Biomaterials (v.31, #12)

Anisotropic dynamic changes in the pore network structure, fluid diffusion and fluid flow in articular cartilage under compression by George W. Greene; Bruno Zappone; Olle Söderman; Daniel Topgaard; Gabriel Rata; Hongbo Zeng; Jacob N. Israelachvili (3117-3128).
A compression cell designed to fit inside an NMR spectrometer was used to investigate the in situ mechanical strain response, structural changes to the internal pore structure, and the diffusion and flow of interstitial water in full-thickness cartilage samples as it was deforming dynamically under a constant compressive load (pressure). We distinguish between the hydrostatic pressure acting on the interstitial fluid and the pore pressure acting on the cartilage fibril network. Our results show that properties related to the pore matrix microstructure such as diffusion and hydraulic conductivity are strongly influenced by the hydrostatic pressure in the interstitial fluid of the dynamically deforming cartilage which differ significantly from the properties measured under static i.e. equilibrium loading conditions (when the hydrostatic pressure has relaxed back to zero). The magnitude of the hydrostatic fluid pressure also appears to affect the way cartilage's pore matrix changes during deformation with implications for the diffusion and flow-driven fluid transport through the deforming pore matrix. We also show strong evidence for a highly anisotropic pore structure and deformational dynamics that allows cartilage to deform without significantly altering the axial porosity of the matrix even at very large strains. The insensitivity of the axial porosity to compressive strain may be playing a critical function in directing the flow of pressurized interstitial fluid in the compressed cartilage to the surface, to support the load, and provide a protective interfacial fluid film that ‘weeps’ out from the deforming tissue and thereby enhances the (elasto)hydrodynamic efficacy of sliding joints. Our results appear to show a close synergy between the structure of cartilage and both the hydrodynamic and boundary lubrication mechanisms.
Keywords: Cartilage; Arthritis; Diffusion; Compression; Lubrication; Porosity;

A functionalizable polyester with free hydroxyl groups and tunable physiochemical and biological properties by Zhengwei You; Haiping Cao; Jin Gao; Paul H. Shin; Billy W. Day; Yadong Wang (3129-3138).
Polyesters with free functional groups allow facile modifications with biomolecules, which can lead to versatile biomaterials that afford controlled interactions with cells and tissues. Efficient synthesis of functionalizable polyesters (Functionalizable polymer is defined as a polymer with functional groups that readily react with biomolecules and functionalized biomaterial as one already modified with biomolecules.) is still a challenge that greatly limits the availability and widespread applications of biofunctionalized synthetic polymers. Here we report a simple route to prepare a functionalizable polyester, poly(sebacoyl diglyceride) (PSeD) bearing free hydroxyl groups. The key synthetic step is an epoxide ring-opening polymerization, instead of the traditional polycondensation that produces poly(glycerol sebacate) (PGS) (Wang YD, Ameer GA, Sheppard BJ, Langer R. A tough biodegradable elastomer. Nat Biotechnol 2002;20(6):602–6). PSeD has a more defined structure with mostly linear backbone, more free hydroxyl groups, higher molecular weight, and lower polydispersity than PGS. Crosslinking PSeD with sebacic acid yields a polymer five times tougher and more elastic than cured PGS. PSeD exhibits good cytocompatibility in vitro. Furthermore, functionalization by glycine proceeds with high efficiency. This versatile synthetic platform can offer a large family of biodegradable, functionalized polymers with tunable physiochemical and biological properties useful for a wide range of biomedical applications.
Keywords: Functionalizable polyester; Elastomer;

The ability to manipulate and control the surface properties, without altering the substrate properties, is of crucial importance in the designing of biomedical materials. In this work, surface-initiated atom transfer radical polymerization (ATRP) is employed to tailor the functionality of polycaprolactone (PCL) film surfaces in a well-controlled manner. Functional polymer brushes of glycidyl methacrylate (GMA) were prepared via surface-initiated ATRPs from the PCL film surfaces. Kinetics study revealed that the chain growth from the PCL films was consistent with a controlled process. The dense and reactive epoxide groups of the grafted P(GMA) brushes were used for the direct coupling of cell-adhesive collagen and Arg-Gly-Asp-Ser (RGDS) peptides to improve the cell-adhesion properties of the PCL film surface. These modified surfaces were evaluated by culturing of a cell line, 3T3 fibroblasts. The cell attachment and proliferation were improved remarkably on the collagen (or RGDS) functionalized PCL film surfaces. The adhesion results also indicated that the collagen-coupled PCL film surface is better for the cell-adhesion process. With the versatility of surface-initiated ATRP and the good biocompatibility nature of biomolecules, the PCL films with desirable surface functionalities can be precisely tailored to cater to various biomedical applications.
Keywords: Surface functionalization; PCL film; ATRP; GMA; Collagen; RGD;

Negative surface charge density gradients were prepared on fused silica slides using selective oxidation of a 3-mercaptopropyltrimethoxysilane (MTS) monolayer converting surface thiol groups (–SH) into negatively charged sulfonate (−SO3 ) groups. The sulfonate-to-thiol gradient samples were characterized by water contact angle and electron spectroscopy for chemical analysis (ESCA). Gradients were pre-adsorbed with proteins from three different solutions: platelet free plasma (PFP), fibrinogen, or albumin in phosphate buffered saline (PBS). Washed platelets were perfused over gradient samples in a parallel plate flow chamber and platelet adhesion was measured across the gradients using differential interference contrast (DIC) microscopy. Gradients pre-adsorbed with PFP showed adhesion contrast inversely related to the negative surface charge density. The magnitude of the adhesion contrast along the gradient was also dependent on PFP concentration. Gradients pre-adsorbed with fibrinogen showed an adhesion maximum in the center of the gradient region. Albumin coating of the gradients resulted in low overall platelet adhesion with increased adhesion in regions of high negative charge density. The effect of gradient orientation with respect to the flow was also investigated. Gradients pre-adsorbed with 10% PFP showed different adhesion contrast when the platelets were perfused in opposite directions. This suggests that platelet adhesion is, in addition to responding to the local surface properties, also dependent on the upstream conditions.
Keywords: Platelet adhesion; Surface gradient; Fibrinogen; Albumin; Plasma proteins; Blood compatibility;

The application of 3D micropatterning of agarose substrate for cell culture and in situ comet assays by Emilie Mercey; Patricia Obeïd; Denise Glaise; Maria-Luisa Calvo-Muñoz; Christiane Guguen-Guillouzo; Brigitte Fouqué (3156-3165).
We report the fabrication of a 3D micropatterned agarose substrate that enables the culture of single or multiple cells. Patterning was performed on dried agarose using deep UV irradiation leading to 6-μm-deep micropatterns of 25–70 μm in diameter. Cell adhesion was facilitated by the specific grafting of ECM (extra cellular matrix) proteins such as fibronectin into the micropatterns. We show that the pattern size induced the adhesion of one or more cells, thus allowing precise control of the cell number used in the assay, and that cells proliferated similarly as in standard culture conditions. Moreover, cell polarity appeared well preserved on this substrate, so polarized cells like hepatoma HepaRG cells might maintain their differentiation status and act as primary human hepatocytes for hepatotoxicity testing. These 3D patterned culture slides have been successfully used for in situ comet assays and there is evidence that the genotoxic effects of sub-cytotoxic concentrations of drugs could be analyzed in a large number of single HeLa cells. Coupled with the parallel-based design of the 3D micropatterning, which allows automated image analysis, these results strongly indicate that this new cell array system is suitable for high-throughput cytotoxicity and genotoxicity screening applications.
Keywords: 3D micropatterning; Agarose; Cell array; Comet assay; HepaRG hepatocytes; Genotoxicity;

Cell encapsulation has long been investigated as a means to achieve transplant immunoprotection as it creates a physical barrier between allograft tissue and host immune cells. Encapsulation with passive barrier materials alone, however, is generally insufficient to protect donor tissue from rejection, because small cytotoxic molecules produced by activated T cells can diffuse readily into the capsule and mediate allograft death. As a means to provide bioactive protection for polymeric encapsulation devices, we investigated a functionalized polymeric coating that mimics a natural T cell regulation pathway. T cells are regulated in vivo via Fas, a well-known ‘death receptor,’ whereby effector cells express Fas ligand and elicit T cell apoptosis upon binding the Fas receptor on a T cell surface. Anti-Fas antibodies are capable of replicating this effect and induce T cell apoptosis in solution. Here, an iniferter-based living radical polymerization was utilized to fabricate surface-anchored polymer chains containing poly(ethylene glycol) with covalently incorporated pendant anti-Fas antibody. Using this reaction mechanism, we demonstrate fabrication conditions that yield surface densities in excess of 1.5 ng/cm2 of incorporated therapeutic, as detected by ELISA. Additionally, we show that coatings containing anti-Fas antibody induced significant T cell apoptosis, 21 ± 2% of cells, after 24 h. Finally, the incorporation of a T cell adhesion ligand, intracellular adhesion molecule-1, along with anti-Fas antibody, yielded even higher levels of apoptosis, 34 ± 1% of T cells, compared to either signal alone.
Keywords: Apoptosis; Immunomodulation; Lymphocyte; Photopolymerization; Surface modification;

The incorporation of strontium and zinc into a calcium–silicon ceramic for bone tissue engineering by Hala Zreiqat; Yogambha Ramaswamy; Chengtie Wu; Angelo Paschalidis; ZuFu Lu; Barbara James; Oliver Birke; Michelle McDonald; David Little; Colin R. Dunstan (3175-3184).
In this study we developed novel scaffolds through the controlled substitution and incorporation of strontium and zinc into a calcium–silicon system to form Sr–Hardystonite (Sr–Ca2ZnSi2O7, Sr–HT). The physical and biological properties of Sr–HT were compared to Hardystonite (Ca2ZnSi2O7) [HT]. We showed that Sr–HT scaffolds are porous with interconnected porous network (interconnectivity: 99%) and large pore size (300–500 μm) and an overall porosity of 78%, combined with a relatively high compressive strength (2.16 ± 0.52 MPa). These properties are essential for enhancing bone ingrowth in load-bearing applications. Sr–HT ceramic scaffolds induced the attachment and differentiation of human bone derived cells (HOB), compared to that for the HT scaffolds. Sr–HT scaffolds enhanced expression of alkaline phosphatase, Runx-2, osteopontin, osteocalcin and bone sialoprotein. The in vivo osteoconductivity of the scaffolds was assessed at 3 and 6 weeks following implantation in tibial bone defects in rats. Histological staining revealed rapid new growth of bone into the pores of the 3D scaffolds with the Sr–HT and HT, relative to the β-tricalcium phosphate (β-TCP). In vivo, HT and Sr–HT produced distinct differences in the patterns of degradation of the materials, and their association with TRAP positive osteoclast-like cells with HT appearing more resistant compared to both Sr–HT and β-TCP.
Keywords: Strontium; Zinc; CaSiO3; Scaffolds; Bone regeneration; Orthopaedic;

A 3-D cardiac muscle construct for exploring adult marrow stem cell based myocardial regeneration by Mani T. Valarmathi; Richard L. Goodwin; John W. Fuseler; Jeffrey M. Davis; Michael J. Yost; Jay D. Potts (3185-3200).
Adult bone marrow stromal cells (BMSCs) are capable of differentiating into cardiomyocyte-like cells in vitro and contribute to myocardial regeneration in vivo. Consequently, BMSCs may potentially play a vital role in cardiac repair and regeneration. However, this concept has been limited by inadequate and inconsistent differentiation of BMSCs into cardiomyocytes along with poor survival and integration of neo-cardiomyocytes after implantation into ischemic myocardium. In order to overcome these barriers and to explore adult stem cell based myocardial regeneration, we have developed an in vitro model of three-dimensional (3-D) cardiac muscle using rat ventricular embryonic cardiomyocytes (ECMs) and BMSCs. When ECMs and BMSCs were seeded sequentially onto a 3-D tubular scaffold engineered from topographically aligned type I collagen-fibers and cultured in basal medium for 7, 14, 21, or 28 days, the maturation and co-differentiation into a cardiomyocyte lineage was observed. Phenotypic induction was characterized at morphological, immunological, biochemical and molecular levels. The observed expression of transcripts coding for cardiomyocyte phenotypic markers and the immunolocalization of cardiomyogenic lineage-associated proteins revealed typical expression patterns of neo-cardiomyogenesis. At the biochemical level differentiating cells exhibited appropriate metabolic activity and at the ultrastructural level myofibrillar and sarcomeric organization were indicative of an immature phenotype. Our 3-D co-culture system sustains the ECMs in vitro continuum of differentiation process and simultaneously induces the maturation and differentiation of BMSCs into cardiomyocyte-like cells. Thus, this novel 3-D co-culture system provides a useful in vitro model to investigate the functional role and interplay of developing ECMs and BMSCs during cardiomyogenic differentiation.
Keywords: Bone marrow stromal cells; Mesenchymal stem cells; Embryonic cardiac myocytes; Myocardial regeneration; Cardiac tissue engineering;

Injectable scaffolds held great promise for the reconstruction of bone defects. We prepared an injectable composite named PTC by combining TCP/chitosan (TC) with platelet-rich plasma (PRP). The objective of this study was to investigate the composite's mechanical and biological properties. First, we found that the introduction of PRP in TC showed no adverse effect on mechanical strength and that there were no significant differences in compressive strength between PTC and TC (P  > 0.05). In cell culture experiments, both cell count and alkaline phosphatase (ALP) activity measurements of PTC were higher than those of TC. The high levels of Cbfa1 and TGF-β were detected early in PTC-induced MSCs by reverse transcriptase polymerase chain reaction. Bone formation following expression of collagen type I, osteocalcin, osteonectin and calcium nodules was also observed in PRP-induced MSCs. Finally, this composite was injected into the tibial bone defect in a goat model, and its ability to induce bone regeneration was observed. Sixteen weeks after the implantation of this composite, the tibial defects had completely recuperated, with significantly better formation of mature bone and less residual material than in the control. These results demonstrate that our composite, with its concomitant mechanical strength, biocompatibility, and osteoinductive properties, has significant potential as an injectable material for the treatment of bone defects.
Keywords: Platelet-rich plasma; Tricalcium phosphate; Chitosan; Bone regeneration;

The performance of bone marrow mesenchymal stem cell – Implant complexes prepared by cell sheet engineering techniques by Wei Zhou; Chun Han; Yingliang Song; Xingrong Yan; Dehua Li; Zhiguo Chai; Zhihong Feng; Yan Dong; Liwen Li; Xing Xie; Fulin Chen; Yimin Zhao (3212-3221).
This study investigated the hypothesis that cell sheets composed of multilayered rabbit bone marrow derived mesenchymal stem cells (MSC) could be assembled with two kinds of implants (surface-modified titanium and zirconia) for the construction of a MSC-implant. The MSC sheets were harvested from culture flasks, wrapped around implants to construct the complexes, and then cultured in osteogenic medium. The layered cell sheets integrated well with implants and remained viable, with small mineralized nodules visible on the implant surfaces for up to four weeks after culture. Cells on the implants underwent classical in vitro osteogenic differentiation with an associated elevation of alkaline phosphatase activity and bone- and vascular-related protein expression. In vivo, two kinds of cell sheet–implant complexes were transplanted under the skin of SCID mice and cultured for eight weeks. For the MSC sheet titanium implant complex, histological examination revealed that new bone tissue that formed around implants followed a predominantly endochondral pathway, exhibiting histological markers of native bone; for the MSC sheet zirconia implant complex, however, intramembranous ossification appeared to occur on the surface of the zirconia implant, as observed with typical osteocytes embedded in dense matrix and accompanied by both microvessels and marrow cavities. These findings demonstrate that MSC-implants possessing osteogenic and vascularization abilities can be produced using cell sheet engineering techniques in conjunction with routine implant materials, which provide a novel technology to modify the implant surface.
Keywords: Mesenchcymal stem cell; Zirconia; Titanium; Implants;

The healing of critical-sized femoral segmental bone defects in rabbits using baculovirus-engineered mesenchymal stem cells by Chin-Yu Lin; Yu-Han Chang; Kun-Ju Lin; Tzu-Chen Yen; Ching-Lung Tai; Chi-Yuan Chen; Wen-Hsin Lo; Ing-Tsung Hsiao; Yu-Chen Hu (3222-3230).
Management of massive segmental bone defects remains a challenging clinical problem and bone marrow-derived mesenchymal stem cells (BMSCs) hold promise for bone regeneration. To explore whether BMSCs engineered by baculovirus (an emerging gene delivery vector) can heal large bone defects, New Zealand White (NZW) rabbit BMSCs were transduced with the BMP2-expressing baculovirus or VEGF-expressing baculovirus, and co-implanted into critical-sized (10 mm) femoral segmental defects in NZW rabbits. X-ray analysis revealed that the baculovirus-engineered BMSCs not only bridged the defects at as early as week 2, but also healed the defects in 100% of rabbits (13/13) at week 4. The osteogenic metabolism, as monitored by positron emission tomography (PET) also suggested the completion of bone healing at week 8. When compared with other control groups, the BMP2/VEGF-expressing BMSCs remarkably enhanced the segmental bone repair and mechanical properties, as evidenced by micro-computed tomography (μCT), histochemical staining and biomechanical testing. The ameliorated bone healing concurred with the augmented angiogenesis. These data demonstrated, that BMSCs engineered to express BMP2 and VEGF accelerate the repair of large femoral bone defects and improve the quality of the regenerated bone, which paves an avenue to utilizing baculovirus as a vector for BMSCs modification and regenerative medicine.
Keywords: Baculovirus; Mesenchymal stem cells; Gene therapy; Segmental bone defect; Tissue engineering;

The effects of Runx2 immobilization on poly (ɛ-caprolactone) on osteoblast differentiation of bone marrow stromal cells in vitro by Ying Zhang; Xiaopei Deng; Erica L. Scheller; Tae-Geon Kwon; Joerg Lahann; Renny T. Franceschi; Paul H. Krebsbach (3231-3236).
In vivo regenerative gene therapy is a promising approach for bone regeneration and can help to address cell-source limitations through surgical implantation of osteoinductive materials and subsequent recruitment of host-derived cells. Localized viral delivery may reduce the risk of virus dispersion, enhance transduction efficiency, and reduce administration/injection dosing, which subsequently increases patient safety. In this manuscript, we present a custom-tailored strategy to immobilize adenovirus expressing runt-related transcription factor 2 (AdRunx2) by using reactive polymer coatings to enhance in vitro osteoblast differentiation of bone marrow stromal cells (BMSCs). A thin polymer film of poly[p-xylylene carboxylic acid pentafluorophenol ester-co-p-xylylene] equipped with amine-reactive active ester groups was deposited on the surface of poly (ɛ-caprolactone) (PCL) using the chemical vapor deposition (CVD) polymerization technique and then anti-adenovirus antibody was conjugated on the material with an amide chemical bond. Following antibody conjugation, AdRunx2 was conjugated to the PCL surface through antibody-antigen interaction. Osteoblast differentiation of BMSCs was induced by incubation in osteogenic medium. Alkaline phosphatase (ALP) activity, calcium deposition, and matrix mineralization were confirmed as markers of osteoblast formation. Incubation of the BMSCs in the presence of AdRunx2 modified PCL resulted in a 6.5-fold increase in ALP activity and significant increases in matrix mineralization when compared to controls. These results demonstrate that adenovirus vectors driving the expression of transcription factors can be delivered directly from biomaterials to direct cell differentiation.
Keywords: Regenerative gene therapy; Local immobilization; Osteogenesis; Bone marrow stromal cells; Biomaterials; Reactive coatings;

Cell surface receptor-specific scaffold requirements for adhesion to laminin-derived peptide–chitosan membranes by Kentaro Hozumi; Dai Otagiri; Yuji Yamada; Ayano Sasaki; Chikara Fujimori; Yuki Wakai; Tatsuya Uchida; Fumihiko Katagiri; Yamato Kikkawa; Motoyoshi Nomizu (3237-3243).
Scaffolds are used for bioengineering to regulate cellular functions. Previously, we developed laminin-derived peptide–conjugated chitosan membranes for cell engineering. Here, we determined whether changes in the chitosan scaffold altered the cellular response. When an αvβ3 integrin-binding peptide A99a (ALRGDN) was conjugated on chitosan membranes of varying density (1.5–1500 ng/mm2), cell adhesion was altered depending on the amount of chitosan. 3 or 30 ng/mm2 of the A99a-chitosan membrane effectively promoted cell attachment, cell spreading with well-organized actin stress fibers, phosphorylation of FAK Tyr397, and neurite outgrowth. In contrast, syndecan-binding peptide AG73 (RKRLQVQLSIRT) conjugated chitosan membranes density (1.5–1500 ng/mm2) promoted similar biological activities at all of the concentrations tested. These results suggest that integrin-mediated cell adhesion is sensitive to the scaffold condition. To improve the function of integrin-mediated biological activities on a large amount of scaffold, we designed an A99a/AG73 mixed peptide–chitosan membrane. The mixed peptide–chitosan membrane promoted the strongest biological activities at 150–1500 ng/mm2 of chitosan membrane. We conclude that the A99a/AG73 mixed peptide–chitosan membrane effectively interacts with both integrins and syndecans and is a useful multi-functional biomaterial.
Keywords: Scaffold; Peptide; Chitin/chitosan; Integrin; ECM (extracellular matrix); Cell adhesion;

Extracellular matrix-mediated osteogenic differentiation of murine embryonic stem cells by Nicholas D. Evans; Eileen Gentleman; Xinyong Chen; Clive J. Roberts; Julia M. Polak; Molly M. Stevens (3244-3252).
Embryonic stem cells (ESCs) are pluripotent and have the ability to differentiate into mineralising cells in vitro. The use of pluripotent cells in engineered bone substitutes will benefit from the development of bioactive scaffolds which encourage cell differentiation and tissue development. Extracellular matrix (ECM) may be a suitable candidate for use in such scaffolds since it plays an active role in cellular differentiation. Here, we test the hypothesis that tissue-specific ECM influences the differentiation of murine ESCs. We induced murine ESCs to differentiate by embryoid body formation, followed by dissociation and culture on ECM prepared by decellularisation of either osteogenic cell (MC3T3-E1) or non-osteogenic cell (A549) cultures, or on defined collagen type I matrix. We assessed osteogenic differentiation by formation of mineralised tissue and osteogenic gene expression, and found it to be significantly greater on MC3T3-E1 matrices than on any other matrix. The osteogenic effect of MC3T3-E1 matrix was reduced by heat treatment and abolished by trypsin, suggesting a bioactive proteinaceous component. These results demonstrate that decellularised bone-specific ECM promotes the osteogenic differentiation of ESCs. Our results are of fundamental interest and may help in tailoring scaffolds for tissue engineering applications which both incorporate tissue-specific ECM signals and stimulate stem-cell differentiation.
Keywords: Embryonic stem cells; Extracellular matrix; Osteogenic; Decellularised matrix; Tissue engineering; Differentiation;

Morphological and mechanical characteristics of the reconstructed rat abdominal wall following use of a wet electrospun biodegradable polyurethane elastomer scaffold by Ryotaro Hashizume; Kazuro L. Fujimoto; Yi Hong; Nicholas J. Amoroso; Kimimasa Tobita; Toshio Miki; Bradley B. Keller; Michael S. Sacks; William R. Wagner (3253-3265).
Although a variety of materials are currently used for abdominal wall repair, general complications encountered include herniation, infection, and mechanical mismatch with native tissue. An approach wherein a degradable synthetic material is ultimately replaced by tissue mechanically approximating the native state could obviate these complications. We report here on the generation of biodegradable scaffolds for abdominal wall replacement using a wet electrospinning technique in which fibers of a biodegradable elastomer, poly(ester urethane)urea (PEUU), were concurrently deposited with electrosprayed serum-based culture medium. Wet electrospun PEUU (wet ePEUU) was found to exhibit markedly different mechanical behavior and to possess an altered microstructure relative to dry processed ePEUU. In a rat model for abdominal wall replacement, wet ePEUU scaffolds (1 × 2.5 cm) provided a healing result that developed toward approximating physiologic mechanical behavior at 8 weeks. An extensive cellular infiltrate possessing contractile smooth muscle markers was observed together with extensive extracellular matrix (collagens, elastin) elaboration. Control implants of dry ePEUU and expanded polytetrafluoroethylene did not experience substantial cellular infiltration and did not take on the native mechanical anisotropy of the rat abdominal wall. These results illustrate the markedly different in vivo behavior observed with this newly reported wet electrospinning process, offering a potentially useful refinement of an increasingly common biomaterial processing technique.
Keywords: Abdomen; Animal model; Biodegradation; Elastomer; Mechanical properties;

The effect of hydrofluoric acid treatment on titanium implant osseointegration in ovariectomized rats by Yunfeng Li; Shujuan Zou; Dazhang Wang; Ge Feng; Chongyun Bao; Jing Hu (3266-3273).
This study aimed to investigate the effects of hydrofluoric acid (HF) treatment of grit-blasted Ti implants on osseointegration in ovariectomized (OVX) rats. After blasting with aluminium oxide particles, half implants were treated with 0.2 vol.% HF, and the other half were kept non-modified as control. The topographical and chemical changes of implant surface were determined by Scanning Electron Microscope, Atomic Force Microscope, and X-ray Photoemission Spectroscopy. 12 Weeks after bilateral ovariectomy, each rat accepted two implants in distal femora, with the control implant on the left and the fluoride-modified on the right. As a result, fluoride modification induced markedly changed surface topography and chemical composition. 12 Weeks after implant insertion, the fluoride-modified implants showed improved osseointegration compared to control, with the bone area ratio and bone-to-implant contact increased by 0.9- and 1.4-fold in histomorphometry, the bone volume ratio and percent osseointegration by 0.8- and 1.3-fold in micro-CT evaluation, and the maximal push-out force and ultimate shear strength by 1.2- and 2.0-fold in biomechanical test. These promising results indicated that HF treatment of Ti surface improved implant osseointegration in OVX rats, and suggested the feasibility of using fluoride modification to improve Ti implant osseointegration in osteoporotic bone.
Keywords: Titanium; Fluoride; Surface modification; Osteoporosis; Osseointegration;

Super-hydrophilic silicone hydrogels with interpenetrating poly(2-methacryloyloxyethyl phosphorylcholine) networks by Takanori Shimizu; Tatsuro Goda; Norihiko Minoura; Madoka Takai; Kazuhiko Ishihara (3274-3280).
We synthesized silicone hydrogels from 2-methacryloyloxyethyl phosphorylcholine (MPC) and bis(trimethylsilyloxy)methylsilylpropyl glycerol methacrylate (SiMA) using two methods: random copolymerization with a small amount of cross-linker (P(SiMA-co-MPC)) and construction of an interpenetration network (IPN) structure composed of cross-linked poly(MPC)(PMPC) chains and cross-linked poly(SiMA)(PSiMA) chains (PSiMA-ipn-PMPC). The polymerization was carried out by photoreaction. The surface hydrophilicity and water absorbability of P(SiMA-co-MPC) increased with an increase in the MPC unit composition. On the other hand, in the case of PSiMA-ipn-PMPC, a super-hydrophilic surface was obtained by the surface enrichment of MPC units. The optical and mechanical properties of PSiMA-ipn-PMPC are suitable for use as a material for preparing contact lenses. In addition, the oxygen permeability of PSiMA-ipn-PMPC remains high because of the PSiMA chains. The MPC units at the surface of the hydrogels reduce protein adsorption effectively. From these results for PSiMA-ipn-PMPC, we confirmed that it has the potential for application to silicone hydrogel contact lenses.
Keywords: Phospholipid polymer; Interpenetrating polymer network; Silicone hydrogel; Hydrophilicity; Contact lenses;

Labels based on mesoporous silica nanoparticles (MSN) loaded with mediator thionine (TH), enzyme horseradish peroxidase (HRP) and secondary anti-human IgG antibody (Ab2) were developed in order to improve the sensitivity and detection limit of an amperometric immunosensor. The sensitivity of the sandwich-type immunosensor using MSN–TH–HRP–Ab2 as labels for human IgG detection was about 100 times higher than that using either MSN–TH–Ab2 or MSN–HRP–Ab2 as labels, indicating the high catalytic efficiency of HRP in the presence of mediator TH toward H2O2. The immunosensor using MSN–TH–Ab2 as labels exhibited a high sensitivity and showed a linear response within the range of 0.01–10 ng/mL human IgG. It also showed good reproducibility and selectivity and acceptable stability. These labels for immunosensors may provide many potential applications for the detection of different biomolecules.
Keywords: Enzyme; Electrochemistry; Mesoporous silica nanoparticle; Immunosensor;

Upconversion luminescence (UCL) imaging is expected to play a significant role in future photoluminescence imaging since it shows advantages of sharp emission lines, long lifetimes, superior photostability and no blinking. To further improve penetration depth, herein, near-infrared to near-infrared (NIR-to-NIR) UCL and magnetic properties were combined into a nanoparticle, and NIR-to-NIR UCL and MRI dual-modal bioimaging in vivo of whole-body animal were developed. Hydrophilic and carboxylic acid-functionalized Tm3+/Er3+/Yb3+ co-doped NaGdF4 upconversion nanophosphors (AA-NPs) were synthesized and showed both NIR-to-visible and NIR-to-NIR luminescence under excitation of 980 nm. Collecting the signal of the upconversion emission from AA-NPs in the visible and NIR range, all UCL imaging of cells, tissues and whole-body animals with different penetration depth showed high contrast. Moreover, AA-NPs showed a high relaxivity of 5.60 s−1 (mm)−1 and were successfully applied as contrast agents for magnetic resonance imaging (MRI) in vivo. By means of the combination of UCL imaging and MRI, the distribution of AA-NPs in living animals was studied, and the results indicated that these particles mainly accumulate in the liver and spleen without undesirable stay in the lungs. Therefore, the concept of UCL and MR dual-modality imaging in vivo of whole-body animals using Tm3+/Er3+/Yb3+ co-doped NaGdF4 with NIR-to-NIR upconversion luminescent and magnetic resonance properties can serve as a platform technology for the next-generation of probes for bioimaging in vivo.
Keywords: NaGdF4; Upconversion luminescence; Magnetic resonance; In vivo;

The use of microgel iron oxide nanoparticles in studies of magnetic resonance relaxation and endothelial progenitor cell labelling by Eddy S.M. Lee; Borys Shuter; Jerry Chan; Mark S.K. Chong; Jun Ding; Swee-Hin Teoh; Olivier Beuf; André Briguet; Kam Chiu Tam; Mahesh Choolani; Shih-Chang Wang (3296-3306).
In vivo tracking of stem cells after transplantation is crucial for understanding cell-fate and therapeutic efficacy. By labelling stem cells with magnetic particles, they can be tracked by Magnetic Resonance Imaging (MRI). We previously demonstrated that microgel iron oxide nanoparticle (MGIO) provide superior tracking sensitivity over commercially available particles. Here, we describe the synthesis of MGIO and report on their morphology, hydrodynamic diameters (87–766 nm), iron oxide weight content (up to 82%) and magnetization characteristics (M s  = 52.9 Am2/kg, M R  = 0.061 Am2/kg and H c  = 0.672 A/m). Their MR relaxation characteristics are comparable to those of theoretical models and represent the first such correlation between model and real particles of varying diameters. A labelling study of primary endothelial progenitor cells also confirms that MGIO is an efficient label regardless of cell type. The facile synthesis of MGIO makes it a useful tool for the studying of relaxation induced by magnetic particles and cellular tracking by MRI.
Keywords: Magnetism; Nanoparticles; MRI; Modelling; Endothelial progenitor cells;

The influence of nanoscale grooved substrates on osteoblast behavior and extracellular matrix deposition by Edwin Lamers; X. Frank Walboomers; Maciej Domanski; Joost te Riet; Falco C.M.J.M. van Delft; Regina Luttge; Louis A.J.A. Winnubst; Han J.G.E. Gardeniers; John A. Jansen (3307-3316).
To fight bone diseases characterized by poor bone quality like osteoporosis and osteoarthritis, as well as in reconstructive surgery, there is a need for a new generation of implantable biomaterials. It is envisioned that implant surfaces can be improved by mimicking the natural extracellular matrix of bone tissue, which is highly a organized nano-composite. In this study we aimed to get a better understanding of osteoblast response to nanometric grooved substrates varying in height, width and spacing. A throughput screening biochip was created using electron beam lithography. Subsequently, uniform large-scale nanogrooved substrates were created using laser interference lithography and reactive ion etching. Results showed that osteoblasts were responsive to nanopatterns down to 75 nm in width and 33 nm in depth. SEM and TEM studies showed that an osteoblast-driven calcium phosphate (CaP) mineralization was observed to follow the surface pattern dimensions. Strikingly, aligned mineralization was found on even smaller nanopatterns of 50 nm in width and 17 nm in depth. A single cell based approach for real time PCR demonstrated that osteoblast-specific gene expression was increased on nanopatterns relative to a smooth control. The results indicate that nanogrooves can be a very promising tool to direct the bone response at the interface between an implant and the bone tissue.
Keywords: Nanotopography; Osteoblast; Interface; Calcification; Cell morphology; Gene expression;

A ternary hybrid CdS/Pt–TiO2 nanotube structure for photoelectrocatalytic bactericidal effects on Escherichia Coli by Qing Kang; Qing Z. Lu; Shao H. Liu; Li X. Yang; Ling F. Wen; Sheng L. Luo; Qing Y. Cai (3317-3326).
A ternary hybrid CdS/Pt–TiO2 nanotube (NT) photoelectrode was developed by dipping and deposition technique as well as successive ionic layer adsorption and reaction (SILAR). Small Pt nanoparticles (NPs) and CdS NPs were effectively deposited on both the inside and outside of the TiO2 NTs. The as-prepared hybrid shows enhanced photon absorption and photocurrent generation efficiency. Higher bactericidal effect to Escherichia coli was observed on the ternary hybrid CdS/Pt–TiO2 NTs as compared with Pt–TiO2 NTs or pure TiO2 NTs.
Keywords: TiO2 NTs; PEC activity; Bactericidal effect;

The effect of a single remote injection of statin-impregnated poly (lactic-co-glycolic acid) microspheres on osteogenesis around titanium implants in rat tibia by Tomohiro Masuzaki; Yasunori Ayukawa; Yasuko Moriyama; Yohei Jinno; Ikiru Atsuta; Yoichiro Ogino; Kiyoshi Koyano (3327-3334).
The aim of this study was to evaluate the effects of newly developed injectable poly (lactic-co-glycolic acid) (PLGA) microspheres containing fluvastatin on osteogenesis around titanium implants in the rat tibia. After confirmation of the sustained-release profile of fluvastatin from the microspheres by an in vitro assay, the microspheres were administered to the back skin of the rats by a single transdermal injection. At 2 and 4 weeks after the implant surgery, the fluvastatin groups showed enhanced new bone formation around the titanium implants without any influence on the serum biochemistry. In addition, the fluvastatin groups showed increased three-point bending strengths of their femurs. The results of this study indicate that a single remote injection of PLGA/fluvastatin microspheres safely and successfully stimulated bone formation around titanium implants and increased the mechanical properties of bone.
Keywords: Poly (lactic-co-glycolic acid); Microsphere; Fluvastatin; Drug delivery system; Bone formation; Dental implant;

An anticancer drug delivery system based on surfactant-templated mesoporous silica nanoparticles by Qianjun He; Jianlin Shi; Feng Chen; Min Zhu; Lingxia Zhang (3335-3346).
Three types of surfactant-templated mesoporous silica nanoparticles (Surf@MSNs) of 150–660 nm in diameter were developed as anticancer drug delivery systems. The Surf@MSNs exhibit the high drug (surfactant) loading capacities, the sustained drug (surfactant) release profiles and the high and long-term anticancer efficacy. The effects of the Surf@MSNs concentration, the type of the contained surfactants and the incubation time on the cytotoxicity and proliferative activity of MCF-7 cells were evaluated. A common anticancer drug CPT-11 was also loaded into surfactant-free MSNs (CPT@MSNs) and used as a reference for estimating the anticancer efficacies of Surf@MSNs. Surfactant-extracted MSNs exhibited neglectable cytotoxicity to MCF-7 cell, and free surfactants exhibited higher cytotoxicity than free CPT-11 at the same concentration. The endocytosis enhanced the drug uptake by MCF-7 cells and the anticancer efficacies of Surf@MSNs and CPT@MSNs, and more surfactants would be released in a longer term, which led to the more significant enhancement of the cytotoxicity, than CPT-11 with the process of incubation. Among the investigated Surf@MSNs, CTAB-contained MSNs (CTAB@MSNs) show remarkably higher long-term anticancer efficacy than CPT-11-loaded surfactant-free MSNs (CPT@MSNs), even at very low concentrations of 2–15 μg mL−1.
Keywords: Mesoporous silica; Nanoparticle; Surfactant; Drug release; Anticancer; Cytotoxicity;

Superporous hydrogel containing poly(acrylic acid-co-acrylamide)/O-carboxymethyl chitosan interpenetrating polymer networks (SPH-IPN) was evaluated as the oral delivery vehicle for insulin, emphasizing on the effect of polymer integrity on insulin absorption mechanisms. The integral SPH-IPN (I-SPH-IPN) and powdered SPH-IPN (P-SPH-IPN) exhibited potent and equivalent in vitro enzymatic inhibition capacities, which were attributed to both enzyme incorporation and Ca2+ deprivation. Nevertheless, I-SPH-IPN showed marked superiority to P-SPH-IPN in in vivo enzymatic inhibition. Through reversible opening of epithelial tight junctions, I-SPH-IPN notably enhanced paracellular permeability of insulin in Caco-2 cell monolayers and excised rat intestine by 4.9 and 4.2 folds, respectively, wherein I-SPH-IPN outperformed P-SPH-IPN by 2.5 and 2.3 folds, respectively. Besides, orally delivered I-SPH-IPN could retain in rat intestine for more than 8 h while P-SPH-IPN was quickly eliminated, suggesting better retentive properties of I-SPH-IPN. Such results were further confirmed by in vivo assessment in that oral administration of insulin-loaded I-SPH-IPN yielded notable insulin absorption and hypoglycemic effect, while P-SPH-IPN was ineffective. Finally, an oral acute and sub-acute toxicity study in mice confirmed biocompatibility of SPH-IPN. Therefore, the detailed mechanism assessment confirmed that I-SPH-IPN was an effective and safe peroral carrier for protein drugs.
Keywords: Interpenetrating polymer networks superporous hydrogel; Polymer integrity; Oral protein delivery; Enzymatic inhibition; Insulin absorption; Biocompatibility;

Creation of a LIGHT mutant with the capacity to evade the decoy receptor for cancer therapy by Tomohiro Morishige; Yasuo Yoshioka; Hiroshi Inakura; Aya Tanabe; Xinglei Yao; Shin-ichi Tsunoda; Yasuo Tsutsumi; Yohei Mukai; Naoki Okada; Shinsaku Nakagawa (3357-3363).
The cytokine LIGHT activates various anti-tumor functions through its two receptors, lymphotoxin β receptor (LTβR) and herpes virus entry mediator (HVEM), and is expected to be a promising candidate for cancer therapy. However, LIGHT is also trapped by decoy receptor 3 (DcR3), which is highly expressed in various tumors. Here, we used phage display technique to create LIGHT mutants that specifically bind LTβR and HVEM, and is not trapped by DcR3 for optimized cancer therapy. We constructed phage library displaying structural variants of LIGHT with randomized amino acid residues. After the affinity panning, we created 6 clones of LIGHT mutants as candidates for DcR3-evading LIGHT. Analysis of binding affinities showed that all candidates had 10-fold lower affinities for DcR3 than wild-type LIGHT, while 5 of the 6 clones had almost the same affinity for LTβR and HVEM. Furthermore, analysis of detailed binding kinetics showed that lower affinity for DcR3 is dependent on their faster off-rate. Further, we showed that the LIGHT mutant had almost the same cytotoxicity via LTβR, and had 62-fold higher DcR3-evading capacity compared to the wild type. Our data provide valuable information for construction of more functional LIGHT mutants that might be powerful tools for cancer therapy.
Keywords: Affinity; Apoptosis; Bioactivity; Cytokine; Cytotoxicity; Immunomodulation;

Adsorption of amphiphilic hyperbranched polyglycerol derivatives onto human red blood cells by Zonghua Liu; Johan Janzen; Donald E. Brooks (3364-3373).
Hydrophobically derivatized hyperbranched polyglycerol (HPG)-polyethylene glycol (PEG) polymers bearing stearoyl chains (HPG–C18–PEG) were originally developed as human serum albumin substitutes and further as a unimolecular drug delivery system. In view of these in vivo applications and the potential for membrane interaction by these materials due to their amphiphilic structure, determining the adsorption of the polymers to human red blood cells (RBCs) is an important issue. This paper reports on the in vitro adsorption to RBCs of tritium-radiolabeled HPG–C18–PEG polymers. The morphological changes of RBCs associated with the adsorption were also examined by light and scanning electron microscopy (SEM). Laser scanning confocal microscopy (LSCM) suggests that the binding site of the polymers on RBCs is the cell membrane. Adsorption experiments show that, in the medium of either saline or plasma, the binding amount of the polymers to RBCs increases with increased polymer concentration in a manner which implies simple Langmurian behavior. The binding amount in saline is of the order of 105 molecules/cell at an equilibrium concentration of 1 mg/mL of HPG–C18–PEG polymer. The RBC morphology depends on the adsorbed amount; the cells become crenated in high concentrations (5 and 10 mg/mL) of the polymer solutions in the absence of plasma proteins. Interestingly, a large amount of polymers remain bound to RBCs even after washes with plasma (of the order of 104 molecules/cell). Thus, the bound polymers might have an extended circulating time by “hitchhiking” on RBCs in the bloodstream. These results provide significant information and insight for related studies of the interaction of amphiphilic molecules with cell membranes and for in vivo applications of biopolymers as drug delivery systems.
Keywords: Adsorption; Hyperbranched polyglycerol; Poly(ethylene glycol); Stearoyl; Red blood cells; Plasma proteins;

Self-activated luminescent and mesoporous strontium hydroxyapatite nanorods for drug delivery by Cuimiao Zhang; Chunxia Li; Shanshan Huang; Zhiyao Hou; Ziyong Cheng; Piaoping Yang; Chong Peng; Jun Lin (3374-3383).
Multifunctional strontium hydroxyapatite (SrHAp) nanorods with luminescent and mesoporous properties have been successfully synthesized by a hydrothermal method. SEM and TEM images indicate that the mesoporous SrHAp samples consist of monodiperse nanorods with lengths of 120–150 nm, diameters of around 20 nm, and the mesopore size of 3–5 nm. The as-obtained SrHAp nanorods show an intense bright blue emission (centered at 432 nm, lifetime 11.6 ns, quantum efficiency: 22%), which might arise from CO2· radical impurities in the crystal lattice under long-wavelength UV-light irradiation. Furthermore, the amount of trisodium citrate has an obvious impact on the particle size and the luminescence properties of the products, respectively. The drug storage/release test indicates that the luminescent SrHAp nanorods show a drug loading and controlled release properties for ibuprofen (IBU). Additionally, the emission intensity of SrHAp in the drug carrier system increases with the cumulative released amount of IBU, making the drug release might be easily tracked and monitored by the change of the luminescence intensity. This luminescent material may be potentially applied in the drug delivery and disease therapy fields.
Keywords: Strontium hydroxyapatite; Nanorod; Luminescence; Mesoporous; Drug delivery;

Enteric-coated capsules filled with freeze-dried chitosan/poly(γ-glutamic acid) nanoparticles for oral insulin delivery by Kiran Sonaje; Yi-Jia Chen; Hsin-Lung Chen; Shiaw-Pyng Wey; Jyuhn-Huarng Juang; Ho-Ngoc Nguyen; Chia-Wei Hsu; Kun-Ju Lin; Hsing-Wen Sung (3384-3394).
A pH-sensitive nanoparticle (NP) system composed of chitosan and poly(γ-glutamic acid) was prepared for the oral delivery of insulin. The biodistribution study in a rat model showed that some of the orally administered NPs were retained in the stomach for a long duration, which might lead to the disintegration of NPs and degradation of insulin. To overcome these problems, we freeze-dried NPs and filled them in an enteric-coated capsule. The small angle X-ray scattering (SAXS) profiles indicated that the freeze-drying process did not significantly disrupt the internal structure of NPs; additionally, their pH-sensitivity was preserved and the insulin release was pH-dependent. The results obtained in the native PAGE analysis indicated that the released insulin molecules were neither fragmented nor aggregated. Upon oral administration, the enteric-coated capsule remained intact in the acidic environment of the stomach, but dissolved rapidly in the proximal segment of the small intestine. Consequently, all the NPs loaded in the capsule were brought into the small intestine, thus enhancing the intestinal absorption of insulin and providing a prolonged reduction in blood glucose levels. The relative bioavailability of insulin was found to be approximately 20%. These results suggest that the formulation developed in the study might be employed as a potential approach for the oral delivery of insulin.
Keywords: Diabetes; SPECT/CT tomography; X-ray imaging; Pharmacodynamic and pharmacokinetic profiles; pH-sensitivity;

PLGA nanoparticles surface decorated with the sialic acid, N-acetylneuraminic acid by Lucia Bondioli; Luca Costantino; Antonio Ballestrazzi; Davide Lucchesi; Diana Boraschi; Federica Pellati; Stefania Benvenuti; Giovanni Tosi; Maria A. Vandelli (3395-3403).
There is a broad interest in the development of nanoparticles (NPs) carrying on their surface carbohydrates such as sialic acids. It is known that these carbohydrates influence the biological and physical properties of biopharmaceutical proteins and living cells. Macromolecular compounds containing these carbohydrates showed an anti-recognition effect, exert an antiviral effect and also are able to be recognized by the cell surface of some kind of cancer cells. Thus, in the present research we performed two different approaches in order to obtain polymeric (poly(d,l-lactide-co-glycolide), PLGA) NPs surface decorated with the sialic acid N-acetylneuraminic acid (Neu5Ac). The first strategy that has been followed is based on the derivatization of the polyester PLGA with the thioderivative of Neu5Ac, starting material for the preparation of the NPs; the second is based on the synthesis of compounds potentially able to insert their lipophilic moiety into the underivatized PLGA NPs during their preparation, and to display their hydrophilic moiety (Neu5Ac) on their surface. The first approach allowed the obtainment of NPs surface decorated with Neu5Ac, as evidenced by ESCA spectroscopy and interaction with the lectin Wheat Germ Agglutinin. Moreover, a formulation of these NPs suitable for in vitro assays showed that they are phagocytosed by human monocytes with an apparently different mechanism with respect of those made of underivatized PLGA. The second strategy led to NPs in which their surface appears to be very different with respect to the NPs obtained following the first strategy, with the carboxylic groups of Neu5Ac markedly shielded. Thus, the new Neu5Ac-modified PLGA polyester represent a useful starting material for the preparation of NPs surface decorated with this sialic acid.
Keywords: Nanoparticle; Poly(lactic-co-glycolic)acid; Surface modification; Monocyte;

Efficient delivery of therapeutic genes requires overcoming key extracellular and intracellular barriers. These include stability during circulation, internalization by target cells, facilitated endosomal escape, and localization of genes in destined intracellular compartments (e.g., nucleus). Micelles that transform their structure in the mildly acidic endosome and release their cargo genes into the cytoplasm were synthesized by self-assembling DNA with PEG-conjugated poly(ketalized serine) [PEG-poly(kSer)]. It was confirmed that, upon acid-hydrolysis of ketal linkages, poly(kSer) converts to neutral and naturally occurring poly(serine), destabilizing PEG-poly(kSer)/DNA micelles. In vitro studies demonstrated that PEG-poly(kSer) micelles were able to transfect NIH 3T3 cells more efficiently than both PEG-poly(Lys)/DNA micelles and poly-L-lysine/DNA polyplexes through efficient DNA dissociation in the cytoplasm. In addition, the core of PEG-poly(kSer)/DNA micelles were cross-linked via acid-cleavable amine-bearing branches, and the resulting cross-linked PEG-poly(kSer)/DNA micelles showed improved transfection capability in the presence of serum. Conjugation of folic acids (FAs) at the PEG termini of the acid-transforming micelles resulted in selectively increased cellular internalization and transfection of FA receptor-expressing HeLa cells over NIH 3T3 cells, implicating the possibility of cancer-targeted gene delivery using FA-PEG-poly(kSer)/DNA micelles. This study demonstrates that the acid-transforming PEG-poly(kSer)/DNA micelles are promising nonviral vectors for stimuli-responsive, efficient, biocompatible, and targeted gene delivery.
Keywords: Gene transfer; Micelle; Peptide; Nanoparticle;

PEG–PLA microparticles for encapsulation and delivery of Tat-EGFP to retinal cells by Mehrdad Rafat; Carolyne A. Cléroux; Wai Gin Fong; Adam N. Baker; Brian C. Leonard; Michael D. O'Connor; Catherine Tsilfidis (3414-3421).
The efficient and controlled delivery of genes and proteins to retinal cells remains a challenge. In this study, we evaluated polyethylene glycol-polylactic acid (PEG–PLA) microparticles for encapsulation and delivery of a Transactivator of transcription-enhanced green fluorescent protein fusion (Tat-EGFP) to retinal cells. Our main objective was to develop a microparticle system that delivers Tat-EGFP with an initial rapid release (within 24 h) followed by a sustained release. We prepared four different formulations of Tat-EGFP encapsulated PEG–PLA particles to investigate the effects of protein and polymer concentrations on particle morphology and protein release, using scanning electron microscopy (SEM) and fluorometry techniques. The optimum formulation was selected based on higher protein release, and smaller particle size. The optimum formulation was then tested in vitro for cell biocompatibility and protein internalization, and in vivo for cellular toxicity following sub-retinal injections into rat eyes. The results suggest that PEG–PLA microparticles can deliver proteins in cell culture allowing protein internalization in as little as 1 h. In vivo, protein was shown to localize within the photoreceptor layer of the retina, and persist for at least 9 weeks with no observed toxicity.
Keywords: Biodegradation; Electron microscopy; Gene therapy; Microencapsulation; Polylactic acid; Retina;

Cell-laden microwells for the study of multicellularity in lymphocyte fate decisions by Junsang Doh; Miju Kim; Matthew F. Krummel (3422-3428).
Cell–cell cooperativity in populations of motile and transiently interacting immune cells has been difficult to assess in the absence of tools to control proximity and communication. Here, we describe the generation of cell-laden microwells that can precisely control contact-mediated interactions and multicellular ‘quorum’ decisions in lymphocytes. Different types of fate decisions for activating T cells can be shown to variously obey ‘binary’ or ‘density’ outcomes, correlated with cell–cell contact, using this new platform.
Keywords: Hydrogel microwell; Multicellularity; T cell; Fate decision;