Biomaterials (v.30, #27)

Blood coagulation on biomaterials requires the combination of distinct activation processes by Claudia Sperling; Marion Fischer; Manfred F. Maitz; Carsten Werner (4447-4456).
The rational design of hemocompatible materials requires a mechanistic understanding of activation processes induced at the blood–material interface. Binary self-assembled monolayers of alkyl thiols (SAMs) with various ratios of –CH3 and –COOH terminations were used to study the relevance of hydrophobic and negatively charged surfaces for the initiation of blood coagulation. Platelet adhesion and activation of the intrinsic coagulation pathway scaled with the surface composition: the numbers of adherent platelets were highest on the 100%-CH3 surface whereas the greatest contact activation was seen on 100%-COOH surfaces. In vitro whole blood incubation assays showed, however, that the surfaces exposing either –CH3 or –COOH groups induced comparably low levels of thrombin formation while the surfaces with intermediate contents of both terminating groups had significantly higher values. These results reveal that contact activation and platelet adhesion have a strong synergistic effect on coagulation on blood-contacting materials even though these events in isolation are not sufficient to induce substantial thrombin formation. Successful surface design strategies for hemocompatible materials therefore need to carefully consider the interplay of both processes.
Keywords: Self-assembled monolayer; Coagulation; Contact system; Platelet adhesion; Hemocompatibility;

Current methods to determine debonding/leakage at the tooth–composite interface are qualitative or semi-quantitative. Our previous work introduced a 3D imaging technique to determine and visualize leakage and its distribution at the interface of cavity wall and composite restoration in model cavities. In this study, an automated program was developed to quantify leakage in terms of area and volume. 3D leakage distribution obtained via the image analysis program was shown to have excellent agreement with leakage visualized by dye penetration. The relationship between leakage and various material performance parameters including processability, shrinkage, stress, and shrinkage strain-rate was determined using a series of experimental composites containing different filler contents. Results indicate that the magnitude of leakage correlated well with polymerization stress, confirming the validity of the common approach utilizing polymerization stress to predict bonding durability. 3D imaging and image analysis provide insight to help understand the relations between leakage and material properties.
Keywords: Dental restorative material; Image analysis; Interface; Infrared spectrum; Photopolymerization; Strain-rate;

Use of polyelectrolyte multi-layers as biomaterials for cell attachment has been limited due to their gel-like characteristics. Herein, we attempt to improve the cellular adhesion properties of multi-layer films, reduce their gel-like nature and rigidify them through chemical cross-linking with genipin; a natural and non-cytotoxic compound. Chitosan (CH), hyaluronan (HA) and alginate (Alg) were used to assemble [CH–HA] n CH and [CH–Alg] n CH films, and the effects of genipin cross-linking on the cell adhesion properties of these multi-layers were investigated. Atomic force microscopy (AFM) confirmed that cross-linking affected each of the films differently. Quartz crystal microbalance with dissipation (QCM-D) revealed that [CH–HA]10 CH films were very viscoelastic, with thicknesses in the range 350–450 nm, while [CH–Alg]10 CH films only grew to thicknesses of ∼100 nm. These differences were a result of the different growth regimes of these two polyelectrolyte systems. Cell adhesion studies using MC3T3 pre-osteoblasts and rat fibroblastic skin cells, carried out on both films demonstrated vast differences in cell adhesion. [CH–HA] n CH cross-linked films proved to be highly non-adhesive for pre-osteoblasts and fibroblastic skin cells. Conversely, cross-linking [CH–Alg] n CH films was shown to dramatically improve pre-osteoblast and rat fibroblastic skin cell adhesion, especially for high bi-layer numbers and using higher concentrations of cross-linker.
Keywords: Genipin cross-linking; Layer-by-layer assembly; Cell adhesion; Viscoelasticity;

Semi-conducting properties of titanium dioxide surfaces on titanium implants by Ingela U. Petersson; Johanna E.L. Löberg; Anette S. Fredriksson; Elisabet K. Ahlberg (4471-4479).
The properties of the TiO2 layer on titanium implant surfaces are decisive for good contact with the surrounding tissue. The oxide properties can be deliberately changed by for example chemical etching, ion incorporation or anodisation. In the present study impedance spectroscopy was used to study the semi-conducting properties of the naturally formed oxide for different pre-treatment of the surface. A turned surface was used as a reference and both physical (blasting) and chemical (hydrofluoric acid etching) treatments were investigated. Blasting of a titanium sample introduces defects in the metal surface and the study clearly shows that also the oxide layer contains defects leading to a higher number of charge carriers (increased conductivity) compared with the oxide on the turned surface. The hydrofluoric acid etching of the blasted surface results in an oxide film with even higher conductivity. Indication of the defect oxide structure for fluoride treated samples was also seen when analysing the TiO+/Ti+ ratio from ToF-SIMS data. The lowest value of this ratio was obtained for the HF etched sample, indicating a less stoichiometric oxide compared to the other surfaces. This is a result of incorporation of fluoride ions in the oxide, as proven by adsorption studies on a TiO2 suspension. The results were treated in the context of surface complexation and two surface complexes were identified. Our results are discussed in relation to pull-out data on rabbit. The pull-out forces depend primarily on surface roughness but the contribution from the hydrofluoric acid etching might be explained by fluoride ion incorporation and the resulting increase in oxide conductivity.
Keywords: Titanium implant; ToF-SIMS; Semi-conducting properties; Electrochemistry; Surface modifications; Impedance spectroscopy;

Dimethacrylate polymers and composites are seeing increased usage in orthopedics. As these applications require the material to integrate with the surrounding tissues, direct contact cytotoxicity assays should be used to assess the biocompatibility. This study utilized a combinatorial testing platform to evaluate the cell response to dimethacrylate composites with a variety of properties on a single sample. MC3T3-E1 pre-osteoblasts were cultured directly on composites with varying filler content, filler type, degree of conversion (DC), and surface topography. Cell viability, density, and area depended on an interplay of the material properties, with low DC causing a reduction in cell area but having minimal effect on cell viability, high filler content causing an increase in cell density, and filler content/type altering the surface roughness as a function of DC. The combinatorial testing platform successfully quantified the effects of numerous material properties on several aspects of the osteoblast response.
Keywords: Composite; Contact angle; Cytotoxicity; Osteoblast; Photopolymerization; Surface topography;

Randomly oriented, upright SiO2 coated nanorods for reduced adhesion of mammalian cells by Jiyeon Lee; Byung Hwan Chu; Ke-Hung Chen; Fan Ren; Tanmay P. Lele (4488-4493).
Cell interactions with nanostructures are of broad interest because of applications in controlling tissue response to biomedical implants. Here we show that dense and upright SiO2 coated nanorods nearly eliminate cell adhesion in fibroblasts and endothelial cells. The lack of adhesion is not due to a decrease in matrix protein adsorption on the nanostructures, but rather an inability of cells to assemble focal adhesions. Using spatially patterned nanorods, we show that cells display a preference for flat regions of the surface. Our results support a model in which interfering with nanoscale spacing of ligated integrins results in reduced cell adhesion and subsequent cell death. We propose that dense monolayers of nanorods are a promising nanotechnology for preventing mammalian cell fouling of biomaterials.
Keywords: Nanorods; Cell adhesion; Cell survival; Anti-fouling;

Nitric oxide-releasing S-nitrosothiol-modified xerogels by Daniel A. Riccio; Kevin P. Dobmeier; Evan M. Hetrick; Benjamin J. Privett; Heather S. Paul; Mark H. Schoenfisch (4494-4502).
The synthesis, material characterization, and in vitro biocompatibility of S-nitrosothiol (RSNO)-modified xerogels are described. Thiol-functionalized xerogel films were formed by hydrolysis and co-condensation of 3-mercaptopropyltrimethoxysilane (MPTMS) and methyltrimethoxysilane (MTMOS) sol–gel precursors at varying concentrations. Subsequent thiol nitrosation via acidified nitrite produced RSNO-modified xerogels capable of generating nitric oxide (NO) for up to 2 weeks under physiological conditions. Xerogels also exhibited NO generation upon irradiation with broad-spectrum light or exposure to copper, with NO fluxes proportional to wattage and concentration, respectively. Xerogels were capable of storing up to ∼1.31 μmol NO mg−1, and displayed negligible fragmentation over a 2-week period. Platelet and bacterial adhesion to nitrosated films was reduced compared to non-nitrosated controls, confirming the antithrombotic and antibacterial properties of the NO-releasing materials. Fibroblast cell viability was maintained on the xerogel surfaces illustrating the promise of RSNO-modified xerogels as biomedical device coatings.
Keywords: Nitric oxide; Nitrosothiol; Xerogel; Antithrombotic; Antimicrobial; Cytotoxicity;

Concentration gradients of matrix-bound guidance cues in the extracellular matrix direct cell growth in native tissues and are of great interest for the design of biomedical scaffolds and on implant surfaces.This study describes effects of covalently immobilized gradients of the 6th Ig-like domain of cell adhesion molecule L1 (TG-L1Ig6) within 3D-fibrin matrices on cell alignment. Linear gradients of TG-L1Ig6 were established and shown to be stable for at least 24 h whereas soluble gradients disappeared completely. Fibroblast alignment along the gradients was observed when cultured on top and within TG-L1Ig6-gradient matrices. Fibroblasts responded to an increase of 0.2 μg TG-L1Ig6/ml per mm matrix, corresponding to a concentration change of <1% per cell. Significant differences were observed when fibroblasts were cultured within the TG-L1Ig6-gradient matrices as the number of aligned cells decreased by 20–30% in the middle of the gradient when compared to cells cultivated on top of the gradient. This finding might be explained by ∼13% reduction in the average cell length of fibroblasts within compared to fibroblasts cultured on top of the gradient matrix. In contrast to fibroblasts endothelial cells did not show any alignment with TG-L1Ig6-gradient matrices. The study indicates that cells exposed to gradients of matrix-bound TG-L1Ig6 are able to respond differentially to 2D- or 3D-environments suggesting the use of gradients for cell guidance within 3D-scaffolds and on implant surfaces to improve their biomedical functions.
Keywords: Covalent gradients; 3D-fibrin matrices; Cell alignment; TG-L1Ig6; Cell guidance cues;

Immobilization of alkaline phosphatase on microporous nanofibrous fibrin scaffolds for bone tissue engineering by Thanaphum Osathanon; Cecilia M. Giachelli; Martha J. Somerman (4513-4521).
Alkaline phosphatase (ALP) promotes bone formation by degrading inorganic pyrophosphate (PPi), an inhibitor of hydroxyapatite formation, and generating inorganic phosphate (Pi), an inducer of hydroxyapatite formation. Pi is a crucial molecule in differentiation and mineralization of osteoblasts. In this study, a method to immobilize ALP on fibrin scaffolds with tightly controllable pore size and pore interconnection was developed, and the biological properties of these scaffolds were characterized both in vitro and in vivo. Microporous, nanofibrous fibrin scaffolds (FS) were fabricated using a sphere-templating method. ALP was covalently immobilized on the fibrin scaffolds using 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC). Scanning electron microscopic observation (SEM) showed that mineral was deposited on immobilized alkaline phosphatase fibrin scaffolds (immobilized ALP/FS) when incubated in medium supplemented with β-glycerophosphate, suggesting that the immobilized ALP was active. Primary calvarial cells attached, spread and formed multiple layers on the surface of the scaffolds. Mineral deposition was also observed when calvarial cells were seeded on immobilized ALP/FS. Furthermore, cells seeded on immobilized ALP/FS exhibited higher osteoblast marker gene expression compared to control FS. Upon implantation in mouse calvarial defects, both the immobilized ALP/FS and FS alone treated group had higher bone volume in the defect compared to the empty defect control. Furthermore, bone formation in the immobilized ALP/FS treated group was statistically significant compared to FS alone group. However, the response was not robust.
Keywords: Alkaline phosphatase; Fibrin; Bone tissue engineering; Phosphate; Pyrophosphate;

Mandibular repair in rats with premineralized silk scaffolds and BMP-2-modified bMSCs by Xinquan Jiang; Jun Zhao; Shaoyi Wang; Xiaojuan Sun; Xiuli Zhang; Jake Chen; David L. Kaplan; Zhiyuan Zhang (4522-4532).
Premineralized silk fibroin protein scaffolds (mSS) were prepared to combine the osteoconductive properties of biological apatite with aqueous-derived silk scaffold (SS) as a composite scaffold for bone regeneration. The aim of present study was to evaluate the effect of premineralized silk scaffolds combined with bone morphogenetic protein-2 (BMP-2) modified bone marrow stromal cells (bMSCs) to repair mandibular bony defects in a rat model. bMSCs were expanded and transduced with adenovirus AdBMP-2, AdLacZ gene in vitro. These genetically modified bMSCs were then combined with premineralized silk scaffolds to form tissue-engineered bone. Mandibular repairs with AdBMP-2 transduced bMSCs/mSS constructs were compared with those treated with AdLacZ-transduced bMSCs/mSS constructs, native (nontransduced) bMSCs/mSS constructs and mSS alone. Eight weeks after post-operation, the mandibles were explanted and evaluated by radiographic observation, micro-CT, histological analysis and immunohistochemistry. The presence of BMP-2 gene enhanced tissue-engineered bone in terms of the most new bone formed and the highest local bone mineral densities (BMD) found. These results demonstrated that premineralized silk scaffold could serve as a potential substrate for bMSCs to construct tissue-engineered bone for mandibular bony defects. BMP-2 gene therapy and tissue engineering techniques could be used in mandibular repair and bone regeneration.
Keywords: Tissue engineering; Silk scaffold; Apatite; Bone morphogenetic proteins; Bone marrow stromal cells;

In vitro evaluation of a multi-layer radial-flow bioreactor based on galactosylated chitosan nanofiber scaffolds by Xue-Hui Chu; Xiao-Lei Shi; Zhang-Qi Feng; Jin-Yang Gu; Hai-Yun Xu; Yue Zhang; Zhong-Ze Gu; Yi-Tao Ding (4533-4538).
Clinical use of bioartificial livers (BAL) strongly relies on the development of bioreactors. In this study, we developed a multi-layer radial-flow bioreactor based on galactosylated chitosan nanofiber scaffolds and evaluated its efficacy in vitro. The bioreactor contains 65 layers of stacked flat plates, on which the nanofiber scaffolds were electrospinned for hepatocyte immobilization and aggregation. Culture medium containing pig red blood cells (RBCs) was perfused from the center to periphery, so that exchange materials are sufficient to afford enough oxygen. We determined the parameters for hepatocyte-specific function and general metabolism and also measured the oxygen consumption rate (OCR). Microscope and scanned electron microscopy observation showed a tight adhesion between cells and scaffolds. Compared with the control (bioreactors without nanofiber scaffolds), the number of adhered cells in our bioreactor was 1.59-fold; the protein-synthesis capacity of hepatocytes was 1.73-fold and urea was 2.86-fold. Moreover, the OCR of bioreactors with RBCs was about 1.91-fold that of bioreactors without RBCs. The galactosylated chitosan nanofiber scaffolds introduced into our new bioreactor greatly enhanced cell adhesion and function, and the RBCs added into the culture medium were able to afford enough oxygen for hepatocytes. Importantly, our new bioreactor showed an exciting efficiency, and it may afford the short-term support of patients with hepatic failure.
Keywords: Bioartificial liver; Bioreactor; Chitosan; Hepatocyte; Nanotopography;

The quantitative assessment of peri-implant bone responses using histomorphometry and micro-computed tomography by Corinne Schouten; Gert J. Meijer; Jeroen J.J.P. van den Beucken; Paul H.M. Spauwen; John A. Jansen (4539-4549).
In the present study, the effects of implant design and surface properties on peri-implant bone response were evaluated with both conventional histomorphometry and micro-computed tomography (micro-CT), using two geometrically different dental implants (Screw type, St; Push-in, Pi) either or not surface-modified (non-coated, CaP-coated, or CaP-coated + TGF-β1). After 12 weeks of implantation in a goat femoral condyle model, peri-implant bone response was evaluated in three different zones (inner: 0–500 μm; middle: 500–1000 μm; and outer: 1000–1500 μm) around the implant. Results indicated superiority of conventional histomorphometry over micro-CT, as the latter is hampered by deficits in the discrimination at the implant/tissue interface. Beyond this interface, both analysis techniques can be regarded as complementary. Histomorphometrical analysis showed an overall higher bone volume around St compared to Pi implants, but no effects of surface modification were observed. St implants showed lowest bone volumes in the outer zone, whereas inner zones were lowest for Pi implants. These results implicate that for Pi implants bone formation started from two different directions (contact- and distance osteogenesis). For St implants it was concluded that undersized implantation technique and loosening of bone fragments compress the zones for contact and distant osteogenesis, thereby improving bone volume at the interface significantly.
Keywords: Animal model; Dental implant; Histomorphometry; Micro-computed tomography; Osteogenesis; Surface modification;

Synthesis of highly porous crosslinked elastin hydrogels and their interaction with fibroblasts in vitro by Nasim Annabi; Suzanne M. Mithieux; Elizabeth A. Boughton; Andrew J. Ruys; Anthony S. Weiss; Fariba Dehghani (4550-4557).
In this study the feasibility of using high pressure CO2 to produce porous α-elastin hydrogels was investigated. α-Elastin was chemically crosslinked with hexamethylene diisocyanate that can react with various functional groups in elastin such as lysine, cysteine, and histidine. High pressure CO2 substantially affected the characteristics of the fabricated hydrogels. The pore size of the hydrogels was enhanced 20-fold when the pressure was increased from 1 bar to 60 bar. The swelling ratio of the samples fabricated by high pressure CO2 was also higher than the gels produced under atmospheric pressure. The compression modulus of α-elastin hydrogels was increased as the applied strain magnitude was modified from 40% to 80%. The compression modulus of hydrogels produced under high pressure CO2 was 3-fold lower than the gels formed at atmospheric conditions due to the increased porosity of the gels produced by high pressure CO2. The fabrication of large pores within the 3D structures of these hydrogels substantially promoted cellular penetration and growth throughout the matrices. The highly porous α-elastin hydrogel structures fabricated in this study have potential for applications in tissue engineering.
Keywords: Elastin; Hydrogel; Crosslinking; Hexamethylene diisocyanate; High pressure CO2; Fibroblast;

The cytocompatibility of amphiphilic, thermoresponsive and chemically crosslinkable macromers was examined in vitro. Macromers synthesized from pentaerythritol diacrylate monostearate, N-isopropylacrylamide, acrylamide and hydroxyethyl acrylate in different molar ratios and with varying molecular weights and lower critical solution temperatures were evaluated for cytocompatibility with rat fibroblasts. Cell viabilities of over 60% for all and over 80% for most formulations were observed after 24-h incubation with macromers with molecular weights in the range of approximately 1500–3000 Da. The chemical modification of the macromers with a (meth)acrylate group was shown to have a time- and dose-dependent effect on cell viability. Uncrosslinked macromers with lower degrees of (meth)acrylation allowed for cell viability of over 60% for up to 6 h. (Meth)acrylated macromers with lower critical solution temperature (LCST) closer to physiological temperature allowed for higher cell viabilities as opposed to those with lower LCST. The data suggest that when the (meth)acrylated macromers are assembled into a physical gel, their cytotoxicity is diminished. After gel phase separation, cytotoxicity increased. This study gives information on the parameters that enable viable cell encapsulation for in situ forming hydrogel systems.
Keywords: Cytotoxicity; Hydrogel; Cell viability; Thermally responsive material; In vitro test;

The promotion of neuronal maturation on soft substrates by Ana I. Teixeira; Shirin Ilkhanizadeh; Jens A. Wigenius; Joshua K. Duckworth; Olle Inganäs; Ola Hermanson (4567-4572).
Microenvironmental mechanical properties of stem cell niches vary across tissues and developmental stages. Accumulating evidence suggests that matching substrate elasticity with in vivo tissue elasticity facilitates stem cell differentiation. However, it has not been established whether substrate elasticity can control the maturation stage of cells generated by stem cell differentiation. Here we show that soft substrates with elasticities commensurable to the elasticity of the brain promote the maturation of neural stem cell-derived neurons. In the absence of added growth factors, neurons differentiated on soft substrates displayed long neurites and presynaptic terminals, contrasting with the bipolar immature morphology of neurons differentiated on stiff substrates. Further, soft substrates supported an increase in astrocytic differentiation. However, stiffness cues could not override the dependency of astrocytic differentiation on Notch signaling. These results demonstrate that substrate elasticity per se can drive neuronal maturation thus defining a crucial parameter in neuronal differentiation of stem cells.
Keywords: Microenvironment; Mechanical properties; Neural stem cells; Neuronal maturation; PDMS;

Neurite infiltration and cellular response to electrospun polycaprolactone scaffolds implanted into the brain by David R. Nisbet; Andrew E. Rodda; Malcolm K. Horne; John S. Forsythe; David I. Finkelstein (4573-4580).
Assessment of axonal infiltration and guidance within neural tissue engineering scaffolds, along with the characterisation of the inflammatory response, is critical in determining these scaffolds' potential for facilitating neural repair. In this study, the extent of microglial and astrocytic response was measured following implantation of electrospun poly(ɛ-caprolactone) (PCL) scaffolds into the caudate putamen of the adult rat brain. The inflammation peaked at around 4 days (microglia) and 7 days (astrocytes) and subsided to homeostatic levels by 60 days. There was no evidence of microglial encapsulation and indeed neurites had infiltrated the implants, evidence of scaffold-neural integration. Whilst the inflammatory response was uninfluenced by the degree of PCL fibre alignment, the extent of neurite entry was. Large porosity, as was the case with the randomly orientated polymer fibres, enabled neurite infiltration and growth within the scaffold. However, neuronal processes could not penetrate scaffolds when fibres were partially aligned and instead, preferentially grew perpendicular to the direction of PCL fibre alignment at the implant-tissue interface i.e. perpendicular, not parallel, contact guidance was provided. This investigation shows that electrospun PCL fibres are compatible with brain tissue and provide preliminary insights regarding the influence of microglia and astrocytes in neural integration within such scaffolds.
Keywords: Electrospinning; Neural tissue engineering; Nerve regeneration; In vivo; Inflammatory response; Neurite;

Cell transplantation is a potential therapy for central nervous disorders such as Parkinson's disease. However, the therapeutic effect is limited by the low viability of transplanted cells. To solve this problem, we synthesized hyaluronic acid (HAc)-based hydrogel to be used as a carrier for neural cells. Another feature of the hydrogel synthesized here is to incorporate brain-derived neurotrophic factor (BDNF) for enhancing cell survival in the hydrogel. The cross-linking of HAc and the incorporation of BDNF were both achieved by employing the ability of a hexahistidine peptide to form stable chelate with metal ions. HAc was reacted with N-(5-amino-1-carboxypentyl) iminodiacetic acid and chelated with Zn(II) ions. An alpha-helical peptide of 70 amino acid residues carrying a hexahistidine peptide (His) at the both termini and BDNF carrying a His at the C-terminus were synthesized by recombinant DNA technology. These polypeptides were coordinated with the Zn(II) ions chelated to HAc chains for both the cross-linking of HAc and the tethering of BDNF. Viscosity measurements revealed that intermolecular cross-links were introduced in the Zn(II)–chelated HAc chains upon mixing with the His-containing helical peptide. It was shown by release tests that the coordinated BDNF was firmly bound to the Zn(II)-chelated HAc for more than 12 days. The results of cell culture experiments combined with live/dead assays demonstrated that a significantly higher fraction of neural cells survived 3 days post-seeding in the HAc-based hydrogel incorporating BDNF than in the control hydrogel lacking BDNF. These results suggest that the HAc-based hydrogel developed here has potential to improve survival of transplanted neural cells.
Keywords: Biodegradable hydrogel; Neurotrophic factor; Cell survival; Transplantation; Chimeric protein;

TiO2 nanoparticles translocation and potential toxicological effect in rats after intraarticular injection by Jiang-Xue Wang; Yu-Bo Fan; Yu Gao; Qing-Hua Hu; Tian-Cheng Wang (4590-4600).
Recently, nanomaterials coating gained much concern in orthopedic implants such as bone, cartilage, joint, etc. The wear particles would generate from coating in living organism due to corrosion. In this study, we demonstrated that the intraarticular injected anatase TiO2 nanoparticles had a potential toxicological effect on major organs and knee joints of rats. The histopathological changes of heart, lung and liver indicated the dissemination of intraarticular TiO2 nanoparticles from joint cavity to system. In the knee joint, the aggregated TiO2 nanoparticles deposited and resulted in the synovium hypotrophy and lymphocytes and plasma cells infiltration, but had no effects on cartilage. In the TiO2-exposed synovium, the oxidative damage was induced because the glutathione peroxidase (GSH-Px), reduced glutathione (GSH), oxidized glutathione (GSSG), and superoxide dismutase (SOD) levels were highly regulated to counteract over-produced free radicals, i.e. hydrogen peroxide (H2O2). Further, the lipid peroxidation was detected in the synovium though the expression of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin (IL-1β) was not much interfered. This research suggested that the amounts of nanocoating in the surface of implants should be controlled and standardized.
Keywords: Nanoparticles; TiO2 biomaterials; Intraarticular injection; Toxicological effect; Oxidative stress;

Modifications of poly(2-hydroxyethyl methacrylate) (PHEMA) with cholesterol and laminin have been developed to design scaffolds that promote cell–surface interaction. Cholesterol-modified superporous PHEMA scaffolds have been prepared by the bulk radical copolymerization of 2-hydroxyethyl methacrylate (HEMA), cholesterol methacrylate (CHLMA) and the cross-linking agent ethylene dimethacrylate (EDMA) in the presence of ammonium oxalate crystals to introduce interconnected superpores in the matrix. With the aim of immobilizing laminin (LN), carboxyl groups were also introduced to the scaffold by the copolymerization of the above monomers with 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA). Subsequently, the MCMEMA moiety in the resulting hydrogel was hydrolyzed to [2-(methacryloyloxy)ethoxy]acetic acid (MOEAA), and laminin was immobilized via carbodiimide and N-hydroxysulfosuccinimide chemistry. The attachment, viability and morphology of mesenchymal stem cells (MSCs) were evaluated on both nonporous and superporous laminin-modified as well as laminin-unmodified PHEMA and poly(2-hydroxyethyl methacrylate-co-cholesterol methacrylate) P(HEMA–CHLMA) hydrogels. Neat PHEMA and laminin-modified PHEMA (LN–PHEMA) scaffolds facilitated MSC attachment, but did not support cell spreading and proliferation; the viability of the attached cells decreased with time of cultivation. In contrast, MSCs spread and proliferated on P(HEMA–CHLMA) and LN-P(HEMA–CHLMA) hydrogels.
Keywords: Cell adhesion; Cell viability; Hydrogel; PolyHEMA; Porosity;

A three-dimensional scaffold with precise micro-architecture and surface micro-textures by Alvaro Mata; Eun Jung Kim; Cynthia A. Boehm; Aaron J. Fleischman; George F. Muschler; Shuvo Roy (4610-4617).
A three-dimensional (3D) structure comprising precisely defined micro-architecture and surface micro-textures, designed to present specific physical cues to cells and tissues, may provide an efficient scaffold in a variety of tissue engineering and regenerative medicine applications. We report a fabrication technique based on microfabrication and soft lithography that permits for the development of 3D scaffolds with both precisely engineered architecture and tailored surface topography. The scaffold fabrication technique consists of three key steps starting with microfabrication of a mold using an epoxy-based photoresist (SU-8), followed by dual-sided molding of a single layer of polydimethylsiloxane (PDMS) using a mechanical jig for precise motion control; and finally, alignment, stacking, and adhesion of multiple PDMS layers to achieve a 3D structure. This technique was used to produce 3D Texture and 3D Smooth PDMS scaffolds, where the surface topography comprised 10 μm diameter/height posts and smooth surfaces, respectively. The potential utility of the 3D microfabricated scaffolds, and the role of surface topography, were subsequently investigated in vitro with a combined heterogeneous population of adult human stem cells and their resultant progenitor cells, collectively termed connective tissue progenitors (CTPs), under conditions promoting the osteoblastic phenotype. Examination of bone-marrow derived CTPs cultured on the 3D Texture scaffold for 9 days revealed cell growth in three dimensions and increased cell numbers compared to those on the 3D Smooth scaffold. Furthermore, expression of alkaline phosphatase mRNA was higher on the 3D Texture scaffold, while osteocalcin mRNA expression was comparable for both types of scaffolds.
Keywords: Micro-architecture; Microfabrication; Surface micro-textures; Scaffolds; Connective tissue progenitor cells; BioMEMS;

Growth factors are a class of signaling proteins that direct cell fate through interaction with cell-surface receptors. Although a myriad of possible cell fates stems from a growth factor binding to its receptor, the signaling cascades that result in one fate over another are still being elucidated. One possible mechanism by which nature modulates growth factor signaling is through the method of presentation of the growth factor – soluble or immobilized (matrix bound). Here we present the methodology to study signaling of soluble versus immobilized VEGF through VEGFR-2. We have designed a strategy to covalently immobilize VEGF using its heparin-binding domain to orient the molecule (bind) and a secondary functional group to mediate covalent binding (lock). This bind-and-lock approach aims to allow VEGF to assume a bioactive orientation before covalent immobilization. Surface plasmon resonance (SPR) demonstrated heparin and VEGF binding with surface densities of 60 ng/cm2 and 100 pg/cm2, respectively. ELISA experiments confirmed VEGF surface density and showed that electrostatically bound VEGF releases in cell medium and heparin solutions while covalently bound VEGF remains immobilized. Electrostatically bound VEGF and covalently bound VEGF phosphorylate VEGFR-2 in both VEGFR-2 transfected cells and VEGFR-2 endogenously producing cells. HUVECs plated on VEGF functionalized surfaces showed different morphologies between surface-bound VEGF and soluble VEGF. The surfaces synthesized in these studies allow for the study of VEGF/VEGFR-2 signaling induced by covalently bound, electrostatically bound, and soluble VEGF and may provide further insight into the design of materials for the generation of a mature and stable vasculature.
Keywords: Angiogenesis; Cell signaling; Endothelial cell; Fibronectin; Growth factors; Heparin;

The influence of tethered epidermal growth factor on connective tissue progenitor colony formation by Nicholas A. Marcantonio; Cynthia A. Boehm; Richard J. Rozic; Ada Au; Alan Wells; George F. Muschler; Linda G. Griffith (4629-4638).
Strategies to combine aspirated marrow cells with scaffolds to treat connective tissue defects are gaining increasing clinical attention and use. In situations such as large defects where initial survival and proliferation of transplanted connective tissue progenitors (CTPs) are limiting, therapeutic outcomes might be improved by using the scaffold to deliver growth factors that promote the early stages of cell function in the graft. Signaling by the epidermal growth factor receptor (EGFR) plays a role in cell survival and has been implicated in bone development and homeostasis. Providing epidermal growth factor (EGF) in a scaffold-tethered format may sustain local delivery and shift EGFR signaling to pro-survival modes compared to soluble ligand. We therefore examined the effect of tethered EGF on osteogenic colony formation from human bone marrow aspirates in the context of three different adhesion environments using a total of 39 donors. We found that tethered EGF, but not soluble EGF, increased the numbers of colonies formed regardless of adhesion background, and that tethered EGF did not impair early stages of osteogenic differentiation.
Keywords: Connective tissue; Mesenchymal stem cell; Stem cell; Bone tissue engineering; Growth factors; Adhesion molecule;

The use of a synthetic oxygen carrier-enriched hydrogel to enhance mesenchymal stem cell-based bone formation in vivo by Nadav Kimelman-Bleich; Gadi Pelled; Dima Sheyn; Ilan Kallai; Yoram Zilberman; Olga Mizrahi; Yamit Tal; Wafa Tawackoli; Zulma Gazit; Dan Gazit (4639-4648).
A major hurdle to surmount in bone-tissue engineering is ensuring a sufficient oxygen supply to newly forming tissue to avoid cell death or delayed development of osteogenic features. We hypothesized that an oxygen-enriched hydrogel scaffold would enhance tissue-engineered bone formation in vivo. To test this, we used a well-characterized mesenchymal stem cell (MSC) line, Tet-off BMP2 MSC, whose cells were engineered to express recombinant human bone morphogenetic protein-2. Cells were suspended in hydrogel supplemented with perfluorotributylamine (PFTBA) and implanted subcutaneously in an ectopic site, a radial bone defect, or a lumbar paravertebral muscle (mouse model of spinal fusion) in C3H/HeN mice. For controls, we used cells suspended in the same gel without PFTBA. In the ectopic site, there were significant increases in bone formation (2.5-fold increase), cell survival, and osteocalcin activity in the PFTBA-supplemented groups. PFTBA supplementation significantly increased structural parameters of bone in radial bone defects and triggered a significant 1.4-fold increase in bone volume in the spinal fusion model. We conclude that synthetic oxygen carrier supplementation of tissue-engineered implants enhances ectopic bone formation and yields better bone quality and volume in bone-repair and spinal fusion models, probably due to increased cell survival.
Keywords: BMP; Bone tissue engineering; Fluorocarbon; Hydrogel; Mesenchymal stem cell; Oxygenation;

The effect of collagen-binding NGF-β on the promotion of sciatic nerve regeneration in a rat sciatic nerve crush injury model by Wenjie Sun; Changkai Sun; Hang Lin; Hui Zhao; Jingyu Wang; Hui Ma; Bing Chen; Zhifeng Xiao; Jianwu Dai (4649-4656).
Nerve growth factor plays a critical role in peripheral nerve regeneration. However, the lack of efficient NGF delivery approach limits its clinical application. It has demonstrated in our previous work that the native human NGF-β (NAT-NGF) fused with a collagen-binding domain (CBD) could bind to collagen specifically. Since collagen is the major component of nerve extracellular matrix, we speculated that the collagen-binding NGF would target to nerve cells and improve their regeneration. In this report, we found that the fusion protein could specifically bind to endogenous collagen of the rat sciatic nerves and maintain NGF activity both in vitro and in vivo. In the rat sciatic nerve crush injury model, we found that collagen-binding NGF could be retained and concentrated at the nerve injured site to promote nerve repair and enhance function recovery following nerve damage. Thus, the collagen-binding NGF could improve the repair of peripheral nerve injury.
Keywords: Collagen; Nerve growth factor; Nerve; Recombinant protein; Wound healing;

In situ chondrogenic differentiation of human adipose tissue-derived stem cells in a TGF-β1 loaded fibrin–poly(lactide-caprolactone) nanoparticulate complex by Youngmee Jung; Yong-Il Chung; Sang Hee Kim; Giyoong Tae; Young Ha Kim; Jong Won Rhie; Sang-Heon Kim; Soo Hyun Kim (4657-4664).
When conducting cartilage tissue engineering with stem cells, it is well known that chemical and physical stimulations are very important for the induction and maintenance of chondrogenesis. In this study, we induced chondrogenic differentiation of human adipose tissue-derived stem cells (hASCs) in situ by effective stimulation via the continuous controlled release of TGF-β1 from a heparin-functionalized nanoparticle–fibrin–poly(lactide-co-caprolactone) (PLCL) complex. PLCL scaffolds were fabricated with 85% porosity and 300–500 μm pore size by a gel-pressing method. Heparin-functionalized nanoparticles were prepared by a solvent-diffusion method, composed of poly(lactide-co-glycolide) (PLGA), Pluronic F-127, and heparin, and then TGF-β1 was loaded to the nanoparticles. A mixture of hASCs, fibrin gels and TGF-β1 loaded nanoparticles was then seeded onto PLCL scaffolds and cultured in vitro, after which they were subcutaneously implanted into nude mice for up to five weeks. The results of in vitro and in vivo studies revealed that chondrogenic differentiation of the hASCs on the complex was induced and sustained by continuous stimulation by TGF-β1 from the heparin-functionalized nanoparticles. In addition, there was no significant difference between the predifferentiation condition prior to incubation in chondrogenic medium and the proliferation condition, which suggests that in situ chondrogenic differentiation of hASCs was induced by the TGF-β1 loaded nanoparticles. Consequently, the hybridization of fibrin and PLCL scaffolds for three-dimensional spatial organization of cells and the effective delivery of TGF-β1 using heparin-functionalized nanoparticles can induce hASCs to differentiate to a chondrogenic lineage and maintain their phenotypes.
Keywords: Human adipose tissue-derived stem cells; Chondrogenic differentiation; TGF-β1; Poly(l-lactide-co-ɛ-caprolactone) scaffold;

The use of three-dimensional nanostructures to instruct cells to produce extracellular matrix for regenerative medicine strategies by Katja Schenke-Layland; Fady Rofail; Sanaz Heydarkhan; Jessica M. Gluck; Nilesh P. Ingle; Ekaterini Angelis; Chang-Hwan Choi; William R. MacLellan; Ramin E. Beygui; Richard J. Shemin; Sepideh Heydarkhan-Hagvall (4665-4675).
Synthetic polymers or naturally-derived extracellular matrix (ECM) proteins have been used to create tissue engineering scaffolds; however, the need for surface modification in order to achieve polymer biocompatibility and the lack of biomechanical strength of constructs built using proteins alone remain major limitations. To overcome these obstacles, we developed novel hybrid constructs composed of both strong biosynthetic materials and natural human ECM proteins. Taking advantage of the ability of cells to produce their own ECM, human foreskin fibroblasts were grown on silicon-based nanostructures exhibiting various surface topographies that significantly enhanced ECM protein production. After 4 weeks, cell-derived sheets were harvested and histology, immunochemistry, biochemistry and multiphoton imaging revealed the presence of collagens, tropoelastin, fibronectin and glycosaminoglycans. Following decellularization, purified sheet-derived ECM proteins were mixed with poly(ɛ-caprolactone) to create fibrous scaffolds using electrospinning. These hybrid scaffolds exhibited excellent biomechanical properties with fiber and pore sizes that allowed attachment and migration of adipose tissue-derived stem cells. Our study represents an innovative approach to generate strong, non-cytotoxic scaffolds that could have broad applications in tissue regeneration strategies.
Keywords: ECM; Nanotopography; Biomimetic material; Tissue engineering; Electrospinning; Multiphoton imaging;

Enhanced bone regeneration via multimodal actions of synthetic peptide SVVYGLR on osteoprogenitors and osteoclasts by Hiroshi Egusa; Yoshitoshi Kaneda; Yoshihiro Akashi; Yoshinosuke Hamada; Takuya Matsumoto; Makio Saeki; Devang K. Thakor; Yasuhiko Tabata; Nariaki Matsuura; Hirofumi Yatani (4676-4686).
Recently, the binding sequence Ser-Val-Val-Tyr-Gly-Leu-Arg (SVVYGLR) was found adjacent to the RGD sequence in osteopontin, suggesting involvement in osteo-immune cross-talk. The aim of this study was to investigate bioactive functions of a synthetic SVVYGLR peptide in osteoprogenitor cells and osteoclasts, and to examine potential applications in bone regeneration. The SVVYGLR peptide significantly enhanced the adhesion and proliferation of several human mesenchymal cells including bone marrow-derived mesenchymal stem cells, and αvβ3 integrin was involved in cell attachment to the peptide. Additionally, the peptide reduced the number of TRAP-positive multinucleated cells during osteoclastogenesis of RAW264.7 cells and normal murine pre-osteoclasts, and also suppressed NFAT activity and expression of osteoclastogenesis-related mRNAs. When standardized bone defects in rat calvariae were filled with a collagen sponge containing the peptide or PBS (control), the number of TRAP-positive osteoclasts in the grafted sites after 3 weeks was significantly lower in the peptide group. By the 5th week, significantly enhanced resorption of the grafted collagen sponge and new bone formation was observed within and surrounding the sponge in the peptide group. These data suggest that SVVYGLR is an effective bioactive peptide for bone tissue regeneration that promotes attachment and proliferation of osteogenic cells while also suppressing osteoclastogenesis.
Keywords: Bone regeneration; Mesenchymal stem cells; Osteoclasts; Peptide; SVVYGLR;

Engineering liver tissue spheroids with inverted colloidal crystal scaffolds by Jungwoo Lee; Meghan J. Cuddihy; George M. Cater; Nicholas A. Kotov (4687-4694).
Multicellular spheroids provide a new three-dimensional (3D) level of control over morphology and function of ex vivo cultured tissues. They also represent a valuable experimental technique for drug discovery and cell biology. Nevertheless, the dependence of many cellular processes on the cluster diameter remains unclear. To provide a tool for the systematic evaluation of such dependences, we introduce here inverted colloidal crystal (ICC) scaffolds. Uniformly sized pores in ICC cell matrixes afford a high yield production of controlled size spheroids in standard 96 well-plates. Transparent hydrogel matrix and ship-in-bottle effect also allows for convenient monitoring of cell processes by traditional optical techniques. Different developmental stages of 46.5–151.6 μm spheroids from HepG2 hepatocytes with vivid morphological similarities to liver tissue (bile canaliculi) were observed. The liver-specific functions of HepG2 cells were systematically investigated and compared for spheroids of different diameters as well as 2D cultures. Clear trends of albumin production and CYP450 activity were observed; diffusion processes and effect of cellular aggregation on metabolic activity were identified to be the primary contributors to the size dependence of the liver functions in HepG2 spheroids in ICC scaffolds. Since the aggregation of cells into clusters is a universal biological process, these findings and scaffolds can be applied to many other relevant cell types.
Keywords: 3D cell culture; Scaffolds; Hepatocytes; Spheroids; Hydrogel;

The influence of hydrogel modulus on the proliferation and differentiation of encapsulated neural stem cells by Akhilesh Banerjee; Manish Arha; Soumitra Choudhary; Randolph S. Ashton; Surita R. Bhatia; David V. Schaffer; Ravi S. Kane (4695-4699).
There has been an increasing interest in understanding how the mechanical properties of the microenvironment influence stem cell fate. We describe studies of the proliferation and differentiation of neural stem cells (NSCs) encapsulated within three-dimensional scaffolds – alginate hydrogels – whose elastic moduli were varied over two orders of magnitude. The rate of proliferation of neural stem cells decreased with increase in the modulus of the hydrogels. Moreover, we observed the greatest enhancement in expression of the neuronal marker β-tubulin III within the softest hydrogels, which had an elastic modulus comparable to that of brain tissues. To our knowledge, this work represents the first demonstration of the influence of modulus on NSC differentiation in three-dimensional scaffolds. Three-dimensional scaffolds that control stem cell fate would be broadly useful for applications in regenerative medicine and tissue engineering.
Keywords: Alginate; Hydrogel; Modulus; Stem cells; Proliferation; Differentiation;

Many different types of magnetic particles have been developed for the purpose of hyperthermia cancer therapy. In this study, a magnetic nanoparticle based on dicalcium phosphate dihydrate (DCPD) was formed by co-precipitation method. Addition of different concentrations of ferrous chloride to DCPD can alter its material properties. Various physical, chemical and magnetic tests of the magnetic DCPD nanoparticles (mDCPD) were performed, including X-ray diffraction (XRD), inductively coupled plasma-optical emission spectrometer (ICP-OES), superconducting quantum interference device (SQUID), and transmission electron microscopy (TEM). The heating efficiency of mDCPD in alternating magnetic field was proved to be suitable for hyperthermia. The results of cytotoxicity tests (WST-1 and LDH assay) showed no harmful effect. The mDCPD showed relative cancer-killing ability without damaging normal cells in vitro.
Keywords: Brushite; Calcium phosphate; Nanoparticle; Nanocomposite; Magnetism; In vitro test;

The incorporation of daunorubicin in cancer cells through the use of titanium dioxide whiskers by Qingning Li; Xuemei Wang; Xiaohua Lu; Honger Tian; Hui Jiang; Gang Lv; Dadong Guo; Chunhui Wu; Baoan Chen (4708-4715).
Porous nanostructure with its unique properties is found to be able to hold promise in drug delivery. We fabricated the one-dimensional titanium dioxide whiskers (TiO2 Ws) and designed a strategy to explore their drug delivery application and anti-tumor function combined with daunorubicin (DNR). We observed good biocompatibility of TiO2 Ws and noted better photocatalytic activity. In human hepatocarcinoma cells (SMMC-7721 cells), TiO2 Ws can obviously increase the intracellular concentration of DNR and enhance its potential anti-tumor efficiency, indicating TiO2 Ws could produce an efficient drug delivery carrier effect importing DNR into target cells. Furthermore, its photocatalysis further led to the enhanced mortality of cancer cells under UV irradiation. These findings reveal that TiO2 Ws-based delivery of anticancer drugs represents a promising approach in cancer therapy.
Keywords: Titanium dioxide whiskers; Titanium dioxide nanoparticles; Ultra violet irradiation; Photocatalysis; Cancer therapy; Drug delivery;

Aside from their superparamagnetic properties exploited in clinical magnetic resonance imaging (MRI), it was recently discovered that magnetic, iron oxide nanoparticles could function as an artificial, inorganic peroxidase. In this paper, we studied the impact of coating on the peroxidase activity of these nanoparticles. Nanoparticles with six different coating structures were synthesized and characterized by FTIR, TGA, TEM, size, zeta potential, and SQUID; and evaluated for peroxidase activity. Catalysis was found to follow Michaelis–Menten kinetics and peroxidase activity varied with respect to electrostatic affinity between nanoparticles and substrates, evidenced by differences in determined kinetic parameters. Glucose detection was selected as a model system because glucose could be indirectly measured from the release of hydrogen peroxide after its oxidation. Nanoparticles with high peroxidase activity exhibited higher sensitivity toward glucose, showing a larger linear slope when compared with those of low activity. A significantly improved linear correlation and detection limit of measured glucose could be readily obtained by manipulating the nanoparticle coating. Our findings suggest that iron oxide nanoparticles can be tailor-made to possess improved peroxidase-like activity. Such enhancements could further widen nanoparticle scope in glucose detection and extend its peroxidase functionality to other biomedical applications.
Keywords: Superparamagnetic nanoparticle; Iron oxide; Peroxidase; Glucose detection;

Whole proteome analysis of osteoprogenitor differentiation induced by disordered nanotopography and mediated by ERK signalling by Fahsai Kantawong; Karl E.V. Burgess; Kamburapola Jayawardena; Andrew Hart; Richard J. Burchmore; Nikolaj Gadegaard; Richard O.C. Oreffo; Matthew J. Dalby (4723-4731).
Topographic features can modulate cell behaviours such as proliferation, migration, differentiation and apoptosis. Biochemical mechanotransduction implies the conversion of mechanical forces (e.g. changes in cell spreading and morphology from changing surface topography) into biochemical signal via biomolecules. Still, little is known concerning which pathways may be directly involved in cell response to changes in the material surface. A number of pathways have been implicated using focused studies of ‘selected’ biomolecules rather than a global analysis of signal pathways. This study used a controlled disorder nanopit topography (NSQ50, fabricated by electron beam lithography) to direct osteoblast differentiation of progenitor cells. This topography is unique as it represents a middle route (from absolute order or random roughness) that allows osteoconversion with similar efficiency as dexamethasone and ascorbate treatment. Two direct-comparison proteomics techniques, firstly gel-based and then chromatography-based, were used to analyse progenitor proteome changes in response to the nanotopography. Many of the changed proteins form part of the Extracellular Signal-regulated Kinase (ERK1/2) pathway.
Keywords: Tissue engineering; Disordered topography; Extracellular-activated protein kinase; Osteoprogenitor cells; Proteomics;

Nanoscale presentation of cell adhesive molecules via block copolymer self-assembly by Peter A. George; Michael R. Doran; Tristan I. Croll; Trent P. Munro; Justin J. Cooper-White (4732-4737).
Precise control over the nanoscale presentation of adhesion molecules and other biological factors represents a new frontier for biomaterials science. Recently, the control of integrin spacing and cellular shape has been shown to affect fundamental biological processes, such as differentiation and apoptosis. Here, we present the self-assembly of maleimide functionalised polystyrene-block-poly (ethylene oxide) copolymers as a simple, yet highly precise method for controlling the position of cellular adhesion molecules. By manipulating the phase separation of the functional PS-PEO block copolymer used in this study, via a simple blending technique, we alter the nanoscale (on PEO domains of 8–14 nm in size) presentation of the adhesion peptide, GRGDS, decreasing lateral spacing from 62 nm to 44 nm and increasing the number density from ∼450 to ∼900 islands per μm2. The results indicate that the spreading of NIH-3T3 fibroblasts increases as the spacing between domains of RGD binding peptides decreases. Further, the same functional PS-PEO surfaces have been utilised to immobilise, via a zinc chelating peptide sequence, poly-histidine tagged proteins and extracellular matrix (ECM) fragments. This method is seen as an ideal platform for investigations into the role of spatial arrangements of cell adhesion molecules and ECM molecules on cell function and, in particular, control of cell phenotype.
Keywords: Nanotechnology; Integrin; Surface modification; Cell spreading; RGD;

A surface-eroding antibiotic delivery system based on poly-(trimethylene carbonate) by Otto S. Kluin; Henny C. van der Mei; Henk J. Busscher; Daniëlle Neut (4738-4742).
Biodegradable delivery systems that do not produce acidic compounds during degradation are preferred for local antibiotic delivery in bone infections in order to avoid adverse bone reactions. Poly(trimethylene carbonate) (PTMC) has good biocompatibility, and is such a polymer. The objective of this in vitro study was to explore the suitability of PTMC as an antibiotic releasing polymer for the local treatment of bone infections. Degradation behaviour and corresponding release profiles of gentamicin and vancomycin from slowly degrading PTMC168 and faster degrading PTMC339 discs were compared in the absence and presence of a lipase solution. Gentamicin release in the absence of lipase was diffusion-controlled, while vancomycin release was limited. Surface erosion of PTMC only occurred in the presence of lipase. Both antibiotics were released in high concentrations from PTMC in the presence of lipase through a combination of surface erosion and diffusion. This illustrates the major advantage of surface-eroding biodegradable polymers, allowing release of larger antibiotic molecules like vancomycin.
Keywords: Poly(trimethylene carbonate); In vitro degradation; Enzymatic surface erosion; Antibiotic release;

The inhibition of glioma growth in vitro and in vivo by a chitosan/ellagic acid composite biomaterial by Sungwoo Kim; Mostafa W. Gaber; Janice A. Zawaski; Feng Zhang; Mekel Richardson; Xin A. Zhang; Yunzhi Yang (4743-4751).
This study has developed a chitosan-based delivery system to locally administer ellagic acid for brain cancer treatment. We fabricated chitosan/ellagic acid composite films with various concentrations of ellagic acid. In vitro release study was performed by using a UV spectrophotometer, and enzymatic degradation rate was determined by analyzing the increased free amino groups. Viability of brain cancer cells (human U87 glioblastomas and rat C6 glioma cells) was measured via direct and indirect cell culture on the films by MTS assay. Caspase-3 activation, Western blot for p53, and anti-angiogenesis assays were also examined. In the in vivo study, GFP-tagged rat C6 glioma cells were implanted subcutaneously at the right flank region of nude mice and treatments were initiated by implanting the films subcutaneously. Tumor growth was evaluated by measuring tumor volume using a caliper, an ultrasound machine, and an optical imaging system. The chitosan/ellagic acid composite films were enzymatically degradable and exhibited a sustained slow release of ellagic acid. These materials could inhibit the cancer cell growth in an ellagic acid concentration-dependent manner by inducing apoptosis of cancer cells as well as suppressing angiogenesis. These materials also significantly suppressed tumor tissue growth in vivo.
Keywords: Brain cancer; U87 glioblastoma; C6 glioma; Ellagic acid; Chitosan; Apoptosis;

Doxorubicin–polyphosphazene conjugate hydrogels for locally controlled delivery of cancer therapeutics by ChangJu Chun; Sun M. Lee; Chang W. Kim; Ki-Yun Hong; Sang Y. Kim; Han K. Yang; Soo-Chang Song (4752-4762).
Poly(organophosphazene)–doxorubicin (DOX) conjugate bearing hydrophobic l-isoleucine ethyl ester (IleOEt) and hydrophilic α-amino-ω-methoxy-poly(ethylene glycol) with molecular weight of 550 Da (AMPEG 550) along with carboxylic acid as a functional group was synthesized to create a drug delivery system, which is based on locally injectable, biodegradable, and thermosensitive hydrogels. In addition to the evaluation of the in vitro and in vivo antitumor activities, the physicochemical properties, hydrolytic degradation, and DOX release profile of the poly(organophosphazene)–DOX conjugate were determined. The aqueous solution of the polymer–DOX conjugate showed a sol–gel transition behavior depending on temperature changes. Based on the in vivo antitumor activities of the locally injected poly(organophosphazene)–DOX conjugate into the tumor-induced nude mice, the conjugate hydrogel after the local injection at the tumor site was shown to inhibit tumor growth more effectively with less toxicity and much longer than doxorubicin and saline as controls, indicating that tumor active DOX from the conjugate hydrogel is released slowly over a longer period of time and effectively accumulated locally in the tumor sites. These results suggest that the poly(organophosphazene)–doxorubicin conjugates hold great potential for use in preclinical and clinical studies as single and/or combination therapies.
Keywords: Localization; Biodegradable; Thermosensitive; Polymer–doxorubicin conjugate; Hydrogel; Antitumor efficacy;

Memory antibody response is the hallmark of long lasting immunity. In this study, we report the generation of memory antibody response while immunizing with single dose of polymer particle entrapped antigens. Immunization with admixture of alum and polylactide (PLA) polymer particles (2–8 μm) entrapping antigens not only elicited long lasting primary antibody response but also very high levels of memory antibody titer upon re-exposure to very small amount of soluble antigen. In the case of tetanus toxoid (TT), the memory antibody titers from PLA particle based immunization were almost four times higher than that achieved from two doses of alum adsorbed antigen and sustained at a higher level for a longer period of time. Memory antibody response was detected even after challenging the animals after 18 months of primary immunization. Similar enhanced memory antibody response was also observed in the case of immunization with PLA particle entrapping diphtheria toxoid (DT). Memory antibody response generated from polymeric formulations was highly antigen specific. Polymer particles with different release profile of antigen were used as a model system to evaluate the role of antigen on immunological memory. The results suggest that slow and continuous release of antigen from polymer particles plays a critical role in eliciting improved memory antibody response from single point immunization.
Keywords: Vaccine delivery; Polylactide; Microparticles; Adjuvants; Memory antibody response Release profile of antigen;

Synergistic anti-tumor activity of paclitaxel-incorporated conjugated linoleic acid-coupled poloxamer thermosensitive hydrogel in vitro and in vivo by Ding-Ding Guo; Cheng-Xiong Xu; Ji-Shan Quan; Chung-Kil Song; Hua Jin; Dae-Duk Kim; Yun-Jaie Choi; Myung-Haing Cho; Chong-Su Cho (4777-4785).
Local delivery of anti-tumor drugs provides a high local concentration and decreases the incidence of side effects commonly observed with systemic therapy. Hydrogel systems are commonly used as a local drug delivery system. In this study, we prepared a novel thermosensitive conjugated linoleic acid (CLA)-coupled poloxamer hydrogel for local delivery of paclitaxel (PTX) to gain the benefits of the pro-drug activity of the CLA-coupled poloxamer and enhanced PTX solubility due to the micellar property of the CLA-coupled poloxamer. To evaluate the anti-tumor activity of the PTX-incorporated CLA-coupled poloxamer hydrogel in vivo, formulations were injected subcutaneously into tumor-bearing mice. Cell cycle and apoptosis markers were examined to determine the mechanism of the anti-tumor activity of PTX. The PTX-incorporated CLA-coupled poloxamer thermosensitive hydrogel showed excellent anti-tumor activity in vivo inducing stronger cell cycle arrest and apoptosis in tumor tissue than the PTX-incorporated poloxamer hydrogel. These results were attributed to the synergistic effect of the anti-tumor property of PTX with released CLA from the CLA-coupled poloxamer as the pro-drug and the enhanced solubility of PTX resulting from the micellar property of the CLA-coupled poloxamer. The CLA-coupled poloxamer designed in this study has great potential as an effective injectable carrier of PTX.
Keywords: Hydrogel; Poloxamer; Conjugated linoleic acid; Paclitaxel; Thermosensitive;

A magnetic, luminescent and mesoporous core–shell structured composite material as drug carrier by Piaoping Yang; Zewei Quan; Zhiyao Hou; Chunxia Li; Xiaojiao Kang; Ziyong Cheng; Jun Lin (4786-4795).
In this paper, hydrothermal synthesized Fe3O4 microspheres have been encapsulated with nonporous silica and a further layer of ordered mesoporous silica through a simple sol–gel process. The surface of the outer silica shell was further functionalized by the deposition of YVO4:Eu3+ phosphors, realizing a sandwich structured material with mesoporous, magnetic and luminescent properties. The multifunctional system was used as drug carrier to investigate the storage and release properties using ibuprofen (IBU) as model drug by the surface modification. X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectra (XPS), Fourier transform infrared spectroscopy (FT-IR), N2 adsorption/desorption, photoluminescence (PL) spectra, and superconducting quantum interference device (SQUID) were used to characterized the samples. The results reveal that the material shows typical ordered mesoporous characteristics, and have monodisperse spherical morphology with smooth surface and narrow size distribution. Additionally, the multifunctional system shows the characteristic emission of Eu3+ (5D07F1–4) even after the loading of drug molecules. Magnetism measurement reveals the superparamagnetic feature of the samples. Drug release test indicates that the multifunctional system shows drug sustained properties. Moreover, the emission intensities of Eu3+ in the drug carrier system increase with the released amount of drug, thus making the drug release be easily tracked and monitored by the change of the luminescence intensity.
Keywords: Drug delivery; Magnetism; Luminescence; Core–shell; Mesoporous;

In this work, a cocktail of dexamethasone (Dex) and transforming growth factor-β3 (TGF-β3) bound with heparin in/on PLGA microspheres was investigated by local dual delivery using rabbit mesenchymal stem cells (rMSCs) for chondrogenic differentiation in in vitro and in vivo study. In this study, we proved that the microsphere constructs were capable of simultaneously releasing TGF-β3-conjugated with cy5.5 and Dex-conjugated with FITC with approximately zero order kinetics determined by Kodak imaging and confocal laser microscope. Microsphere constructs containing TGF-β3 and Dex showed a significantly higher number of specific lacunae phenotypes at the end of the 4 week study irrespective of the presence of dexamethasone and TGF-β3. Thus, dual delivery of TGF and Dex can be used to engineer inflammation-free and cartilage-associate tissue in the transplanted PLGA construct into nude mouse. These heparin-bound TGF-β3-coated and Dex-loaded PLGA microsphere constructs show promise as coatings for implantable biomedical devices to improve biocompatibility and ensure both in vitro and in vivo performance.
Keywords: Cy5.5; FITC; Microsphere; Mesenchymal stem cells; Dual delivery;

Envelope-type lipid nanoparticles incorporating a short PEG-lipid conjugate for improved control of intracellular trafficking and transgene transcription by Tomoya Masuda; Hidetaka Akita; Kenichi Niikura; Takashi Nishio; Masami Ukawa; Kaoru Enoto; Radostin Danev; Kuniaki Nagayama; Kuniharu Ijiro; Hideyoshi Harashima (4806-4814).
Lipid envelope-type nanoparticles are promising carriers for gene delivery. The modification of liposomes with polyethyleneglycol (PEG) can often be useful in liposomal formation and pharmacokinetics. However, there is a dilemma concerning the use of PEG because of its poor intracellular trafficking properties. To overcome this problem, in the present study, we report on a strategy for improving the intracellular trafficking of PEG-modified lipid particles by incorporating a short PEG lipid. The findings presented here show that the incorporation of tetra(ethylene)glycol (TEG)-conjugated cholesterol into a liposome composition is useful in controlling the number of lipid envelopes, resulting in an improvement in particle uniformity with a reduced particle size. The TEG-modified lipid particles were found to enhance transfection activity by more than 100-fold. This increase is attributed to an enhancement of cellular uptake, and nuclear transcription by improving intracellular decoating. Moreover, the use of a various short PEG lipids in lipid particle formation showed a clear threshold polymerization degree (less or equal 25: PEG1100), for achieving stimulated transfection activity. Collectively, the use of short PEG lipid promises to be useful in developing an efficient non-viral gene vector.
Keywords: Non-viral vector; Gene delivery; Short PEG; Intracellular trafficking; Envelope-type lipid nanoparticle;

A chitosan hydrogel delivery system for osteosarcoma gene therapy with pigment epithelium-derived factor combined with chemotherapy by Hang T. Ta; Crispin R. Dass; Ian Larson; Peter F.M. Choong; Dave E. Dunstan (4815-4823).
Osteosarcoma (OS) is the most common type of malignant bone cancer, and the sixth most common type of cancer in children and young adults. Currently, gene therapy is being evaluated as a novel method for OS treatment. Here we report on an in situ gelling chitosan-based hydrogel system that sustains the release of a potential anti-cancer gene (pigment epithelium-derived factor) to the tumor site. A significant reduction of the primary OS in a clinically relevant orthotopic model was measured. The combination of plasmid treatment and chemotherapy together with the use of this delivery system led to the highest suppression of tumor growth without side effects. The results obtained from this study demonstrate the potential application of a biodegradable hydrogel technology as an anti-cancer drug delivery system for successful chemo-gene therapy.
Keywords: Chitosan; Orthophosphate; Drug delivery; Hydrogel; Osteosarcoma; Cancer;

Polyethylenimine-grafted polycarbonates as biodegradable polycations for gene delivery by Chang-Fang Wang; Yan-Xin Lin; Tao Jiang; Feng He; Ren-Xi Zhuo (4824-4832).
Polycations as one of non-viral vectors have gained increasing attentions. In this paper, polyethylenimine(PEI)-grafted polycarbonates (PMAC-g-PEIx) were synthesized as a kind of biodegradable polycations for gene delivery. Backbone polymer, poly(5-methyl-5-allyloxycarbonyl-trimethylene carbonate) (PMAC), was synthesized in bulk catalyzed by immobilized porcine pancreas lipase (IPPL). Then, PMAC–O, the allyl epoxidation product of PMAC, was further modified by PEIx with low molecular weight (x = 423, 800 and 1800). The MWs of PMAC-g-PEIx, measured by GPC–MALLS, were 81,900, 17,9900 and 200,600 g/mol with polydispersities of 1.2, 1.4 and 1.7, respectively. PMAC-g-PEIx could form positively charged nano-sized particles (30–90 nm) with pDNA, and all the three PAMC-g-PEIx/DNA polyplexes had similar buffer capabilities. In vitro experiments demonstrated that the PAMC-g-PEIx showed much low cytotoxicity and enhanced transfection efficiency could be found in comparison with PEI25K in 293T cells. Furthermore, pre-incubation of PMAC-g-PEI1800 showed a weakening binding capacity with DNA. The biodegradability of PMAC-g-PEIx can facilitate the efficient release of pDNA from polyplexes and reduce cell cytotoxicity. These results suggested that PMAC-g-PEIx would be a promising non-viral biodegradable vector for gene delivery system.
Keywords: Gene delivery; PEI-grafted; Biodegradable polycarbonates; Enzymatic ring-opening polymerization; Cytotoxicity; Transfection;

Control of 3-dimensional collagen matrix polymerization for reproducible human mammary fibroblast cell culture in microfluidic devices by Kyung Eun Sung; Gui Su; Carolyn Pehlke; Steven M. Trier; Kevin W. Eliceiri; Patricia J. Keely; Andreas Friedl; David J. Beebe (4833-4841).
Interest in constructing a reliable 3-dimensional (3D) collagen culture platform in microfabricated systems is increasing as researchers strive to investigate reciprocal interaction between extracellular matrix (ECM) and cells under various conditions. However, in comparison to conventional 2-dimensional (2D) cell culture research, relatively little work has been reported about the polymerization of collagen type I matrix in microsystems. We, thus, present a study of 3D collagen polymerization to achieve reproducible 3D cell culture in microfluidic devices. Array-based microchannels are employed to efficiently examine various polymerization conditions, providing more replicates with less sample volume than conventional means. Collagen fibers assembled in microchannels were almost two-times thinner than those in conventional gels prepared under similar conditions, and the fiber thickness difference influenced viability and morphology of embedded human mammary fibroblast (HMF) cells. HMF cells contained more actin stress fibers and showed increased viability in 3D collagen matrix composed of thicker collagen fibers. Relatively low pH of the collagen solution within a physiological pH range (6.5–8.5) and pre-incubation at low temperature (∼4 °C) before polymerization at 37 °C allow sufficient time for molecular assembly, generating thicker collagen fibers and enhancing HMF cell viability. The results provide the basis for improved process control and reproducibility of 3D collagen matrix culture in microchannels, allowing predictable modifications to provide optimum conditions for specific cell types. In addition, the presented method lays the foundation for high throughput 3D cellular screening.
Keywords: Collagen polymerization; Microchannel; 3D cell culture; Array-based microsystem;

Dimethyl sulfoxide (DMSO) has been used for several decades as the most efficient cryoprotective agent (CPA) for many types of cells and tissues in spite of its cytotoxicity and its effects on differentiation. Here we report that polyampholytes with an appropriate ratio of amino and carboxyl groups show higher cryopreservation efficiency and lower cytotoxicity than DMSO. Culture medium solutions of ɛ-poly-l-lysine (PLL) with more than 50 mol% of amino groups carboxylated showed excellent post-thaw survival efficiency of 95% murine L929 cells, and rat mesenchymal stem cells fully retained the potential for differentiation without serum. We also found that carboxylated PLLs showed antifreeze protein properties, such as ice recrystallization inhibition, which may contribute to successful cryopreservation by membrane protection. Thus, these polyampholytes can replace DMSO as new materials for CPAs in various preserving functions and will also be useful in studies elucidating the mechanisms of cryopreservation.
Keywords: Polyampholyte; Polyamino acid; Cryopreservation; Antifreeze protein; Mesenchymal stem cell; Cell viability;