Biomaterials (v.31, #8)

Genome-wide pathways analysis of nickel ion-induced differential genes expression in fibroblasts by Xiaoying Lü; Huiqin Lu; Lifeng Zhao; Yamin Yang; Zuhong Lu (1965-1973).
To reveal molecular mechanisms of the interaction between Ni2+ and cells, cDNA microarray technology and GenMAPP analysis were utilized to investigate changes of gene expression profile and identify significant biological pathways in mouse fibroblast cells (L-929) treated by 100 μm Ni2+ for 12, 24, 48 and 72 h, respectively. The microarray data was validated by real-time PCR. Methylthiazoltetrazolium (MTT) analysis and flow cytometry experiment were used to assess the cellular response of L-929 cells to Ni2+. It was found that six main biological pathways were affected by Ni2+ with 118 differentially expressed genes involved. Further analysis illuminated that the exposure of cells to Ni2+ may evoke series of cellular responses to hypoxia by regulating hypoxia-inducible gene expression and cause irreversible DNA damage. Cell cycle pathway analysis results showed DNA replication in S phase could be inhibited by Ni2+ which was consistent with the data gained from flow cytometry experiment. Compared to previous researches based on conventional molecular biology experiments, the present work has not only indirectly validated the findings of other groups but also obtained several discoveries related to cell-Ni2+ interaction, such as inhibition of electron transport chain and accumulation of extracellular matrix (ECM) collagens. The routine of the present study not only can analyze gene expression profile but also may provide a more convenient and efficient approach to explain molecular mechanisms of cell–biomaterial interaction.
Keywords: Gene expression microarray; Real-time PCR; Pathway analysis; Molecular mechanism; Nickel ion;

Physical surface and electromechanical properties of doped polypyrrole biomaterials by Amy Gelmi; Michael J. Higgins; Gordon G. Wallace (1974-1983).
Conducting polymers doped with biomolecules (biodopants) are becoming more widely evaluated for use as biomaterials. The use of biodopants is intended to enhance the compatibility of the polymers, however their effect on the physical properties of the final composite material has generally been less of a consideration. Here, we have characterised the physical surface properties of polypyrrole substrates doped with extracellular matrix and non-biological molecules using Atomic Force Microscopy (AFM) and Electrochemical AFM (EC-AFM) techniques. The physical parameters of the differently doped films ranged between 5 and 32 nm for the RMS roughness, 30–1000 MPa for the Young's modulus, and 1.6–4.7% for the actuation strain. It was found that irrespective of whether the dopant was biologically derived, the physical properties tended to group together with films having either a low roughness, low modulus and high strain, or vice versa. When compared to our previous study, which investigated these polymers as potential biomaterials for supporting the growth and differentiation of skeletal muscle cells, these two groupings of the parameters correlated with the differing ability of the polypyrrole substrates to support the cells. Thus, in addition to the chemical advantage gained from using biodopants, the resulting physical properties of the polymer material should also be considered in their design as biomaterials for tissue engineering applications.
Keywords: Conducting polymer; Atomic force microscopy; Electrochemical AFM; Polypyrrole; Extracellular matrix; Hyaluronic acid;

The potential for bio-optical imaging of biomaterial-associated infection in vivo by Jelmer Sjollema; Prashant K. Sharma; Rene J.B. Dijkstra; Gooitzen M. van Dam; Henny C. van der Mei; Anton F. Engelsman; Henk J. Busscher (1984-1995).
This review presents the current state of Bioluminescence and Fluorescent Imaging technologies (BLI and FLI) as applied to Biomaterial-Associated Infections (BAI). BLI offers the opportunity to observe the in vivo course of BAI in small animals without the need to sacrifice animals at different time points after the onset of infection. BLI is highly dependent on the bacterial cell metabolism which makes BLI a strong reporter of viable bacterial presence. Fluorescent sources are generally more stable than bioluminescent ones and specifically targeted, which renders the combination of BLI and FLI a promising tool for imaging BAI. The sensitivity and spatial resolution of both imaging tools are, however, dependent on the imaging system used and the tissue characteristics, which makes the interpretation of images, in terms of the location and shape of the illuminating source, difficult. Tomographic reconstruction of the luminescent source is possible in the most modern instruments, enabling exact localization of a colonized implant material, spreading of infecting organisms in surrounding tissue and immunological tissue reactions. BLI studies on BAI have successfully distinguished between different biomaterials with respect to the development and clearance of BAI in vivo, simultaneously reducing animal use and experimental variation. It is anticipated that bio-optical imaging will become an indispensable technology for the in vivo evaluation of antimicrobial coatings.
Keywords: Biofilm; Infection; Cell viability; Fluorescence; Bioluminescence;

Protection against titanium particle induced osteolysis by cannabinoid receptor 2 selective antagonist by Dechun Geng; Yaozeng Xu; Huilin Yang; Junhua Wang; Xuesong Zhu; Guangming Zhu; Xianbin Wang (1996-2000).
Osteolysis and subsequent aseptic loosening are the most common causes of failure of total joint arthroplasty. Osteolysis is initiated by inflammatory response to wear debris, resulting in localized, osteoclastic peri-implant bone loss. However, there were no effective measures for prevention and treatment of periprosthetic osteolysis. The aim of the current study was to determine whether CB2 selective antagonist (AM630) inhibits wear debris-induced osteolysis in a murine osteolysis model. Titanium (Ti) particles were introduced into established air pouches on BALB/c mice, followed by implantation of calvaria bone from syngeneic littermates. AM630 was given to mice intraperitoneally 2 days before Ti particles introduction and maintained until the sacrifice of the mice. Mice without drug treatment, as well as mice injected with saline alone, were included. Each group contains 10 mice. Pouch tissues were harvested 14 days after bone implantation for histological and molecular analysis. Ti particles stimulation significantly increased CB2 expression. However, less CB2 was observed in AM630 treatment group. AM630 inhibited Ti particle-induced osteolysis associated gene activity of RANK, RANKL and CPK, and diminished RANKL expression in Ti particle stimulated pouches. AM630 markedly reduced the number of TRAP+ cells in pouch tissues. In conclusion, this study provides the evidence that blockage of CB2 with AM630 can markedly reduce Ti particle induced osteolysis in a murine air pouch model. This finding points to the possibility that CB2 selective antagonists like AM630 may have potential value for prevention and treatment of wear particle induced osteolysis.
Keywords: Cannabinoid receptor 2; AM630; Air pouch; Osteolysis; Wear particle;

The effect of zinc on hydroxyapatite-mediated activation of human polymorphonuclear neutrophils and bone implant-associated acute inflammation by Frédéric Velard; Dominique Laurent-Maquin; Julien Braux; Christine Guillaume; Sylvie Bouthors; Edouard Jallot; Jean-Marie Nedelec; Abderrazzaq Belaaouaj; Patrice Laquerriere (2001-2009).
Hydroxyapatite (HA) is widely used as coating biomaterial for prosthesis metal parts and as bone substitute. The release of HA particles induces an inflammatory response and, if uncontrolled, could result in implant loss. At the inflamed site, the polymorphonuclear cells (PMNs) represent the earliest phagocytic cells that predominate the cellular infiltrate. We have recently proposed that HA wear debris activate polymorphonuclear cells (PMNs) initiating and/or amplifying thereby the acute inflammatory response. Previous studies have shown that activation of monocytes by HA could be modulated by supplementing this latter with the divalent cation, Zinc. The purpose of this work was to investigate the modulation of PMNs activation following exposure to zinc-substituted HA. Our study demonstrate that addition of zinc to HA particles resulted in decreased levels of the pro-inflammatory mediator interleukin-8 (IL-8) and the matrix metallo-proteinase-9. We also show that these changes involve IL-8 receptors (CXCR-1 and CXCR-2).
Keywords: Inflammation; Neutrophil; Interleukin-8; Hydroxyapatite; Zinc;

Induction of DNA double-strand breaks in primary gingival fibroblasts by exposure to dental resin composites by Ebru Urcan; Harry Scherthan; Marianthi Styllou; Uschi Haertel; Reinhard Hickel; Franz-Xaver Reichl (2010-2014).
Dental resin composites and their reactive monomers/co-monomers have been shown to elicit cytotoxic responses in human gingival fibroblasts (HGF), and their metabolic radical intermediates have the potential to attack the DNA backbone, which may induce DNA double-strand breaks (DSBs). In this study we have tested the cytotoxicity and induction of DSBs by the most common composite resin monomers/co-monomers: BisGMA, HEMA, TEGDMA, and UDMA in gingival fibroblasts using the sensitive γ-H2AX DNA repair focus assay. Our results show increasing monomer cytotoxicities in the order of BisGMA > UDMA > TEGDMA > HEMA, an order that was also observed for their capacity to induce DSBs. BisGMA at the EC50 concentration of 0.09 mm evoked the highest rate of γ-H2AX foci-formation that was 11-fold higher DNA DSBs as compared to the negative controls that ranged between 0.25 and 0.5 γ-H2AX foci/HGF cell. Our results for the first time show that exposure to dental resin monomers can induce DSBs in primary human oral cavity cells, which underscores their genotoxic capacity.
Keywords: Dental resin composites; HGFs; DNA damage; DNA double-strand break; γ-H2AX;

Osteoblast differentiation on tissue culture polystyrene (TCPS) requires Wnt/beta-catenin signaling, regulating modulators of the Wnt pathway like Dickkopf-1 (Dkk1) and Dkk2. Osteoblast differentiation is increased on microstructured titanium (Ti) surfaces compared to TCPS; therefore, we hypothesized that surface topography and hydrophilicity affect Dkk1 and Dkk2 expression and that their roles in osteoblast differentiation on Ti differs depending on cell maturation state. Human osteoblast-like MG63 cells, normal human osteoblasts (HOBs), and human mesenchymal stem cells (MSCs), as well as MG63 cells stably silenced for Dkk1 or Dkk2 were grown for 6 days on TCPS and Ti surfaces (PT [Ra<0.2 μm], SLA [Ra = 4 μm], modSLA [hydrophilic-SLA]). Dkk1 and Dkk2 mRNA and protein increased on SLA and modSLA for all cell types, but exogenous rhDkk1 and rhDkk2 affected MSCs differently than MG63 cells and HOBs. Silencing Dkk1 reduced MG63 cell number on TCPS and PT, but increased differentiation on these substrates. Silencing Dkk2 reduced stimulatory effects of SLA and modSLA on osteoblast differentiation; Dkk2 but not Dkk1 restored these effects. Antibodies to Dkk1 or Dkk2 specifically blocked substrate-dependent changes caused by the proteins, demonstrating their autocrine action. This indicates major roles for Dkk1 and the canonical Wnt pathway in early-stage differentiation, and for Dkk2 and Wnt/Ca2+-dependent signaling in late-stage differentiation on microstructured and hydrophilic surfaces, during osseointegration.
Keywords: Osseointegration; Titanium; Osteoblast; Mesenchymal stem cell; Surface roughness; Cell signaling;

The inhibition of prions through blocking prion conversion by permanently charged branched polyamines of low cytotoxicity by Yong-beom Lim; Charles E. Mays; Younghwan Kim; William B. Titlow; Chongsuk Ryou (2025-2033).
Branched polyamines are effective in inhibiting prions in a cationic surface charge density dependent manner. However, toxicity associated with branched polyamines, in general, often hampers the successful application of the compounds to treat prion diseases. Here, we report that constitutively maintained cationic properties in branched polyamines reduced the intrinsic toxicity of the compounds while retaining the anti-prion activities. In prion-infected neuroblastoma cells, quaternization of amines in polyethyleneimine (PEI) and polyamidoamine (PAMAM) dendrimers markedly increased the nontoxic concentration ranges of the compounds and still supported, albeit reduced, an appreciable level of anti-prion activity in clearing prions from the infected cells. Furthermore, quaternized PEI was able to degrade prions at acidic pH conditions and inhibit the in vitro prion propagation facilitated by conversion of the normal prion protein isoform to its misfolded counterpart, although such activities were decreased by quaternization. Quaternized PAMAM was least effective in degrading prions but efficiently inhibited prion conversion with the same efficacy as unmodified PAMAM. Our results suggest that quaternization represents an effective strategy for developing nontoxic branched polyamines with potent anti-prion activity. This study highlights the importance of polyamine structural control for developing polyamine-based anti-prion agents and understanding of an action mechanism of quaternized branched polyamines.
Keywords: Prion; Branched polyamines; Quaternization; Cytotoxicity; Prion protein conversion; Therapy;

Biodistribution of gold nanoparticles and gene expression changes in the liver and spleen after intravenous administration in rats by Suresh K. Balasubramanian; Jinatta Jittiwat; Jayapal Manikandan; Choon-Nam Ong; Liya E. Yu; Wei-Yi Ong (2034-2042).
Biodistribution of gold nanoparticles (AuNPs) in more than 25 organs were examined on 1 day, 1 week, 1 month and 2 months after a single intravenous (i.v.) injection in rats. Au was rapidly and consistently accumulated in liver (49.4 ± 50.4–72.2 ± 40.5 ng/g) and spleen (8.4 ± 5.0–9.5 ± 6.4 ng/g) throughout the entire timeframe of the study (2 months). Significant accumulation of Au in kidney (up to 5.5 ± 2.5 ng/g) and testis (up to 0.6 ± 0.1 ng/g) occurred from 1 month post-injection when Au level in urine and feces decreased. Significant increase of Au in blood occurred 2 months after injection, coincident with the delayed accumulation in kidney. Au accumulation in lungs was found at 1 day post-injection but decreased within a week. No accumulation of Au was found in the brain. Microarray results of liver and spleen point to significant effects on genes related to detoxification, lipid metabolism, cell cycle, defense response, and circadian rhythm. These results demonstrate that significant biodistribution of Au occurs in the body over 2 months after a single i.v. injection of AuNPs, accompanied by gene expression changes in target organs.
Keywords: Gold nanoparticles; Intravenous injection; Biodistribution; Microarray; Liver; Toxicity;

In vitro and in vivo evaluation of an alumina–zirconia composite for arthroplasty applications by Olivier Roualdes; Marie-Eve Duclos; Dan Gutknecht; Lucien Frappart; Jérôme Chevalier; Daniel J. Hartmann (2043-2054).
In order to improve the reliability and the mechanical properties of orthopaedic hip prosthesis, new ceramic composites starting with nanosized powders of alumina and zirconia have been recently developed. The aim of the present study was to investigate the biological tolerance of one of these sintered ceramics and of its alumina and zirconia constitutive nanosized powders with both in vitro and in vivo approaches. At first, osteoblasts and fibroblasts were cultured either upon sintered ceramic discs with polished or rough surfaces or in the presence of the corresponding alumina or zirconia powders at various concentrations. Thereafter, we chronically injected these powders in the knee articulation of rats. In vitro, the materials showed no deleterious effect on cell proliferation, extra-cellular matrix production (human type I collagen and fibronectin) or on cell morphology. In vivo, the histological examination showed only a very moderate and non-specific granulomatous response of the synovial membrane but no major inflammation as clinically described with metals or polyethylene wear debris. Besides its improved physical properties, this recently developed alumina–zirconia composite showed satisfactory biocompatibility.
Keywords: Ceramic; Composite; Alumina; Zirconia; Nanoparticles; Biocompatibility;

The role of sterilization in the cytocompatibility of titania nanotubes by Lingzhou Zhao; Shenglin Mei; Wei Wang; Paul K. Chu; Zhifen Wu; Yumei Zhang (2055-2063).
Titiania nanotubes have large potential in medical implant applications but their tissue compatibility is still controversial. Since the sterilization methods may impact the biocompatibility of titania nanotubes and be the source of the controversy, we investigate the influence of three commonly used sterilization methods, autoclaving, ultraviolent irradiation and ethanol immersion, on the cytocompatibility of titania nanotubes. Two titania nanostructures, namely nanonets with an average pore diameter of 25 nm and nanotubes with an average diameter of 80 nm, are used in this study. The results show that the sterilization methods significantly affect the cytocompatibility of these titania surfaces. UV and ethanol sterilization give rise to a higher surface free energy inducing higher initial cell adhesion and proliferation compared to autoclaving, whereas UV irradiation produces the best cell functions including adhesion, proliferation, as well as differentiation represented by related gene expressions. The cytocompatibility results obtained from the nanoscale surfaces are compared to those acquired from the polished surface demonstrating the significant effects. Our results suggest that the sterilization process plays an important role in the observed cytocompatibility of titania nanotubes and may be the reason for the controversial results so far. UV sterilization is found to be the best method from the viewpoint of surface contamination elimination.
Keywords: Nanotopography; Sterilization; Osteoblast; Cytocompatibility; Real-time PCR;

Effect of functionalised fluorescence-labelled nanoparticles on mesenchymal stem cell differentiation by Andrea Tautzenberger; Steffen Lorenz; Ludwika Kreja; Anke Zeller; Anna Musyanovych; Hubert Schrezenmeier; Katharina Landfester; Volker Mailänder; Anita Ignatius (2064-2071).
The combined use of nanoparticles and mesenchymal stem cells (MSC) in regenerative medicine requires the incorporation of the particles and, at the same time, undisturbed cell viability and maintenance of the multi-lineage potential of MSC. The aim of this study was to investigate the uptake of novel phosphonate-functionalised polystyrene nanoparticles prepared by miniemulsion polymerisation. After exposition of human MSC to the particles, their uptake and localisation were analysed by flow cytometry, confocal laser scanning microscopy (CLSM), and transmission electron microscopy (TEM). The osteogenic, adipogenic and chondrogenic differentiation potential was examined by analysing representative marker genes by RT-PCR. Flow cytometry revealed that after 5 and 16 days more than 98% of the MSC and of the cells, which underwent osteogenic and adipogenic differentiation were positive for particle association. CLSM and TEM demonstrated the successful intracellular incorporation of the particles without using any transfection agents and their presence over the cultivation period. The cell viability was found to be unaffected. Particle treated MSC maintained their potential for osteogenic, adipogenic and chondrogenic differentiation. It was concluded that the surface functionalisation with phosphonate groups provides a promising basis for the development of nanoparticles with high intracellular uptake rates for drug delivery or cell labelling.
Keywords: Nanoparticle; Mesenchymal stem cell; Differentiation; Cellular uptake;

The covalent immobilization of heparin to pulsed-plasma polymeric allylamine films on 316L stainless steel and the resulting effects on hemocompatibility by Zhilu Yang; Jin Wang; Rifang Luo; Manfred F. Maitz; Fengjuan Jing; Hong Sun; Nan Huang (2072-2083).
For an improved hemocompatibility of 316L stainless steel (SS), we develop a facile and effective approach to fabricating a pulsed-plasma polymeric allylamine (P-PPAm) film that possesses a high cross-linking degree and a high density of amine groups, which is used for subsequent bonding of heparin. The P-PPAm film as a stent coating shows good resistance to the deformation behavior of compression and expansion of a stent. Using deionized water as an aging medium, it is demonstrated that the heparin-immobilized P-PPAm (Hep-P-PPAm) surface has a good retention of heparin. The systematic in vitro hemocompatibility evaluation reveals lower platelet adhesion, platelet activation and fibrinogen activation on the Hep-P-PPAm surface, and the activated partial thromboplastin time prolongs for about 15 s compared with 316L SS. The P-PPAm surface significantly promotes adhesion and proliferation of endothelial cells (ECs). For the Hep-P-PPAm, although EC adhesion and proliferation is slightly suppressed initially, after cultivation for 3 days, the growth behavior of ECs is remarkably improved over 316L SS. In vivo results indicate that the Hep-P-PPAm surface successfully restrain thrombus formation by growing a homogeneous and intact shuttle-like endothelium on its surface. The Hep-P-PPAm modified 316L SS shows a promising application for vascular devices.
Keywords: 316L stainless steel; Plasma polymerization; Stability; Stent; Heparin; Hemocompatibility;

A biodegradable polymer-based coating to control the performance of magnesium alloy orthopaedic implants by Hoi Man Wong; Kelvin W.K. Yeung; Kin On Lam; Vivian Tam; Paul K. Chu; Keith D.K. Luk; Kenneth M.C. Cheung (2084-2096).
Magnesium and its alloys may potentially be applied as degradable metallic materials in orthopaedic implantations due to their degradability and resemblance to human cortical bone. However, the high corrosion rate and accumulation of hydrogen gas upon degradation hinders its clinical application. In this study, we adopt a new approach to control the corrosion rate by coating a controllable polymeric membrane fabricated by polycaprolactone and dichloromethane onto magnesium alloys, in which the pore size was controlled during the manufacturing process. The addition of the polymeric membrane was found to reduce the degradation rate of magnesium, and the bulk mechanical properties were shown to be maintained upon degradation. The in-vitro studies indicated good cytocompatibility of eGFP and SaOS-2 osteoblasts with the polymer-coated samples, which was not observed for the uncoated samples. The in-vivo study indicated that the uncoated sample degraded more rapidly than that of the polymer-coated samples. Although new bone formation was found on both samples, as determined by Micro-CT, higher volumes of new bone were observed on the polymer-coated samples. Histological analysis indicated no inflammation, necrosis or hydrogen gas accumulation on either of the samples during degradation. Collectively, these data suggest that the use of polymeric membrane may be potentially applied for future clinical use.
Keywords: Magnesium; Polycaprolactone; Biodegradable; Corrosion; Biocompatibility;

The use of nanodiamond monolayer coatings to promote the formation of functional neuronal networks by Agnes Thalhammer; Robert J. Edgington; Lorenzo A. Cingolani; Ralf Schoepfer; Richard B. Jackman (2097-2104).
Nanostructured materials provide a new dimension of interaction with biological systems that takes place on a sub-cellular level with a high degree of specificity. In the field of neuroscience the nanoscale corresponds to the size of synapses; the specific connections between brain cells. In this context, diamond-based materials have attracted much attention due to their extreme mechanical and electrical properties and their chemical inertness. Here the suitability of nanodiamond (ND) monolayers to act as a platform for neuronal growth is investigated. Neurons cultured on various ND-coated substrates perform remarkably well, and similar to those grown on standard protein-coated materials with respect to their initial cell attachment, sustained neurite outgrowth, cell-autonomous neuronal excitability and functionality of the resulting electrical networks. ND layering provides an excellent growth substrate on various materials for functional neuronal networks and bypasses the necessity of protein coating, which promises great potential for chronic medical implants.
Keywords: Diamond; Electrophysiology; Nanoparticle; Neural network;

Adult cell therapy for brain neuronal damages and the role of tissue engineering by Gaëtan J.-R. Delcroix; Paul C. Schiller; Jean-Pierre Benoit; Claudia N. Montero-Menei (2105-2120).
No long term effective treatments are currently available for brain neurological disorders such as stroke/cerebral ischemia, traumatic brain injury and neurodegenerative disorders. Cell therapy is a promising strategy, although alternatives to embryonic/foetal cells are required to overcome ethical, tissue availability and graft rejection concerns. Adult cells may be easily isolated from the patient body, therefore permitting autologous grafts to be performed. Here, we describe the use of adult neural stem cells, adrenal chromaffin cells and retinal pigment epithelium cells for brain therapy, with a special emphasis on mesenchymal stromal cells. However, major problems like cell survival, control of differentiation and engraftment remain and may be overcome using a tissue engineering strategy, which provides a 3D support to grafted cells improving their survival. New developments, such as the biomimetic approach which combines the use of scaffolds with extracellular matrix molecules, may improve the control of cell proliferation, survival, migration, differentiation and engraftment in vivo. Therefore, we later discuss scaffold properties required for brain cell therapy as well as new tissue engineering advances that may be implemented in combination with adult cells for brain therapy. Finally, we describe an approach developed in our laboratory to repair/protect lesioned tissues: the pharmacologically active microcarriers.
Keywords: Brain disorders; Mesenchymal stromal cells; Adult cells; Scaffolds; Biomimetic surface; Pharmacologically active microcarriers;

The recruitment of two consecutive and different waves of host stem/progenitor cells during the development of tissue-engineered bone in a murine model by Roberta Tasso; Franco Fais; Daniele Reverberi; Federico Tortelli; Ranieri Cancedda (2121-2129).
Angiogenesis plays a central role in bone regeneration, not only for the transport of nutrients, but also for locally directing skeletal stem/progenitor cells. Following ectopic implantation of porous ceramic cubes seeded with mouse GFP-labeled mesenchymal stem cells (MSC) into syngenic mice, we investigated the cascade of events leading to bone formation. Implants harvested at different times were enzymatically digested to generate single-cell suspensions. Recovered cells were sorted to separate GFP + implanted MSC and host recruited GFP- cells. We isolated and characterized two different waves of cells, migrating from the host to the MSC-seeded ceramic. The first migrated cell population, recovered 7 days after implantation, was enriched in CD31 + endothelial progenitors, while the second one, recruited at day 11, was enriched in CD146 + pericyte-like cells. Both populations were not recruited into the scaffold following implantation of a non-MSC seeded ceramic. Pericyte-like cell mobilization was dependent on the first migrated endothelial cell population. Pericyte-like cells retained properties distinctive of stem cells, such as capacity of performing a high number of in vitro cell divisions and showed an osteogenic potential. Studies on the cross talk between implanted exogenous MSC and resident stem/progenitor cells could open new perspectives for future clinical applications.
Keywords: Bone; Tissue engineering; Endothelial progenitor; Mesenchymal stem cell; Angiogenesis; Osteogenesis;

Deep magnetic capture of magnetically loaded cells for spatially targeted therapeutics by Zheyong Huang; Ning Pei; Yanyan Wang; Xinxing Xie; Aijun Sun; Li Shen; Shuning Zhang; Xuebo Liu; Yunzeng Zou; Juying Qian; Junbo Ge (2130-2140).
Magnetic targeting has recently demonstrated potential in promoting magnetically loaded cell delivery to target lesion, but its application is limited by magnetic attenuation. For deep magnetic capture of cells for spatial targeting therapeutics, we designed a magnetic pole, in which the magnetic field density can be focused at a distance from the pole. As flowing through a tube served as a model of blood vessels, the magnetically loaded mesenchymal stem cells (MagMSCs) were highly enriched at the site distance from the magnetic pole. The cell capture efficiency was positively influenced by the magnetic flux density, and inversely influenced by the flow velocity, and well-fitted with the deductive value by theoretical considerations. It appeared to us that the spatially-focused property of the magnetic apparatus promises a new deep targeting strategy to promote homing and engraftment for cellular therapy.
Keywords: Magnetism; In vitro test; Mesenchcymal stem cell; Nanoparticle;

The influence of structural design of PLGA/collagen hybrid scaffolds in cartilage tissue engineering by Wenda Dai; Naoki Kawazoe; Xiaoting Lin; Jian Dong; Guoping Chen (2141-2152).
3D biodegradable porous scaffold plays a very important role in articular cartilage tissue engineering. We developed hybrid structures of 3D scaffolds that combined the advantages of natural type I collagen and synthetic PLGA knitted mesh. The mechanically strong PLGA mesh served as a skeleton while the collagen microsponges facilitated cell seeding and tissue formation. The scaffolds were divided into 3 groups: (1) THIN: collagen microsponge formed in interstices of PLGA mesh; (2) SEMI: collagen microsponge formed on one side of PLGA mesh; (3) SANDWICH: collagen sponge formed on both sides of PLGA mesh. Bovine chondrocytes were cultured in these scaffolds and transplanted subcutaneously into nude mice for 2, 4, and 8 weeks. All three groups of transplants showed homogeneous cell distribution, natural chondrocyte morphology, and abundant cartilaginous ECM deposition. Production of GAGs per DNA and the expression of type II collagen and aggrecan mRNA were much higher in the SEMI and SANDWICH groups than in the THIN group. When compared to native articular cartilage, the mechanical strength of the engineered cartilage reached 54.8%, 49.3% in Young's modulus and 68.8%, 62.7% in stiffness, respectively, in SEMI and SANDWICH. These scaffolds could be used for the tissue engineering of articular cartilage with adjustable thickness. The design of the hybrid structures provides a strategy for the preparation of 3D porous scaffolds.
Keywords: Porous scaffold; Hybrid scaffold; Synthetic polymer; Collagen; Tissue engineering; Cartilage;

The use of surface modified poly(glycerol-co-sebacic acid) in retinal transplantation by Christopher D. Pritchard; Karin M. Arnér; Rebekah A. Neal; William L. Neeley; Peter Bojo; Erika Bachelder; Jessica Holz; Nicki Watson; Edward A. Botchwey; Robert S. Langer; Fredrik K. Ghosh (2153-2162).
Retinal transplantation experiments have advanced considerably during recent years, but remaining diseased photoreceptor cells in the host retina and inner retinal cells in the transplant physically obstruct the development of graft-host neuronal contacts which are required for vision. Recently, we developed methods for the isolation of donor photoreceptor layers in vitro, and the selective removal of host photoreceptors in vivo using biodegradable elastomeric membranes composed of poly(glycerol-co-sebacic acid) (PGS). Here, we report the surface modification of PGS membranes to promote the attachment of photoreceptor layers, allowing the resulting composite to be handled surgically as a single entity. PGS membranes were chemically modified with peptides containing an arginine-glycine-aspartic acid (RGD) extracellular matrix ligand sequence. PGS membranes were also coated with electrospun nanofiber meshes, containing laminin and poly(epsilon-caprolactone) (PCL). Following in vitro co-culture of biomaterial membranes with isolated embryonic retinal tissue, composites were tested for surgical handling and examined with hematoxylin and eosin staining and immunohistochemical markers. Electrospun nanofibers composed of laminin and PCL promoted sufficient cell adhesion for simultaneous transplantation of isolated photoreceptor layers and PGS membranes. Composites developed large populations of recoverin and rhodopsin labeled photoreceptors. Furthermore, ganglion cells, rod bipolar cells and AII amacrine cells were absent in co-cultured retinas as observed by neurofilament, PKC and parvalbumin labeling respectively. These results facilitate retinal transplantation experiments in which a composite graft composed of a biodegradable membrane adhered to an immature retina dominated by photoreceptor cells may be delivered in a single surgery, with the possibility of improving graft-host neuronal connections.
Keywords: Cell adhesion; Elastomer; Laminin; Nanotopography; Polycaprolactone; Retina;

The regulation of tendon stem cell differentiation by the alignment of nanofibers by Zi Yin; Xiao Chen; Jia Lin Chen; Wei Liang Shen; Thi Minh Hieu Nguyen; Ling Gao; Hong Wei Ouyang (2163-2175).
Tendon is a specific connective tissue composed of parallel collagen fibers. The effect of this tissue-specific matrix orientation on stem cell differentiation has not been investigated. This study aimed to determine the effects of nanotopography on the differentiation of human tendon stem/progenitor cells (hTSPCs) and develop a biomimetic scaffold for tendon tissue engineering. The immuno-phenotype of fetal hTSPCs was identified by flow cytometry. The multipotency of hTSPCs toward osteogenesis, adipogenesis, and chondrogenesis was confirmed. Then, the hTSPCs were seeded onto aligned or randomly-oriented poly (l-lactic acid) nanofibers. Scanning electron micrographs showed that hTSPCs were spindle-shaped and well orientated on the aligned nanofibers. The expression of tendon-specific genes was significantly higher in hTSPCs growing on aligned nanofibers than those on randomly-oriented nanofibers in both normal and osteogenic media. In addition, alkaline phosphatase activity and alizarin red staining showed that the randomly-oriented fibrous scaffold induced osteogenesis, while the aligned scaffold hindered the process. Moreover, aligned cells expressed significantly higher levels of integrin α1, α5 and β1 subunits, and myosin II B. In in vivo experiments, the aligned nanofibers induced the formation of spindle-shaped cells and tendon-like tissue. In conclusion, the aligned electrospun nanofiber structure provides an instructive microenvironment for hTSPC differentiation and may lead to the development of desirable engineered tendons.
Keywords: Tendon stem cells; Aligned nanofiber; Differentiation; Tendon regeneration; Mechanotransduction;

In vitro engineering of human ear-shaped cartilage assisted with CAD/CAM technology by Yu Liu; Lu Zhang; Guangdong Zhou; Qiong Li; Wei Liu; Zheyuan Yu; Xusong Luo; Ting Jiang; Wenjie Zhang; Yilin Cao (2176-2183).
Due to the lack of appropriate scaffolds, the in vitro engineering of cartilage tissue with a sophisticated structure, such as a human ear, remains a great challenge. Although polyglycolic acid (PGA) has become one of the most successful scaffolds for cartilage regeneration, how to overcome its limitations in achieving desirable mechanical strength and accurate control over shape remains an unsolved problem. In this study, the mechanical strength of PGA scaffold was enhanced by coating with polylactic acid (PLA). The content of PLA was optimized by balancing the scaffold's biocompatibility and mechanical strength. The PLA/PGA scaffold was then fabricated into a human ear-shape mirror-symmetrical to a normal ear by pressing the scaffold in the ear negative molds, which were fabricated by the computer aided design and manufacturing (CAD/CAM) technique according to the CT scan data from the normal ear. The ear-shaped scaffold reached a similarity level of over 97% compared to the positive ear mold by the shape analysis using a 3D laser scan system. Most importantly, after chondrocyte seeding, the constructs largely retained the original shape during culture with a similarity level of over 84%. Furthermore, the constructs formed ear-shaped cartilage-like tissues at 12 weeks, which revealed a tissue structure with abundant cartilage extracellular matrices and mature lacuna. Additionally, the ear-shaped cartilage at 12 weeks also exhibited fine elasticity and good mechanical strength. These results may provide a useful strategy for reconstructing cartilage tissue with complicated shapes such as a human ear by an in vitro engineering approach.
Keywords: Cartilage; Human ear; CAD/CAM; Polyglycolic acid; Polylactic acid; 3D surface laser scan;

The repair of the injured adult rat hippocampus with NT-3-chitosan carriers by Linhong Mo; Zhaoyang Yang; Aifeng Zhang; Xiaoguang Li (2184-2192).
The injury of the CA1 region of the adult rat hippocampus causes cognitive impairment. In this study, animal models were established by mechanically injuring the CA1 region of the adult rat hippocampus, and into the injured area were implanted chitosan carriers loaded either with or without NT-3. Immunohistochemical and nerve tracer methods were adopted to observe the role of the above-mentioned carriers in repairing the injured brain and to observe the scar formation after the injury, and Morris water maze (MWM) tests were performed to evaluate the recovery degree of the cognitive function. The results showed that NT-3-chitosan carriers stimulated regeneration of a large amount of NF-positive nerve fiber and neuron-like cells into the injured area. The newly regenerated NF-positive nerve fibers in the injured area rebuilt a neural circuit with the contralateral CA1 region via corpus callosum. Comparison of the lesion control rats and the treated rats indicates that the chitosan carriers loaded either with or without NT-3 may significantly improve the cognitive function after the hippocampus injury.
Keywords: Traumatic brain injury; Chitosan scaffold; Transplantation; Neurotrophin-3; Hippocampus; Cognitive function;

In vitro generation of mechanically functional cartilage grafts based on adult human stem cells and 3D-woven poly(ɛ-caprolactone) scaffolds by Piia K. Valonen; Franklin T. Moutos; Akihiko Kusanagi; Matteo G. Moretti; Brian O. Diekman; Jean F. Welter; Arnold I. Caplan; Farshid Guilak; Lisa E. Freed (2193-2200).
Three-dimensionally woven poly(ɛ-caprolactone) (PCL) scaffolds were combined with adult human mesenchymal stem cells (hMSC) to engineer mechanically functional cartilage constructs in vitro. The specific objectives were to: (i) produce PCL scaffolds with cartilage-like mechanical properties, (ii) demonstrate that hMSCs formed cartilage after 21 days of culture on PCL scaffolds, and (iii) study effects of scaffold structure (loosely vs. tightly woven), culture vessel (static dish vs. oscillating bioreactor), and medium composition (chondrogenic additives with or without serum). Aggregate moduli of 21-day constructs approached normal articular cartilage for tightly woven PCL cultured in bioreactors, were lower for tightly woven PCL cultured statically, and lowest for loosely woven PCL cultured statically (p < 0.05). Construct DNA, total collagen, and glycosaminoglycans (GAG) increased in a manner dependent on time, culture vessel, and medium composition. Chondrogenesis was verified histologically by rounded cells within a hyaline-like matrix that immunostained for collagen type II but not type I. Bioreactors yielded constructs with higher collagen content (p < 0.05) and more homogenous matrix than static controls. Chondrogenic additives yielded constructs with higher GAG (p < 0.05) and earlier expression of collagen II mRNA if serum was not present in medium. These results show feasibility of functional cartilage tissue engineering from hMSC and 3D-woven PCL scaffolds.
Keywords: Cartilage repair; Stem cell; Bioreactor; Biomechanics; Growth factors;

The cavity-to-cavity migration of leukaemic cells through 3D honey-combed hydrogels with adjustable internal dimension and stiffness by Joakim da Silva; Franziska Lautenschläger; Easan Sivaniah; Jochen R. Guck (2201-2208).
Whilst rigid, planar surfaces are often used to study cell migration, a physiological scenario requires three-dimensional (3D) scaffolds with tissue-like stiffness. This paper presents a method for fabricating periodic hydrogel scaffolds with a 3D honeycomb-like structure from colloidal crystal templates. The scaffolds, made of hydrogel-walled cavities interconnected by pores, have separately tuneable internal dimensions and adjustable gel stiffness down to that of soft tissues. In conjunction with confocal microscopy, these scaffolds were used to study the importance of cell compliance on invasive potential. Acute promyelocytic leukaemia (APL) cells were differentiated with all-trans retinoic acid (ATRA) and treated with paclitaxel. Their migration ability into the scaffolds' size-restricted pores, enabled by cell softening during ATRA differentiation, was significantly reduced by paclitaxel treatment, which interferes with cell shape recovery. These findings demonstrate the usability of the scaffolds for investigating factors that affect cell migration, and potentially other cell functions, in a realistic 3D tissue model.
Keywords: Inverted colloidal crystal; Poly-acrylamide; Cell mechanics; Tissue invasion; Metastasis;

Engineering of capillary-like structures in tissue constructs by electrochemical detachment of cells by Yuki Seto; Rina Inaba; Tomoaki Okuyama; Fumihiro Sassa; Hiroaki Suzuki; Junji Fukuda (2209-2215).
A major challenge in the development of functional thick tissues is the formation of vascular networks for oxygen and nutrient supply throughout the engineered tissue constructs. This study describes an electrochemical approach for fabrication of capillary-like structures, precisely aligned within micrometer distances, whose internal surfaces are covered with vascular endothelial cells. In this approach, an oligopeptide containing a cell adhesion domain (RGD) in the center and cysteine residues at both ends was designed. Cysteine has a thiol group that adsorbs onto a gold surface via a gold–thiolate bond. The cells attached to the gold surface via the oligopeptide were readily and noninvasively detached by applying a negative electrical potential and cleaving the gold–thiolate bond. This approach was applicable not only for a flat surface but also for various configurations, including cylindrical structures. By applying this approach to thin gold rods aligned in a spatially controlled manner in a perfusion culture device, human umbilical vein endothelial cells (HUVECs) were transferred onto the internal surface of capillary structures in collagen gel. In the subsequent perfusion culture, the HUVECs grew into the collagen gel and formed luminal structures, thereby forming vascular networks in vitro.
Keywords: Tissue engineering; Blood vessel; Peptide; Electrochemistry; Gold–thiolate bond; Collagen;

Strategies for cell manipulation and skeletal tissue engineering using high-throughput polymer blend formulation and microarray techniques by Ferdous Khan; Rahul S. Tare; Janos M. Kanczler; Richard O.C. Oreffo; Mark Bradley (2216-2228).
A combination of high-throughput material formulation and microarray techniques were synergistically applied for the efficient analysis of the biological functionality of 135 binary polymer blends. This allowed the identification of cell-compatible biopolymers permissive for human skeletal stem cell growth in both in vitro and in vivo applications. The blended polymeric materials were developed from commercially available, inexpensive and well characterised biodegradable polymers, which on their own lacked both the structural requirements of a scaffold material and, critically, the ability to facilitate cell growth. Blends identified here proved excellent templates for cell attachment, and in addition, a number of blends displayed remarkable bone-like architecture and facilitated bone regeneration by providing 3D biomimetic scaffolds for skeletal cell growth and osteogenic differentiation. This study demonstrates a unique strategy to generate and identify innovative materials with widespread application in cell biology as well as offering a new reparative platform strategy applicable to skeletal tissues.
Keywords: Polymer blends; Scaffolds; Tissue regeneration; Skeletal stem cells;

Control of cell attachment through polyDNA hybridization by Yuji Teramura; Hao Chen; Takuo Kawamoto; Hiroo Iwata (2229-2235).
Cell–cell interactions play vital roles in embryo development and in homeostasis maintenance. Such interactions must be stringently controlled for cell-based tissue engineering and regenerative medicine therapies, and methods for studying and controlling cell–cell interactions are being developed using both biomedical and engineering approaches. In this study, we prepared amphiphilic PEG-lipid polymers that were attached to polyDNA with specific sequences. Incubation of cells with the polyDNA–PEG-lipid conjugate transferred some of the polyDNA to the cells' surfaces. Similarly, polyDNA–PEG-lipid conjugate using polyDNA with a complementary sequence was introduced to the surfaces of other cells or to a substrate surface. Cell–cell or cell–substrate attachments were subsequently mediated via hybridization between the two complementary polyDNAs and monitored using fluorescence microscopy.
Keywords: Cell–cell attachment; Poly(ethylene glycol)-lipid (PEG-lipid); DNA hybridization; Surface modification;

Engineering cardiac tissue in vivo from human adipose-derived stem cells by Yu Suk Choi; Ken Matsuda; Gregory J. Dusting; Wayne A. Morrison; Rodney J. Dilley (2236-2242).
Cardiac tissue engineering offers promise as a surgical approach to cardiac repair, but requires an adequate source of cardiomyocytes. Here we evaluate the potential for generating human cardiac muscle cells in vivo from adipose-derived stem cells (ASC) by co-implanting in a vascularised tissue engineering chamber with inducing rat cardiomyocytes (rCM). Co-implantation (ASC–rCM) was compared with rCM or ASC controls alone after 6 weeks. Immunostaining using human nucleus specific antibody and cardiac markers revealed several fates for ASC in the chamber; (1) differentiation into cardiomyocytes and integration with co-implanted rCM; (2) differentiation into smooth muscle cells and recruitment into vascular structures; (3) adipogenic differentiation. ASC–rCM and ASC groups grew larger tissue constructs than rCM alone (212 ± 25 μl, 171 ± 16 μl vs. 137 ± 15 μl). ASC–rCM and rCM groups contracted spontaneously at up to 140 bpm and generated a 10–15-fold larger volume of cardiac muscle (14.5 ± 4.8 μl and 18.5 ± 2.6 μl) than ASC alone group (1.3 ± 0.5 μl). Vascular volume in ASC–rCM group was twice that of the rCM group (28.7 ± 5.0 μl vs. 14.8 ± 1.8 μl). The cardiac tissue engineered by co-implanting human ASC with neonatal rCM showed in vivo plasticity of ASC and their cardiomyogenic potential in tissue engineering. ASC contribution to vascularisation also promoted the growth of engineered tissue, confirming their utility in this setting.
Keywords: Cardiac tissue engineering; Adipose-derived stem cells; Cardiomyocyte; Cardiomyogenic differentiation;

The development of a three-dimensional scaffold for ex vivo biomimicry of human acute myeloid leukaemia by Teresa Mortera Blanco; Athanasios Mantalaris; Alexander Bismarck; Nicki Panoskaltsis (2243-2251).
Acute myeloid leukaemia (AML) is a cancer of haematopoietic cells that develops in three-dimensional (3-D) bone marrow niches in vivo. The study of AML has been hampered by lack of appropriate ex vivo models that mimic this microenvironment. We hypothesised that fabrication and optimisation of suitable biomimetic scaffolds for culturing leukaemic cells ex vivo might facilitate the study of AML in its native 3-D niche. We evaluated the growth of three leukaemia subtype-specific cell lines, K-562, HL60 and Kasumi-6, on highly porous scaffolds fabricated from biodegradable and non-biodegradable polymeric materials, such as poly (L-lactic-co-glycolic acid) (PLGA), polyurethane (PU), poly (methyl-methacrylate), poly (d, l-lactade), poly (caprolactone), and polystyrene. Our results show that PLGA and PU supported the best seeding efficiency and leukaemic growth. Furthermore, the PLGA and PU scaffolds were coated with extracellular matrix (ECM) proteins, collagen type I (62.5 or 125 μg/ml) and fibronectin (25 or 50 μg/ml) to provide biorecognition signals. The 3 leukaemia subtype-specific lines grew best on PU scaffolds coated with 62.5 μg/ml collagen type I over 6 weeks in the absence of exogenous growth factors. In conclusion, PU-collagen scaffolds may provide a practical model to study the biology and treatment of primary AML in an ex vivo mimicry.
Keywords: Three-dimensional culture; Scaffold; Leukaemia culture; Haematopoiesis;

Engineering human cell-based, functionally integrated osteochondral grafts by biological bonding of engineered cartilage tissues to bony scaffolds by Celeste Scotti; Dieter Wirz; Francine Wolf; Dirk J. Schaefer; Vivienne Bürgin; Alma U. Daniels; Victor Valderrabano; Christian Candrian; Marcel Jakob; Ivan Martin; Andrea Barbero (2252-2259).
In this study, we aimed at developing and validating a technique for the engineering of osteochondral grafts based on the biological bonding of a chondral layer with a bony scaffold by cell-laid extracellular matrix. Osteochondral composites were generated by combining collagen-based matrices (Chondro-Gide®) containing human chondrocytes with devitalized spongiosa cylinders (Tutobone®) using a fibrin gel (Tisseel®). We demonstrate that separate pre-culture of the chondral layer for 3 days prior to the generation of the composite allows for (i) more efficient cartilaginous matrix accumulation than no pre-culture, as assessed histologically and biochemically, and (ii) superior biological bonding to the bony scaffold than 14 days of pre-culture, as assessed using a peel-off mechanical test, developed to measure integration of bilayered materials. The presence of the bony scaffold induced an upregulation in the infiltrated cells of the osteoblast-related gene bone sialoprotein, indicative of the establishment of a gradient of cell phenotypes, but did not affect per se the quality of the cartilaginous matrix in the chondral layer. The described strategy to generate osteochondral plugs is simple to be implemented and – since it is based on clinically compliant cells and materials – is amenable to be readily tested in the clinic.
Keywords: Chondrocyte; Cartilage tissue engineering; Biomimetic material; Integration; Interface;

The inhibition of advanced glycation end-products-induced retinal vascular permeability by silver nanoparticles by Sardarpasha Sheikpranbabu; Kalimuthu Kalishwaralal; Kyung-jin Lee; Ramanathan Vaidyanathan; Soo Hyun Eom; Sangiliyandi Gurunathan (2260-2271).
The increased permeability of the blood–retinal barrier is known to occur in patients with diabetes, and this defect contributes to retinal edema. This study aimed to determine the effects of silver nanoparticles (Ag-NPs) on advanced glycation end-products (AGEs)-induced endothelial cell permeability. Cultured porcine retinal endothelial cells (PRECs) were exposed to AGE-modified bovine serum albumin (AGE-BSA) and the endothelial cell permeability was detected by measuring the flux of RITC-dextran across the PREC monolayers. We found that AGE-BSA increased the dextran flux across a PREC monolayer and Ag-NPs blocked the solute flux induced by AGE-BSA. In order to understand the underlying signaling mechanism of Ag-NPs on the inhibitory effect of AGE-BSA-induced permeability, we demonstrated that Ag-NPs could inhibit the AGE-BSA-induced permeability via Src kinase pathway. AGE-BSA also increased the PREC permeability by stimulating the expression of intracellular adhesion molecule-1 (ICAM-1) and decreased the expression of occludin and ZO-1. Further, Ag-NPs inhibited the AGE-BSA-induced permeability by increased expression of tight junction proteins occludin and ZO-1, co-incident with an increase in barrier properties of endothelial monolayer. Together, our results indicate that Ag-NPs could possibly act as potent anti-permeability molecule by targeting the Src signaling pathway and tight junction proteins and it offers potential targets to inhibit the ocular related diseases.
Keywords: AGE-BSA; Silver nanoparticles; Vascular permeability; ICAM-1; Src kinase; Occludin;

The second harmonic generation (SHG) active nanocrystals have been demonstrated as attractive imaging probes in nonlinear microscopy due to their coherent, non-bleaching and non-blinking signals with a broad flexibility in the choice of excitation wavelength. For the use of these nanocrystals as biomarkers, it is essential to prepare a chemical interface for specific labeling. We developed a specific labeling scheme for barium titanate (BaTiO3) nanocrystals which we use as second harmonic radiation imaging probes. The specificity was achieved by covalently coupling antibodies onto the nanocrystals. We demonstrate highly specific labeling of the nanocrystal conjugates in an antibody microarray and also the membrane proteins of live biological cells in vitro. The development of surface functionalization and bioconjugation of SHG active nanocrystals provides the opportunities of applying them to biological studies.
Keywords: Confocal microscopy; Cross-linking; Immunochemistry; In vitro test; Nanoparticle; Surface modification;

Peptide-conjugated biodegradable nanoparticles as a carrier to target paclitaxel to tumor neovasculature by De-Hong Yu; Qin Lu; Jing Xie; Chao Fang; Hong-Zhuan Chen (2278-2292).
Antiangiogenic cancer therapy can be achieved through the targeted delivery of antiangiogenic agents to the endothelial cells of tumor neovasculature. In the present study, we developed a drug delivery system (DDS), nanoparticles conjugated with K237-(HTMYYHHYQHHL) peptides for tumor neovasculature targeting drug delivery. Paclitaxel, a chemotherapeutic agent with potent antiangiogenic activity, was used as a prototype drug. We synthesized the aldehyde poly(ethylene glycol)–poly(lactide) (aldehyde-PEG–PLA) block copolymer by ring opening polymerization. The nanoparticles loading paclitaxel (PTX-NP) were fabricated using the O/W emulsion and evaporation technique. K237 ligand, a peptide that can bind to the KDR receptors predominantly expressed on the surface of tumor neovasculature endothelial cells with high affinity and specificity and inhibit the VEGF-KDR angiogenic signal pathway, was conjugated to the aldehyde group of PEG chain using the N-terminal PEGylation technique. The K237 conjugated paclitaxel-loaded nanoparticles (K237-PTX-NP) had a hydrodynamic diameter of 150 nm. The K237 density on nanoparticle surface was 474 and the mean distance between two neighboring PEG chains linked to K237 peptide was 12 nm. The K237 conjugated nanoparticles could be significantly internalized by human umbilical vein endothelial cells (HUVEC) through the K237-KDR interaction, and this facilitated uptake led to the expected enhanced antiangiogenic activity shown by HUVEC proliferation, migration and tube formation compared to cells treated with the commercial formulation Taxol® and PTX-NP. The long-circulating property and the K237 ligand of K237-PTX-NP warranted rapid, long-term, and accurate in vivo tumor neovasculature targeting, and thereafter the significant apoptosis of tumor neovasculature endothelial cells and necrosis of tumor tissues of MDA-MB-231 breast tumors implanted in female BLAB/c nude mice. This nanoparticulate DDS offers a new strategy for paclitaxel chemotherapy application and it could also be used to carry other chemotherapeutic drugs, genes, and proteins with antiangiogenic activity for antiangiogenic cancer therapy.
Keywords: Nanoparticles; Paclitaxel; K237; KDR; Tumor neovasculature; Antiangiogenic cancer therapy;

Graft and diblock copolymer multifunctional micelles for cancer chemotherapy and imaging by Hsieh-Chih Tsai; Wei-Hsiang Chang; Chun-Liang Lo; Cheng-Hung Tsai; Che-Hau Chang; Ta-Wei Ou; Tzu-Chen Yen; Ging-Ho Hsiue (2293-2301).
Multifunctional mixed micelles that constructed from poly(HEMA-co-histidine)-g-PLA and diblock copolymer PEG–PLA with functional moiety was developed in this study. The mixed micelles had well defined core shell structure which was evaluated by TEM. The functional inner core of poly(HEMA-co-histidine)-g-PLA exhibited pH stimulate to enable intracellular drug delivery and outer shell of PEG-b-PLA with functional moiety Cy5.5 for biodistribution diagnosis and folate for cancer specific targeting were synthesized at the end of the polymer chain. The graft and diblock copolymer self assembled to nanospheres against water with an average diameter below 120 nm without doxorubicin, and an average diameter of around 200 nm when loaded with drug. From drug released study, a change in pH destroy the inner core to lead a significant doxorubicin(Dox) release from mixed micelles. Cellular uptake of folate–micelles was found to be higher than that of non-folate–micelles due to the folate-binding effect on the cell membrane, thereby providing a similar cytotoxic effect to drug only against the HeLa cell line. In vivo study revealed that specific targeting of folate–micelles exhibited cancer targeting and efficiency expression on tumor growth, indicating that multifunctional micelles prepared from poly(HEA-co-histidine)-g-PLA and folate–PEG–PLA have great potential in cancer chemotherapy and diagnosis.
Keywords: Micelles; Drug delivery; Biocompatibility; Copolymer; In vivo test;

Anti-HIF-1α antibody-conjugated pluronic triblock copolymers encapsulated with Paclitaxel for tumor targeting therapy by Hua Song; Rong He; Kan Wang; Jing Ruan; Chenchen Bao; Na Li; Jiajia Ji; Daxiang Cui (2302-2312).
Targeted uptake of nanoscale controlled release polymer micelles encapsulated with drugs represents a potential powerful therapeutic technology. Herein we reported the development of anti-HIF-1α antibody-conjugated unimolecular polymer nano micelles filled with Paclitaxel for cancer targeting therapy. Pluronic triblock copolymers(Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), PEO-block-PPO-block-PEO) P123 were functionalized with terminal carboxylic groups, and were characterized by infrared (IR) spectroscopy, nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), and differential scanning calorimetric (DSC). The amphiphilic copolymer nano micelles encapsulated with Paclitaxel were fabricated by self-assembly means, and then were conjugated with anti-HIF-1α antibody, the resultant anti-HIF-1α conjugated nano micelles filled with PTX (anti-HIF-1α-NMs-PTX nanocomposites) were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM), and incubated with stomach cancer MGC-803 cells and HDF fibroblast cells, these treated cells were analyzed by MTT and cell-ELISA. The nanocomposites composed of anti-HIF-1α conjugated nano micelles filled with CdTe quantum dots were also prepared, and incubated with stomach cancer MGC-803 cells and HDF fibroblast cells for 24 h, then were observed by fluorescent microscope. Results showed that the anti-HIF-1α-NMs-PTX nanocomposites were successfully prepared, bound with stomach cancer MGC-803 cells specifically, were internalized, and released PTX inside cancer cells, and selectively killed cancer cells. In conclusion, unique anti-HIF-1α antibody-conjugated nano micelles filled with Paclitaxel can target and selectively kill cancer cells with over-expression of HIF-1α, and has great potential in clinical tumor targeting imaging and therapy.
Keywords: Polymer nanomicelle; Carboxyl-functionalized Pluronic triblock copolymers; Paclitaxel; MTT; Quantum dots;

The purpose of this study was to develop a biodegradable polymer nerve guide that locally delivers bioactive neurotrophic factors in physiologically relevant concentrations for the period required by transected peripheral nerves to cross from the proximal to distal nerve stump. Delivery of a neurotrophic factor may enhance nerve regeneration and could potentially be used to overcome the current limitations in nerve repair across large defects. Glial Cell Line-Derived Neurotrophic Factor (GDNF) is a known promoter of axonal elongation and branching and has shown promising pre-clinical results in analysis of nerve regeneration with nerve guides. In addition, GDNF has been shown to promote Schwann cell proliferation and migration. In this study we have created a double-walled microsphere delivery system for bioactive GDNF with a sustained release profile >50 days in vitro. Microspheres were incorporated within degradable poly(caprolactone) nerve guides in a reproducible distribution. Implantation of nerve guides across a 1.5 cm defect in a rat sciatic nerve gap resulted in an increase in tissue integration in both the proximal and distal segments of the lumen of the nerve guide after 6 weeks. In addition, transverse sections of the distal region of the explanted guides showed the presence of Schwann cells while none were detectable in negative control guides. Migration of Schwann cells to double-walled microspheres indicated that bioactive GDNF was encapsulated and delivered to the internal environment of the nerve guide. Because GDNF increased tissue formation within the nerve guide lumen and also promoted the migration and proliferation of Schwann cells, the nerve guides presented within this study show promise toward the development of an off-the-shelf product alternative that promotes nerve regeneration beyond that capable with currently available nerve guides.
Keywords: Double-walled microspheres; Nerve guide; Nerve conduit; Poly(caprolactone); GDNF;

Artificial nerve conduits (NC) can be used as an alternative to autologous nerve grafts to enhance the repair of small nerve gaps. Current NC lack adequate molecular and structural functionalities. Thus, we developed silk fibroin NC (SF NC) that were loaded with glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) and topographically functionalized with aligned and non-aligned SF nanofibers. The SF NC were produced from fully functionalized SF membranes on which initial experiments were performed. DRG (dorsal root ganglions) sensory neurons and spinal cord (SpC) motor neurons, both from chicken embryos, exhibited an augmented length and rate of axonal outgrowth parallel to the aligned nanofibers. In addition, glial cells from DRG proliferated and migrated in close association and even slightly ahead of the outgrowing axons. On the contrary, axonal and glial growth was slower and randomly oriented on non-aligned nanofibers. The DRG and SpC explants were also inserted into the lumen of the finished SF NC. The unidirectional orientation of axo-glial outgrowth from the explants evidenced the preservation of the trophic and topographical functionalities in the SF NC. Bioactive GDNF and NGF were released in vitro from SF NC over 4 weeks. Thus, the developed functionalized SF NC hold promise to enhance functional recovery of injured peripheral nerves.
Keywords: Nanofibers; DRG; Spinal cord; Glial guidance;

Cyclodextrin-based device coatings for affinity-based release of antibiotics by Thimma R. Thatiparti; Andrew J. Shoffstall; Horst A. von Recum (2335-2347).
Cyclodextrin-based hydrogels were synthesized to create robust networks with tunable mechanical properties capable of serving as device coatings. The CD networks were able to swell and load drug in aqueous and organic solvents. The rheological properties of the swollen gels were investigated using stress and frequency sweeps, with both demonstrating high storage modulus, indicating strong elastic gels. The ability of the gels to swell in numerous solvents allowed for the separate loading and release of different antibiotic drug molecules with varying hydrophilicities. Based on FTIR and TGA studies, each drug was found to form an inclusion complex with CD. For comparison, dextran gels were prepared similarly. As expected for affinity-based mechanisms, the release of drugs from the CD-based gels was slower than diffusion-based release from the dextran gels, and could be sustained for more than 200 h. Coating potential was tested by coating two different medical devices: metal screws and polymer meshes. The meshes were characterized by SEM, revealing that CD-based coatings resulted in a uniform thin film, whereas the dextran gels only partly coated the device and showed delamination. Considerably longer bactericidal activity against Staphylococcus aureus was observed for both the CD hydrogels and coatings, as compared to dextran-based ones. The slow, sustained, affinity-based release of antibiotics from the CD-based networks reflects their potential as a delivery platform.
Keywords: Hydrogel; Polysaccharide; Drug delivery; Controlled release; Affinity; Antibiotic;

Controlling the release of peptide antimicrobial agents from surfaces by Anita Shukla; Kathleen E. Fleming; Helen F. Chuang; Tanguy M. Chau; Christopher R. Loose; Gregory N. Stephanopoulos; Paula T. Hammond (2348-2357).
Medical conditions are often exacerbated by the onset of infection caused by hospital dwelling bacteria such as Staphylococcus aureus. Antibiotics taken orally or intravenously can require large and frequent doses, further contributing to the sharp rise in resistant bacteria observed over the past several decades. These existing antibiotics are also often ineffective in preventing biofilm formation, a common cause of medical device failure. Local delivery of new therapeutic agents that do not allow bacterial resistance to occur, such as antimicrobial peptides, could alleviate many of the problems associated with current antibacterial treatments. By taking advantage of the versatility of layer-by-layer assembly of polymer thin films, ponericin G1, an antimicrobial peptide known to be highly active against S. aureus, was incorporated into a hydrolytically degradable polyelectrolyte multilayer film. Several film architectures were examined to obtain various drug loadings that ranged from 20 to 150 μg/cm2. Release was observed over approximately ten days, with varying release profiles, including burst as well as linear release. Results indicated that film-released peptide did not suffer any loss in activity against S. aureus and was able to inhibit bacteria attachment, a necessary step in preventing biofilm formation. Additionally, all films were found to be biocompatible with the relevant wound healing cells, NIH 3T3 fibroblasts and human umbilical vein endothelial cells. These films provide the level of control over drug loading and release kinetics required in medically relevant applications including coatings for implant materials and bandages, while eliminating susceptibility to bacterial resistance.
Keywords: Layer-by-layer assembly; Polyelectrolyte multilayer film; Antimicrobial peptides; Local drug delivery;

Bicalutamide is the most widely used non-steroidal antiandrogen for treating early stage prostate cancer, but suffers variable oral absorption due to its limited aqueous solubility. Thus, our objective was to synthesize novel biodegradable copolymers for the systemic micellar delivery of bicalutamide. Flory–Huggins interaction parameter (χ FH) was used to assess compatibility between bicalutamide and poly(l-lactide) or poly(carbonate-co-lactide) polymer pairs. Polyethylene glycol-b-poly(carbonate-co-lactide) [PEG-b-P(CB-co-LA)] copolymers were synthesized and characterized by NMR and gel permeation chromatography. These micelles had average diameter of 100 nm and had a smooth surface and distinct spherical shape. Drug loading studies revealed that adding the carbonate monomer could increase bicalutamide loading. Among the series, drug loading of micelles formulated with PEG-b-P(CB-co-LA) copolymer containing 20 mol% carbonate was about four-fold higher than PEG-b-PLLA and aqueous solubility of bicalutamide increased from 5 to 4000 μg/mL. CMC values for PEG-b-P(CB-co-LA) copolymers was up to 10-fold lower than those of PEG-b-PLLA. In vitro release experiments showed PEG-b-P(CB-co-LA) copolymers to be more efficient in sustaining the release of bicalutamide compared to PEG-b-PLLA. Bicalutamide-loaded PEG-b-P(CB-co-LA) micelles showed significant inhibition of LNCaP cell growth in a dose-dependent manner which was similar to the methanol solution of free drug.
Keywords: Micelles; Copolymer; Polyethylene glycol; Polycarbonate; Poly lactic acid; Bicalutamide;

The effects of mixed MPEG–PLA/Pluronic® copolymer micelles on the bioavailability and multidrug resistance of docetaxel by Chao-Feng Mu; Prabagar Balakrishnan; Fu-De Cui; Yong-Mei Yin; Yong-Bok Lee; Han-Gon Choi; Chul Soon Yong; Suk-Jae Chung; Chang-Koo Shim; Dae-Duk Kim (2371-2379).
A mixed micelle that comprised of MPEG–PLA (MPP) and Pluronic® copolymers was developed for enhanced bioavailability and to overcome multidrug resistance of docetaxel in cancer therapy. The mixed micelles that sufficiently solubilized docetaxel were evaluated for the effect of Pluronic® copolymers weight ratio on the mixed micelles with respect to drug loading and drug release. In vitro, cell viability and cytotoxicity studies in KB and KBv cells revealed that the mixed micellar formulations were more potent than the commercial docetaxel formulation (Taxotere®). In vivo pharmacokinetics study in rats showed that the mixed micelles significantly enhanced the bioavailability of docetaxel (3.6 fold) than Taxotere®. Moreover, antitumor activity assessed in KBv cancer xenograft BALB/C nude mice models showed that the mixed micelles significantly reduced the tumor size than the control (Taxotere®). Clear differences in the intracellular uptake of docetaxel between MPP and mixed micelles were observed using confocal laser scanning microscopy. This study presents not only a new micelle structure for a diblock–triblock copolymer system, but also a method for enhanced bioavailability of docetaxel and to overcome some of the limitations on its multidrug resistance in cancer therapy.
Keywords: Docetaxel; Mixed micelles; Solubilization; Enhanced bioavailability; Multidrug resistance;

Nanoscaled materials are normally engulfed in endosomes by energy-dependent endocytosis and fail to access the cytosolic cell machinery. Although some biomolecules may penetrate non-endocytically or fuse with plasma membranes without overt membrane disruption, to date no synthetic macromolecule of comparable size has been shown to exhibit this property. Here, we discovered mechanism of direct cell membrane penetration using synthetic phospholipid polymers. These water-soluble amphiphilic phospholipid polymers enter the cytoplasm of living mammalian cells in vitro within a few minutes without overt bilayer disruption even under conditions where energy-dependent endocytic uptakes are blocked. Furthermore, targeted cytosolic distribution to cell organelles was achieved by selecting specific fluorescent tags to the polymers. Thus, the phospholipid polymers can provide a new way of thinking about access to the cellular interior, namely direct membrane penetration.
Keywords: Phospholipid polymer; Amphiphilicity; Non-endocytosis; Drug delivery; Molecular imaging;

Enhanced drug targeting by attachment of an anti αv integrin antibody to doxorubicin loaded human serum albumin nanoparticles by Sylvia Wagner; Florian Rothweiler; Marion G. Anhorn; Daniel Sauer; Iris Riemann; Eike C. Weiss; Alisa Katsen-Globa; Martin Michaelis; Jindrich Cinatl; Daniel Schwartz; Jörg Kreuter; Hagen von Briesen; Klaus Langer (2388-2398).
Specific transport of anti-cancer drugs into tumor cells may result in increased therapeutic efficacy and decreased adverse events. Expression of αvβ3 integrin is enhanced in various types of cancer and monoclonal antibodies (mAbs) directed against αvβ3 integrins hold promise for anti-cancer therapy. DI17E6 is a monoclonal antibody directed against αv integrins that inhibits growth of melanomas in vitro and in vivo and inhibits angiogenesis due to interference with αvβ3 integrins. Here, DI17E6 was covalently coupled to human serum albumin nanoparticles. Resulting nanoparticles specifically targeted αvβ3 integrin positive melanoma cells. Moreover, doxorubicin loaded DI17E6 nanoparticles showed increased cytotoxic activity in αvβ3-positive melanoma cells than the free drug. Therefore, DI17E6-coupled human serum albumin nanoparticles represent a potential delivery system for targeted drug transport into αvβ3-positive cells.
Keywords: Albumin; Chemotherapy; Drug delivery; ECM (extracellular matrix); Integrin; Nanoparticles;

Effect of delivery of MMP inhibitors from PDMS as a model IOL material on PCO markers by Diana Morarescu; Judy A. West-Mays; Heather D. Sheardown (2399-2407).
Posterior capsule opacification (PCO) or secondary cataract formation, following intraocular lens implantation, is a significant complication affecting an estimated 28% of cataract patients. Matrix metalloproteinases (MMPs) have been demonstrated to play a role in the formation of anterior subcapsular cataracts and it has been shown that the presence of MMP inhibitors (MMPI) decreases subcapsular cataract formation ex vivo. Since the mechanisms responsible for anterior subcapsular cataract formation and posterior capsule opacification are similar, it is reasonable to suggest that MMP inhibitors may also mitigate PCO. One of the most effective ways of delivering the inhibitors may be from the implanted intraocular lens (IOL) material itself. In the current work, delivery of three different MMP inhibitors from silicone rubber as a model IOL material was examined. Loading methods were developed which allowed continuous release of active MMPI for periods of over 5 months in some cases. Reduced migration rates were observed in human lens epithelial cells in vitro, suggesting that an effect on PCO may be possible. While further studies are necessary to tune the systems to achieve the desired rates of release, this work demonstrates that delivery of MMPI from silicone IOL materials has the potential to decrease the incidence of PCO.
Keywords: Matrix metalloproteinase inhibitor; Polydimethylsiloxane; Posterior capsule opacification; Controlled drug release; Lens epithelial cells; Migration;

Co-delivery of siRNA and paclitaxel into cancer cells by biodegradable cationic micelles based on PDMAEMA–PCL–PDMAEMA triblock copolymers by Caihong Zhu; Sooyeon Jung; Sibin Luo; Fenghua Meng; Xiulin Zhu; Tae Gwan Park; Zhiyuan Zhong (2408-2416).
Biodegradable cationic micelles were prepared from PDMAEMA–PCL–PDMAEMA triblock copolymers and applied for the delivery of siRNA and paclitaxel into cancer cells. PDMAEMA–PCL–PDMAEMA copolymers were readily obtained by reversible addition-fragmentation chain transfer (RAFT) polymerization of dimethylaminoethyl methacrylate (DMAEMA) using CPADN–PCL–CPADN (CPADN: 4-cyanopentanoic acid dithionaphthalenoate; PCL: 3600 Da) as a macro-RAFT agent. The molecular weights of PDMAEMA blocks, controlled by monomer/CPADN–PCL–CPADN mole ratios, varied from 2700, 4800 to 9100 (denoted as polymer 1, 2 and 3, respectively). These triblock copolymers formed nano-sized micelles in water with positive surface charges ranging from +29.3 to +35.5 mV. Both micelles 1 and 2 revealed a low cytotoxicity. Gel retardation assay showed that micelles 1 and 2 could effectively complex with siRNA at and above N/P ratios of 4/1 and 2/1, respectively. Notably, GFP siRNA complexed with micelle 1 exhibited significantly enhanced gene silencing efficiency as compared to that formulated with 20 kDa PDMAEMA or 25 kDa branched PEI in GFP-expressed MDA-MB-435-GFP cells. Moreover, micelle 1 loaded with paclitaxel displayed higher drug efficacy than free paclitaxel in PC3 cells, due to most likely improved cellular uptake. The combinatorial delivery of VEGF siRNA and paclitaxel showed an efficient knockdown of VEGF expression. Confocal laser scanning microscope studies on GFP siRNA complexed with nile red-loaded micelle revealed that nile red was delivered into GFP-expressed MDA-MB-435-GFP cells and that GFP expression was significantly inhibited. These results demonstrated that cationic biodegradable micelles are highly promising for the combinatorial delivery of siRNA and lipophilic anti-cancer drugs.
Keywords: Micelles; siRNA; Gene delivery; Paclitaxel; Drug delivery;

Targeted nonviral delivery vehicles to neural progenitor cells in the mouse subventricular zone by Ester J. Kwon; Jurate Lasiene; Berit E. Jacobson; In-Kyu Park; Philip J. Horner; Suzie H. Pun (2417-2424).
Targeted gene therapy can potentially minimize undesirable off-target toxicity due to specific delivery. Neuron-specific gene delivery in the central nervous system is challenging because neurons are non-dividing and also outnumbered by glial cells. One approach is to transfect dividing neural stem and progenitor cells (NSCs and NPCs, respectively). In this work, we demonstrate cell-specific gene delivery to NPCs in the brains of adult mice using a peptide-modified polymeric vector. Tet1, a 12-amino acid peptide which has been shown to bind specifically to neuronal cells, was utilized as a neuronal targeting ligand. The cationic polymer polyethylenimine (PEI) was covalently modified with polyethylene glycol (PEG) for in vivo salt stability and Tet1 for neuron targeting to yield a Tet1–PEG–PEI conjugate. When plasmid DNA encoding the reporter gene luciferase was complexed with Tet1–PEG–PEI and delivered in vivo via an injection into the lateral ventricle, Tet1–PEG–PEI complexes mediated increased luciferase expression levels in brain tissue when compared to unmodified PEI–PEG complexes. In addition, cells transfected by Tet1–PEG–PEI complexes were found to be exclusively adult NPCs whereas untargeted PEG–PEI complexes were found to transfect a heterogenous population of cells. Thus, we have demonstrated targeted, nonviral delivery of nucleic acids to adult NPCs using the Tet1 targeting ligand. These materials could potentially be used to deliver therapeutic genes for the treatment of neurodegenerative diseases.
Keywords: Gene therapy; Nanoparticle; Peptide;

Multifunctional core-shell polymeric nanoparticles for transdermal DNA delivery and epidermal Langerhans cells tracking by Po-Wei Lee; Sheng-Hsiang Hsu; Jin-Sheng Tsai; Fu-Rong Chen; Pei-Jai Huang; Cherng-Jyh Ke; Zi-Xian Liao; Chun-Wen Hsiao; Hao-Jan Lin; Hsing-Wen Sung (2425-2434).
Skin is a highly immune-reactive tissue containing abundant antigen-presenting cells such as Langerhans cells (LCs), and thus is a favorable site for DNA immunization. This study developed a multifunctional core-shell nanoparticle system, which can be delivered transdermally into the epidermis via a gene gun, for use as a DNA carrier. The developed nanoparticles comprised a hydrophobic PLGA core and a positively-charged glycol chitosan (GC) shell. The core of the nanoparticles was used to load fluorescent quantum dots (QDs) for ultrasensitive detection of Langerhans cell migration following transdermal delivery, while a reporter gene was electrostatically adsorbed onto the GC shell layer of the nanoparticles. Results of fluorescence spectrophotometry, transmission electron microscopy, energy dispersive X-ray analysis, and X-ray diffraction measurement confirmed that the prepared nanoparticles had a core-shell structure with QDs in their core area. The surface charge of nanoparticles depended strongly on pH environment, enabling the intracellular release of the loaded DNA via a pH-mediated mechanism. Using a mouse model, this study demonstrated that bombardment of nanoparticles transfected DNA directly into LCs present in the epidermis; the transfected LCs then migrated and expressed the encoded gene products in the skin draining lymph nodes. These observation results suggest that the developed nanoparticle system is suitable for monitoring and fine-tuning important functional aspects of the immune system, in conjunction with the loaded fluorescence, and thus has potential for use in immunotherapy and vaccine development.
Keywords: Quantum dots; Cell tracking; Gene delivery; Immunotherapy; Stimuli-responsive materials;

The therapeutic efficiency of FP-PEA/TAM67 gene complexes via folate receptor-mediated endocytosis in a xenograft mice model by Rohidas B. Arote; Soon-Kyung Hwang; Hwang-Tae Lim; Tae-Hee Kim; Dhananjay Jere; Hu-Lin Jiang; You-Kyoung Kim; Myung-Haing Cho; Chong-Su Cho (2435-2445).
To circumvent carrier related obstacles, we developed a biodegradable, folate conjugated poly (ester amine) (FP-PEA) that mediates high level folate receptor (FR) mediated endocytosis in vitro as well as in vivo. We report the efficacy of a therapeutic strategy that combines the potency of FP-PEA based on polycaprolactone (PCL) and low molecular weight polyethylenimine (LMW-PEI) with the tumor targeting potential of receptor mediated endocytosis. When tested on cells in culture, FP-PEA was found to retain high affinity for FR-positive cells compared with PEA without folate moiety (P-PEA). The FR specific activity of FP-PEA was drastically decreased in the presence of an excess free folic acid and very less significant transfection was detected against FR-negative cells. FP-PEA showed marked anti-tumor activity against FR-positive human KB tumors in nude mice with no evidence of toxicity during and after therapy using TAM67 gene. Furthermore, the therapeutic effect occurred in the apparent absence of weight loss or noticeable tumor apoptosis. In contrast, no significant anti-tumor activity was observed in P-PEA treated mice which were co dosed with an excess of FR, thus demonstrating the target specific gene delivery. Furthermore, anti-tumor activity with PEA without folic acid moiety (P-PEA) proved not to be effective against xenograft mice model with KB cells when administered at the same dose to that of FP-PEA. Taken together, these results indicate that FP-PEA is highly effective gene carrier capable of producing therapeutic benefit in xenograft mice model without any sign of toxicity.
Keywords: Biodegradable; Poly(ester amine); Folate receptor-mediated endocytosis; Angiogenesis;

Mechanical stimuli are one of the factors that influence tissue differentiation. In the development of biomaterials for bone tissue engineering, mechanical stimuli and formation of a vascular network that transport oxygen to cells within the pores of the scaffolds are essential. Angiogenesis and cell differentiation have been simulated in scaffolds of regular porosity; however, the dynamics of differentiation can be different when the porosity is not uniform. The objective of this study was to investigate the effect of the mechanical stimuli and the capillary network formation on cell differentiation within a scaffold of irregular morphology. A porous scaffold of calcium phosphate based glass was used. The pores and the solid phase were discretized using micro computed tomography images. Cell activity was simulated within the interconnected pore domain of the scaffold using a lattice modeling approach. Compressive strains of 0.5 and 1% of total deformation were applied and two cases of mesenchymal stem cells initialization (in vitro seeding and in vivo) were simulated. Similar capillary networks were formed independently of the cell initialization mode and the magnitude of the mechanical strain applied. Most of vessels grew in the pores at the periphery of the scaffolds and were blocked by the walls of the scaffold. When 0.5% of strain was applied, 70% of the pore volume was affected by mechano-regulatory stimuli corresponding to bone formation; however, because of the lack of oxygen, only 40% of the volume was filled with osteoblasts. 40% of volume was filled with chondrocytes and 3% with fibroblasts. When the mechanical strain was increased to 1%, 11% of the pore volume was filled with osteoblasts, 59% with chondrocytes, and 8% with fibroblasts. This study has shown the dynamics of the correlation between mechanical load, angiogenesis and tissue differentiation within a scaffold with irregular morphology.
Keywords: Tissue engineering; Calcium phosphates; Mechanoregulation; Micro computer tomography; Finite element modeling;