-rich fluids as saliva and blood plasma, despite evidence from X-ray photoelectron spectroscopy that the coating integrity was not affected by exposure to these fluids. We conclude that this new coating chemistry provides beneficial properties to prevent or hinder bacterial adhesion and colonization in applications where low protein-conditions prevail.
Keywords: Bacterial adhesion; Poly(ethylene glycol) coating; Parallel plate flow chamber; Physiological fluids; Infection; Device;

This paper reports the hydrolytic degradation and protein release studies for a series of newly synthesized thermogelling tri-component multi-block poly(ether ester urethane)s consisting of poly[(R)-3-hydroxybutyrate] (PHB), poly(propylene glycol) (PPG), and poly(ethylene glycol) (PEG). The poly(PEG/PPG/PHB urethane) copolymer hydrogels were hydrolytically degraded in phosphate buffer at pH 7.4 and 37 °C for a period of up to 6 months. The mass loss profiles of the copolymer hydrogels were obtained. The hydrogel residues at different time periods of hydrolysis were visualized by scanning electron microscopy, which exhibited increasing porosity with time of hydrolysis. The degradation products in the buffer were characterized by GPC, 1H NMR, MALDI-TOF, and TGA. The results showed that the ester backbone bonds of the PHB segments were broken by random chain scission, resulting in a decrease in the molecular weight. In addition, the constituents of degradation products were found to be 3-hydroxybutyric acid monomer and oligomers of various lengths (n=1–5). The protein release profiles of the copolymer hydrogels were obtained using BSA as model protein. The results showed that the release rate was controllable by varying the composition of the poly(ether ester urethane)s or by adjusting the concentration of the copolymer in the hydrogels. Finally, we studied the correlation between the protein release characteristics of the hydrogels and their hydrolytic degradation. This is the first example that such a correlation has been attempted for a biodegradable thermogelling copolymer system.
Keywords: Poly(ether ester urethane); Poly[(R)-3-hydroxybutyrate]; Poly(ethylene glycol); Poly(propylene glycol); Hydrolytic degradation; Drug release;

Pulsatile release of parathyroid hormone from an implantable delivery system by Xiaohua Liu; Glenda J. Pettway; Laurie K. McCauley; Peter X. Ma (4124-4131).
Intermittent (pulsatile) administration of parathyroid hormone (PTH) is known to improve bone micro-architecture, mineral density and strength. Therefore, daily injection of PTH has been clinically used for the treatment of osteoporosis. However, this regimen of administration is not convenient and is not a favorable choice of patients. In this study, an implantable delivery system has been developed to achieve pulsatile release of PTH. A well-defined cylindrical device was first fabricated with a biodegradable polymer, poly(l-lactic acid) (PLLA), using a reverse solid-free form fabrication technique. Three-component polyanhydrides composed of sebacic acid, 1,3-bis(p-carboxyphenoxy) propane and poly(ethylene glycol) were synthesized and used as isolation layers. The polyanhydride isolation layers and PTH-loaded alginate layers were then stacked alternately within the delivery device. The gap between the stacked PTH-releasing core and the device frame was filled with PLLA to seal. Multi-pulse PTH release was achieved using the implantable device. The lag time between two adjacent pulses were modulated by the composition and the film thickness of the polyanhydride. The released PTH was demonstrated to be biologically active using an in vitro assay. Timed sequential release of multiple drugs has also been demonstrated. The implantable device holds promise for both systemic and local therapies.
Keywords: PTH; Pulsatile release; Controlled drug delivery; Osteoporosis; Polyanhydride; Bone tissue engineering;

A series of amphiphilic cationic graft polymers (PEC) were synthesized by coupling poly(ε-caprolactone) of differing molecular weights (MW) to low MW branched polyethylenimine via an amide group. IR, 1H-NMR and GPC were employed to characterize the graft copolymers. The self-assembly characteristics of these copolymers in an aqueous solution were studied by fluorescence techniques. The critical micelle concentration (CMC) varied from 0.044 to 0.032 g/L when the MW of poly(ε-caprolactone) increased from 1800 to 5500. The micelles formed electrostatic complexes with a reporter gene (pCMV-Luc) after an anticancer drug, Doxorubicin (DOX), was loaded by dialysis method. Gel retardation studies proved that micelles with or without DOX were able to complex with DNA completely at an equivalent N/P ratio of around 2.0, indicating that drug loading did not interfere in the interaction between the PEI shell and DNA. Particle size slightly decreased at higher N/P ratios of polyplexes, but increased with drug encapsulation. It was also noted that DNA/micelle complexes were significantly less toxic to HepG2 cells than blank PEC micelles, and improved gene transfection efficiency (about 3 orders of magnitude greater than PEI 25K alone at most) whether DOX was present in the system or not. These results suggest that this group of cationic graft polymers may be a potential candidate for the development of a drug delivery system that can examine the synergistic effects of combined drug and gene therapy.
Keywords: Gene therapy; Doxorubicin; Polyethylenimine; Poly(ε-caprolactone); Graft copolymer;

The in vitro kinetics of the interactions between PEG-ylated magnetic-fluid-loaded liposomes and macrophages by Marie-Sophie Martina; Valerie Nicolas; Claire Wilhelm; Christine Ménager; Gillian Barratt; Sylviane Lesieur (4143-4153).
Binding and uptake kinetics of magnetic-fluid-loaded liposomes (MFL) by endocytotic cells were investigated in vitro on the model cell-line J774. MFL consisted of unilamellar phosphatidylcholine vesicles (mean hydrodynamic diameter close to 200 nm) encapsulating 8-nm nanocrystals of maghemite (γ-Fe2O3) and sterically stabilized by introducing 5 mol% of distearylphosphatidylcholine poly(ethylene glycol)2000 (DSPE-PEG2000) in the vesicle bilayer. The association processes with living macrophages were followed at two levels. On one hand, the lipid vesicles were imaged by confocal fluorescence microscopy. For this purpose 1 mol% of rhodamine-marked phosphatidylethanolamine was added to the liposome composition. On the other hand, the iron oxide particles associated with cells were independently quantified by magnetophoresis. All the experiments were similarly performed with PEG-ylated or conventional MFL to point out the role of polymer coating. The results showed cell association with both types of liposomes resulting from binding followed by endocytosis. Steric stabilization by PEG chains reduced binding efficiency limiting the amount of MFL internalized by the macrophages. In contrast, PEG coating did not change the kinetics of endocytosis which exhibited the same first-order rate constant for both conventional and PEG-ylated liposomes. Moreover, lipids and iron oxide particle uptakes were perfectly correlated, indicating that MFL vesicle structure and encapsulation rate were preserved upon cell penetration.
Keywords: Magnetoliposomes; Ferrofluid; Endocytosis; Poly(ethylene glycol) coating; Confocal fluorescence microscopy; Magnetophoresis;

Methoxy polyethylene glycol–polylactide (MPEG–PLA) nanoparticles (NPs) were prepared by the nanoprecipitation method with particle size of 140±21 nm in diameter and drug encapsulation efficiency of 87.6±3.1%. In vitro cytotoxicity of the drug formulated in the NPs was investigated with MCF-7 cancer cells in close comparison with that of Taxol®. The in vitro cytotoxicity with MCF-7 cells showed that the NP formulation could be 33.3, 10.7, 7.7 times more effective than Taxol® after 24, 48, 72 h culture at the same drug concentration of 1 μg/ml. Confocal laser scanning microscopy (CLSM) visualized cellular internalization of the coumarin 6-loaded MPEG–PLA NPs. The in vitro results were further confirmed by the in vivo pharmacokinetic analysis with SD rats. The total area-under-the-curve (AUC0−∞), which determines the therapeutic effects of a dose, was found to be 29,600±1690 ng-h/ml for the NP formulation, which is 3.09 times of 9570±1480 ng-h/l for Taxol® with 10 mg/kg dose i.v. injection. The half-life (t 1/2) of the drug formulated in the NPs was found to be 18.80±3.14 h, which is 2.75 times of 6.84±1.39 h for Taxol®. The distribution volume at steady state for the drug loaded in the NPs was 7.21±2.17 l/kg, which was 2.93 times of 2.46±1.41 l/kg for Taxol®. Our proof-of-concept in vitro and in vivo valuation shows that our MPEG–PLA NP formulation could have great advantages versus the original drug in small-molecule drug chemotherapy as well as in various applications in nanomedicine.
Keywords: Biodegradable polymers; Cancer nanotechnology; Chemotherapeutic engineering; Nanobiotechnology; Nanomedicine; Oral drug delivery;

Silk coatings on PLGA and alginate microspheres for protein delivery by Xiaoqin Wang; Esther Wenk; Xiao Hu; Guillermo R. Castro; Lorenz Meinel; Xianyan Wang; Chunmei Li; Hans Merkle; David L. Kaplan (4161-4169).
Bombyx mori silk fibroin self-assembles on surfaces to form ultrathin nanoscale coatings based on our prior studies using layer-by-layer deposition techniques driven by hydrophobic interactions between silk fibroin protein molecules. In the present study, poly(lactic-co-glycolic acid) (PLGA) and alginate microspheres were used as substrates and coated with silk fibroin. The coatings were visualized by confocal laser scanning microscopy using fluorescein-labeled silk fibroin. On PLGA microspheres, the coating was ∼1 μm and discontinuous, reflecting the porous surface of these microspheres determined by SEM. In contrast, on alginate microspheres the coating was ∼10 μm thick and continuous. The silk fibroin penetrated into the alginate gel matrix. The silk coating on the PLGA microspheres delayed PLGA degradation. The silk coating on the alginate microspheres survived ethylenediamine tetraacetic acid (EDTA) treatment used to remove the Ca2+-cross-links in the alginate gels to solubilize the alginate. This suggests that alginate microspheres can be used as templates to form silk microcapsules. Horseradish peroxidase (HRP) and tetramethylrhodamine-conjugated bovine serum albumin (Rh-BSA) as model protein drugs were encapsulated in the PLGA and alginate microspheres with and without the silk fibroin coatings. Drug release was significantly retarded by the silk coatings when compared to uncoated microsphere controls, and was retarded further by methanol-treated silk coating when compared to silk water-based coatings on alginate microspheres. Silk coatings on PLGA and alginate microspheres provide mechanically stable shells as well as a diffusion barrier to the encapsulated protein drugs. This coating technique has potential for biosensor and drug delivery applications due to the aqueous process employed, the ability to control coating thickness and crystalline content, and the biocompatibility of the silk fibroin protein used in the process.
Keywords: Silk; Fibroin; Alginate; Polylactic acid; Polyglycolic acid; Controlled release;