Biomaterials (v.28, #19)

Highly oriented hydroxyapatite coatings (HACs) were obtained on titanium substrates through a radio-frequency thermal plasma spraying (TPS) method. XRD patterns showed that the HACs had crystallites with [0 0 l] preferred orientation vertical to the coating's surface. XRD results also indicated that tetracalcium phosphate crystallites in the as-sprayed HAC were oriented in the (1 0 0) direction. XRD peaks corresponding to tetracalcium phosphate, tricalcium phosphate and calcium oxide were absent after heat and hydrothermal treatment. The orientation degree of the HAC was influenced little by such post-heat treatments. Considering the crystallographic relationship between the tetracalcium phosphate in the as-sprayed HAC and the HA crystallites formed in the heat-treated HAC, these XRD results indicate that the tetracalcium phosphate in the as-prepared coatings transformed topotaxially into HA during the post-heat treatment. TEM and SEM analyses of the highly oriented HAC were conducted. The characteristic lamellar structure of TPS deposits was observed in cross-sections of the HAC. A prismatic texture was also observed in magnified SEM images. TEM observation showed that 200–800-nm-wide prismatic crystallites were formed in HA splats, and their longitudinal axis was oriented vertically to the coating's surface. SAD patterns showed that the longitudinal axis of the prismatic crystallites corresponded to the [0 0 1] zone axis of the HA crystal.
Keywords: Hydroxyapatite; Plasma spraying; Crystal orientation; Crystallization; Phase transformation; Topotaxy;

Behaviour of SH-SY5Y neuroblastoma cell line grown in different media and on different chemically modified substrates by M. Buttiglione; F. Vitiello; E. Sardella; L. Petrone; M. Nardulli; P. Favia; R. d’Agostino; R. Gristina (2932-2945).
Among the parameters that can be tested in experiments on neuronal cell culture the use of different culture media and substrates represents a powerful assay to influence cell adhesion and differentiation. In this work, plasma-enhanced-chemical vapour depositions (PE-CVD) from acrylic acid and allylamine vapours have been performed to deposit coatings bearing oxygen (O)- and nitrogen (N)-containing functional groups on polyethylenetherephtalate (PET) surface. Human neuroblastoma SH-SY5Y cells were grown on plasma modified substrates and in presence of media containing different amount of fetal calf serum (FCS) or in serum-free medium containing cAMP. Our results showed that N-containing substrates improved cell adhesion, while the neurites sprouting was influenced by cell culture media. Interestingly, the presence of carboxylic groups on the modified surface can influence the expression of a differentiation marker, neurofilament-200 (NF-H), in cells grown in serum-containing media.
Keywords: Neural cell; Surface modification; Plasma polymerisation; Cell morphology;

The effect of nano- and micron-sized particles of cobalt–chromium alloy on human fibroblasts in vitro by I. Papageorgiou; C. Brown; R. Schins; S. Singh; R. Newson; S. Davis; J. Fisher; E. Ingham; C.P. Case (2946-2958).
Wear debris from metal on polyethylene joint replacements causes asceptic loosening as a result of an inflammatory reaction of macrophages to micron-sized particles. Metal on metal implants, which generate nanoparticles, have been reintroduced into surgical practise in order to avoid this problem. There is a current concern about possible long-term effects of exposure to metal particles. In this study, the cytotoxic and genotoxic effects of nanoparticles and micron-sized particles of cobalt chrome alloy have been compared using human fibroblasts in tissue culture. Nanoparticles, which caused more free radicals in an acellular environment, induced more DNA damage than micron-sized particles using the alkaline comet assay. They induced more aneuploidy and more cytotoxicity at equivalent volumetric dose. Nanoparticles appeared to disintegrate within the cells faster than microparticles with the creation of electron dense deposits in the cell, which were enriched in cobalt. The mechanism of cell damage appears to be different after exposure to nanoparticles and microparticles. The concept of nanotoxicology is, therefore, an important consideration in the design of future surgical devices.
Keywords: Cell viability; Cobalt alloy; Electron microscopy; Genotoxicity; Mutagenicity; Nanoparticle;

The effect of surface charge on the uptake and biological function of mesoporous silica nanoparticles in 3T3-L1 cells and human mesenchymal stem cells by Tsai-Hua Chung; Si-Han Wu; Ming Yao; Chen-Wen Lu; Yu-Shen Lin; Yann Hung; Chung-Yuan Mou; Yao-Chang Chen; Dong-Ming Huang (2959-2966).
Cellular uptake of nanoparticles for stem cell labeling/tracking is considered as the most promising method. Recently mesoporous silica nanoparticles (MSNs) are emerging as an idea agent for efficient stem cell labeling. The objective of this study was to evaluate the effect of surface charge on the highly efficient cellular uptake and in vitro cytotoxicity of MSNs in human mesenchymal stem cells (hMSCs). The surface charge was varied by the degree of surface modification with N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride and the uptake of MSNs was detected by flow cytometry. 3T3-L1 cells were also used to compare the uptake behavior of MSNs between cell types. A clear correlation of positive surface charge and the number of fluorescence-labeled cells was mainly observed in 3T3-L1 cells. In both cells, uptake of unmodified MSNs was inhibited by phenylarsine oxide (PAO) and cytochalasin D (Cyt D) suggesting a clathrin- and an actin-dependent endocytosis were involved. With strongly positive-charged MSNs, the inhibitory effects were observed in 3T3-L1 cells but not in hMSCs. Without regard to the surface charge, uptake of MSNs into both cells did not affect their viability, proliferation, and differentiation. Our results show that MSNs uptake by hMSCs can be regulated by a threshold of positive surface charge but also imply that the modulation of surface charge on MSNs uptake is specific to cell type.
Keywords: Nanoparticle; Silica; Cellular uptake; Surface modification; Mesenchymal stem cell; Biocompatibility;

In vitro bioactivity and gene expression by cells cultured on titanium dioxide doped phosphate-based glasses by Ensanya Ali Abou Neel; Toshihide Mizoguchi; Michio Ito; Malak Bitar; Vehid Salih; Jonathan Campbell Knowles (2967-2977).
In our previous study, glasses with 50 P2O5-(20–15) Na2O-30 CaO-(0–5 mol%) TiO2 have been prepared by the conventional melt-quenching process. MG63 cell proliferation, gene expression, in vivo biocompatibility, and bioactivity of these glasses is the concern of this study. The results showed that addition of TiO2 in small amounts up to 5 mol% enhanced the biocompatibility of these glasses. The cell metabolic activity was conspicuous, on 3 and 5 mol% TiO2 compositions in particular, with no significant difference from Thermanox® control over a period of 21 days. The findings from the gene expression study showed that, at day 1 and on 5 mol% TiO2 glass, core binding protein factor alpha 1 (Cbfa1) and alkaline phosphatase (ALP) showed significantly lower transcription level; however, collagen type I alpha subunit I (COLIAI) and Osteonectin (Sparc) showed no significant differences compared to the control. At day 7, all these genes transcription levels were not significantly different form the control, but at day 14, they were significantly higher than the control. Moreover, there were no significant differences detected in these genes on both 3 and 5 mol% TiO2 glasses up to 7 days. At day 14; however, 5 mol% TiO2 glasses showed significantly higher level than 3 mol% TiO2 composition. This was also correlated by the presence of new bone tissue at the bone–particles interface for 5 mol% TiO2 composition after 5 weeks of implantation in rat calvarium. Regardless of this favourable cell response and gene up-regulation, these glasses showed no evidence of apatite layer formation after 14 days incubation in SBF.
Keywords: Phosphate glass; Titanium doped; Bioactivity; Simulated body fluid;

Photo-patterning of porous hydrogels for tissue engineering by Stephanie J. Bryant; Janet L. Cuy; Kip D. Hauch; Buddy D. Ratner (2978-2986).
Since pore size and geometry strongly impact cell behavior and in vivo reaction, the ability to create scaffolds with a wide range of pore geometries that can be tailored to suit a particular cell type addresses a key need in tissue engineering. In this contribution, we describe a novel and simple technique to design porous, degradable poly(2-hydroxyethyl methacrylate) hydrogel scaffolds with well-defined architectures using a unique photolithography process and optimized polymer chemistry. A sphere-template was used to produce a highly uniform, monodisperse porous structure. To create a patterned and porous hydrogel scaffold, a photomask and initiating light were employed. Open, vertical channels ranging in size from 360±25 to 730±70 μm were patterned into ∼700 μm thick hydrogels with pore diameters of 62±8 or 147±15 μm. Collagen type I was immobilized onto the scaffolds to facilitate cell adhesion. To assess the potential of these novel scaffolds for tissue engineering, a skeletal myoblast cell line (C2C12) was seeded onto scaffolds with 147 μm pores and 730 μm diameter channels, and analyzed by histology and digital volumetric imaging. Cell elongation, cell spreading and fibrillar formation were observed on these novel scaffolds. In summary, 3D architectures can be patterned into porous hydrogels in one step to create a wide range of tissue engineering scaffolds that may be tailored for specific applications.
Keywords: Scaffold; Hydrogel; Photopolymerization; Photolithography; Tissue engineering;

Integration of layered chondrocyte-seeded alginate hydrogel scaffolds by Christopher S.D. Lee; Jason P. Gleghorn; Nak Won Choi; Mario Cabodi; Abraham D. Stroock; Lawrence J. Bonassar (2987-2993).
Motivated by the necessity to engineer appropriately stratified cartilage, the shear mechanics of layered, bovine chondrocyte-seeded 20 mg/mL alginate scaffolds were investigated and related to the structure and biochemical composition. Chondrocyte-seeded alginate scaffolds were exposed to a calcium-chelating solution, layered, crosslinked in CaCl2, and cultured for 10 weeks. The shear mechanical properties of the layered gels were statistically similar to those of the non-layered controls. Shear modulus of layered gels increased by approximately six-fold while toughness and shear strength increased by more than two-fold during the culture period. Hydroxyproline content in both layered gels and controls had statistically significant increases after 6 weeks. Glycosaminoglycan (GAG) content of controls increased throughout culture while GAG content in layered gels leveled off after 4 weeks. Hematoxylin and eosin histological staining showed tissue growth at the interface over the first 4 weeks. Shear mechanical properties in the engineered tissues showed significant correlations to hydroxyproline content. Dependence of interfacial mechanical properties on hydroxyproline content was most evident for layered gels when compared to controls, especially for toughness and shear strength. Additionally, interfacial properties showed almost no dependence on GAG content. These findings demonstrate the feasibility of creating stratified engineered tissues through layering and that collagen deposition is necessary for interfacial integrity.
Keywords: Cartilage tissue engineering; Interface; Alginate; Micropatterning; Mechanical properties;

Ectopic neocartilage formation from predifferentiated human adipose derived stem cells induced by adenoviral-mediated transfer of hTGF beta2 by Xiao bing Jin; Yong sheng Sun; Ke Zhang; Jing Wang; Tai ping Shi; Xiao dong Ju; Si quan Lou (2994-3003).
Chondrogenic potential of human adipose derived stem cells (hASCs) makes them a possible source of seeding cells for cartilage tissue engineering. In this study, chondrogenic differentiation of hASCs induced by transduction with replication-deficient adenovirus carrying human transforming growth factor beta2 (Ad5-hTGF beta2) was demonstrated by RT-PCR, immunohistochemistry staining, biochemical and western blot analysis. To evaluate if the in vitro differentiated hASCs could keep their chondrocytic phenotype and produce neo-cartilage in vivo, predifferentiated hASCs were seeded in different scaffolds and implanted in subcutaneous pockets on the dorsum of nude mice. After 4 and 12 weeks culture in vivo, specimens were harvested and examined by histological and immunohistochemical analysis, cartilage-like tissue formation was only found in alginate gel and PLGA/alginate compound groups, in PLGA group, fibrous tissues and angiogenesis ingrowth were observed. These findings demonstrated that adenovirus-mediated hTGF beta2 gene transfer could induce hASCs into a chondrogenic lineage in vitro, however, this predifferentiation did not guarantee ectopic cartilage formation in vivo unless appropriate three-dimensional scaffolds were used as the cell carry vehicles.
Keywords: Chondrogenic; Adenovirus; Human transforming beta 2 (hTGF beta2); Tissue engineering; Alginate; PLGA;

The influence of matrix-derived adhesive peptide sequences on encapsulated β-cell survival and glucose-stimulated insulin release was explored by covalently incorporating synthetic peptide sequences within a model encapsulation environment. Photopolymerized poly(ethylene glycol) (PEG) hydrogels were functionalized via the addition of acrylate-PEG-peptide conjugates to the polymer precursor solution prior to β-cell photoencapsulation. Individual MIN6 β-cells were encapsulated in the presence of the laminin-derived recognition sequences, IKLLI, IKVAV, LRE, PDSGR, RGD, and YIGSR, and the collagen type I sequence, DGEA. In the absence of cell–cell and cell–matrix contacts, encapsulated MIN6 β-cell survival diminishes within one week; however, in PEG hydrogel derivatives including the laminin sequences IKLLI and IKVAV, encapsulated β-cells exhibit preserved viability, reduced apoptosis, and increased insulin secretion. Interactions with the laminin sequences LRE, PDSGR, RGD, and YIGSR contribute to improved viability, but insulin release from these samples was not statistically greater than that from controls. MIN6 β-cells were also encapsulated with various concentrations of IKLLI and IKVAV (0.05–5.0 mm), individually, and the peptide combinations IKLLI–IKVAV, IKVAV–YIGSR, and PDSGR–YIGSR to explore synergistic effects. The presented results give evidence that synthetic peptide epitopes may be useful in the design of an islet encapsulation environment that promotes cell survival and function via targeted cell–matrix interactions.
Keywords: Cell encapsulation; Peptide; Hydrogel; β-cell; Laminin;

Guidance of glial cell migration and axonal growth on electrospun nanofibers of poly-ε-caprolactone and a collagen/poly-ε-caprolactone blend by Eva Schnell; Kristina Klinkhammer; Simone Balzer; Gary Brook; Doris Klee; Paul Dalton; Jörg Mey (3012-3025).
Our long-term goal is to develop an artificial implant as a conduit for axonal regeneration after peripheral nerve injury. In this study, biodegradable, aligned poly-ε-caprolactone (PCL) and collagen/PCL (C/PCL) nanofibers designed as guidance structures were produced by electrospinning and tested in cell culture assays. We compared fibers of 100% PCL with fibers consisting of a 25:75% C/PCL blend. To test their biocompatibility, assays of cell adhesion, survival, migration, effects on cell morphology, axonal growth and axonal guidance were performed. Both types of eletrospun fibers supported oriented neurite outgrowth and glial migration from dorsal root ganglia (DRG) explants. Schwann cell migration, neurite orientation, and process formation of Schwann cells, fibroblasts and olfactory ensheathing cells were improved on C/PCL fibers, when compared to pure PCL fibers. While the velocity of neurite elongation from DRG explants was higher on PCL fibers, analysis of isolated sensory neurons showed significantly better axonal guidance by the C/PCL material. The data demonstrate that electrospun fibers composed of a collagen and PCL blend represent a suitable substrate for supporting cell proliferation, process outgrowth and migration and as such would be a good material for artificial nerve implants.
Keywords: Electrospinning; Schwann cells; DRG; Regeneration; Collagen; Polycaprolactone;

Chitosan microparticles encapsulating PEDF plasmid demonstrate efficacy in an orthotopic metastatic model of osteosarcoma by Crispin R. Dass; Karla G. Contreras; David E. Dunstan; Peter F.M. Choong (3026-3033).
The major stumbling block for most therapies against deep-seated disease, including tumours, is inefficient drug delivery. Such a concern is particularly important for osteosarcoma, the predominant form of bone cancer, and the largest cancer of its type in the paediatric age group. Pigment epithelium-derived factor (PEDF) is the most potent anti-angiogenic factor found endogenously in the body, with an increasing number of reports pointing to its direct antitumour activity. In this report, when a plasmid expressing PEDF (pPEDF) was encapsulated within two types of chitosan microparticles, anti-invasion and increased adhesion of the osteosarcoma cell line SaOS-2 was noted. Microparticles were formulated using two methods of complex coacervation and were ∼400–600 nm in diameter. The plasmids were strongly attached to the particles which were polymorphic in shape as determined by electron microscopy. Preliminary experiments with the green fluorescent protein (GFP) reporter plasmid revealed that cells were efficiently transfected with the particles, with particles outlasting transfection with lipofectamine cationic liposomes at 5 days. In vivo, the better pPEDF microparticle resulted in a decrease in primary tumour growth, reduced bone lysis and reduced establishment of lung metastases in a clinically relevant orthotopic model of osteosarcoma. Thus, this new mode of localised gene delivery may hold promise for molecular therapy of osteosarcoma.
Keywords: Chitosan; Microparticle; PEDF; Cancer; Gene delivery;

Enamel inspired nanocomposite fabrication through amelogenin supramolecular assembly by Yuwei Fan; Zhi Sun; Rizhi Wang; Christopher Abbott; Janet Moradian-Oldak (3034-3042).
Fabricating the structures similar to dental enamel through the in vitro preparation method is of great interest in the fields of dentistry and material sciences. Developing enamel is composed of calcium phosphate mineral, water, and enamel matrix proteins, mainly amelogenins. To prepare a material mimicking such composition a novel approach of simultaneously assembling amelogenin and calcium phosphate precipitates by electrolytic deposition (ELD) was established. It was found that recombinant full-length amelogenin (rP172) self-assembled into nanochain structures during ELD (following increase in solution pH), and had significant effect on the induction of the parallel bundles of calcium phosphate nanocrystals, grown on semiconductive silicon wafer surface. When a truncated amelogenin (rP148) was used; no nanochain assembly was observed, neither parallel bundles were formed. The coating obtained in the presence of rP172 had improved elastic modulus and hardness when compared to the coating incorporated with rP148. Our data suggest that the formation of organized bundles in amelogenin–apatite composites is mainly driven by amelogenin nanochain assembly and highlights the potential of such composite for future application as dental restorative materials.
Keywords: Electrolytic deposition; Amelogenin; Self-assembly; Enamel; Nanoindentation; Elastic modulus;

Micro-Raman and FTIR studies of synthetic and natural apatites by Anastasios Antonakos; Efthymios Liarokapis; Theodora Leventouri (3043-3054).
B-type synthetic carbonate hydroxyapatite (CHAp), natural carbonate fluorapatite (CFAp) and silicon-substituted hydroxyapatite (SiHAp) have been studied by using micro-Raman and infrared (IR) spectroscopy. It was found that while B-type carbonate substitution predominates in carbonate apatites (CAps), A-type is also detected. B-type carbonate substitution causes a broadening of the v 1 P–O stretching mode that is associated with the atomic disorder and lowering of the local symmetry in CAps from C 6 h 2 to C 3h. An ∼15 cm−1 shift of the v 3c PO4 stretching IR mode was observed upon decarbonation of the CFAp. Such shift which was confirmed by lattice dynamics calculations points out that the P–O bond lengths on the mirror plane increase when carbonate leaves the apatite structure. The present results support the substitution mechanism proposed on the basis of neutron powder diffraction studies of the same samples whereby carbonate substitutes on the mirror plane of the phosphate tetrahedron. The intensity ratios of the v 2 IR CO3 and v 1 PO4 bands in samples of various carbonate contents provide a measure of the degree of carbonation for CAps.
Keywords: Apatite structure; Carbonate substitution; FTIR; Hydroxyapatite; Micro-Raman spectroscopy;