Biomaterials (v.32, #14)

In vivo molecular evidence of delayed titanium implant osseointegration in compromised bone by Katleen Vandamme; Xavier Holy; Morad Bensidhoum; Delphine Logeart-Avramoglou; Ignace E. Naert; Joke A. Duyck; Hervé Petite (3547-3554).
Optimization of implant osseointegration in patients with reduced bone healing potential is a challenge remaining in implant dentistry. Identification of the genes that are modulated during implant osseointegration in normal versus osteopenic bone is needed to successfully address these pertinent clinical needs. The present study aimed to assess the initial and early molecular events following titanium implant installation in normal and compromised bone in a rat tibia model. Peri-implant tissue from a well-defined tissue regeneration compartment was analyzed at 2 and 7 days post-surgery for the expression of select markers of inflammation, angiogenesis, bone resorption and bone formation. Impaired bone was induced by hindlimb unloading and validated using μCT. The essential step of angiogenesis preceding bone regeneration was evidenced for the peri-implant setting in healthy bone. Compromised bone significantly affected the angiogenesis-osteogenesis coupling in the initial phase (2 days post-surgery), with altered expressions of Vegfa and Epas1 coinciding with downregulated expressions of Col1a1, Bmp2, Bmp4, Alpl and Bglap. At 7 days post-implantation, differences between normal and compromised peri-implant bone were no longer observed. This in vivo molecular evidence of delayed implant osseointegration in compromised bone reassert modern strategies in implant development, such as surface modifications and bioengineered approaches, to improve implant osseointegration in compromised conditions.
Keywords: Titanium; Implant; Osseointegration; Bone; Gene expression; Animal model;

The use of leukemia inhibitory factor immobilized on virus-derived polyhedra to support the proliferation of mouse embryonic and induced pluripotent stem cells by Naoki Nishishita; Hiroshi Ijiri; Chiemi Takenaka; Kenichiro Kobayashi; Kohei Goto; Eiji Kotani; Tohru Itoh; Hajime Mori; Shin Kawamata (3555-3563).
Human leukemia inhibitory factor (LIF) was immobilized into insect virus-derived microcrystals (polyhedra) to generate LIF polyhedra (LIF-PH) that can slowly release LIF into embryonic stem (ES) cell culture media and thus maintain ES cells in an undifferentiated state. Assays of the biological activities of LIF-PH indicated that a single addition of LIF-PH to the ES cell culture medium can support the proliferation of mouse ES and induced pluripotent stem (iPS) cells continuously for 14 days, and suggest that LIF-PH can be successfully used in the place of a periodic addition of recombinant LIF to the media every 2–3 days. The release of LIF protein from LIF-PH was determined by enzyme-linked immunosorbent assay (ELISA). Maintenance of undifferentiated state of mouse ES and iPS cells cultured with LIF-PH was determined by the detection of pluripotency-related biomarkers Oct3/4 and stage-specific embryonic antigen-1 (SSEA-1) through immunostaining and measurement of alkaline phosphatase activity. In this paper, we propose a closed culture system for mass production of ES and iPS cells that utilize a slow-releasing agent of LIF.
Keywords: LIF; Polyhedra; Slow release; ES cells; iPS cells;

The performance of human mesenchymal stem cells encapsulated in cell-degradable polymer-peptide hydrogels by Sarah B. Anderson; Chien-Chi Lin; Donna V. Kuntzler; Kristi S. Anseth (3564-3574).
Thiol-ene photopolymerization offers a unique platform for the formation of peptide-functionalized poly(ethylene glycol) hydrogels and the encapsulation, culture and differentiation of cells. Specifically, this photoinitiated polymerization scheme occurs at neutral pH and can be controlled both spatially and temporally. Here, we have encapsulated human mesenchymal stem cells (hMSCs) in matrix metalloproteinase (MMP) degradable and cell-adhesive hydrogels using thiol-ene photopolymerization. We find that hMSCs survive equally well in this system, regardless of MMP-degradability. When hMSCs are encapsulated in these cell-degradable hydrogels, they survive and are able to proliferate. In classic hMSC differentiation medias, hMSCs locally remodel their microenvironment and take on characteristic morphologies; hMSCs cultured in growth or osteogenic differentiation media are less round, as measured by elliptical form factor, and are smaller than hMSCs cultured in chondrogenic or adipogenic differentiation media. In addition, hMSCs encapsulated in completely cell-degradable hydrogels and cultured in osteogenic, chondrogenic, or adipogenic differentiation media generally express increased levels of specific differentiation markers as compared to cells in hydrogels that are not cell-degradable. These studies demonstrate the ability to culture and differentiate hMSCs in MMP-degradable hydrogels polymerized via a thiol-ene reaction scheme and that increased cell-mediated hydrogel degradability facilitates directed differentiation of hMSCs.
Keywords: Mesenchymal stem cell; Hydrogel; Peptide; Photopolymerisation; Metalloproteinase;

The role of extracellular matrix composition in structure and function of bioengineered skeletal muscle by Sara Hinds; Weining Bian; Robert G. Dennis; Nenad Bursac (3575-3583).
One of the obstacles to the potential clinical utility of bioengineered skeletal muscle is its limited force generation capacity. Since engineered muscle, unlike most native muscle tissue, is composed of relatively short myofibers, we hypothesized that, its force production and transmission would be profoundly influenced by cell-matrix interactions. To test this hypothesis, we systematically varied the matrix protein type (collagen I/fibrin/Matrigel) and concentration in engineered, hydrogel-based neonatal rat skeletal muscle bundles and assessed the resulting tissue structure, generation of contractile force, and intracellular Ca2+ handling. After two weeks of culture, the muscle bundles consisted of highly aligned and cross-striated myofibers and exhibited standard force–length and force–frequency relationships achieving tetanus at 40 Hz. The use of 2 mg/ml fibrin (control) yielded isometric tetanus amplitude of 1.4 ± 0.3 mN as compared to 0.9 ± 0.4 mN measured in collagen I-based bundles. Higher fibrin and Matrigel concentrations synergistically yielded further increase in active force generation to 2.8 ± 0.5 mN without significantly affecting passive mechanical properties, tetanus-to-twitch ratio, and twitch kinetics. Optimized matrix composition yielded significant cellular hypertrophy (protein/DNA ratio = 11.4 ± 4.1 vs. 6.5 ± 1.9 μg/μg in control) and a prolonged Ca2+ transient half-width (Ca50 = 232.8 ± 33.3 vs. 101.7 ± 19.8 ms). The use of growth-factor-reduced Matrigel, instead of standard Matrigel did not alter the obtained results suggesting enhanced cell-matrix interactions rather than growth factor supplementation as an underlying cause for the measured increase in contractile force. In summary, biomaterial-based manipulation of cell-matrix interactions represents an important target for improving contractile force generation in engineered skeletal muscle.
Keywords: Skeletal myoblasts; Myogenesis; Hydrogel; Contractile force;

The effect of degradable polymer surfaces on co-cultures of monocytes and smooth muscle cells by Joanne E. McBane; Kyle G. Battiston; Aman Wadhwani; Soroor Sharifpoor; Rosalind S. Labow; J. Paul Santerre (3584-3595).
Strategies to optimize biomaterial chemistry for applications in vascular tissue engineering attempt to promote endothelial and smooth muscle cell recruitment into porous material constructs. The primary objective is to facilitate relevant tissue formation in a wound healing versus pro-inflammatory manner. The present work investigated the interactive co-cellular response of human monocytes and human vascular smooth muscle cells (VSMCs) with a novel degradable, polar/hydrophobic/ionic (D-PHI) polyurethane and compared it to a commercially available biomaterial, poly-lactic-glycolic acid (PLGA) as well as tissue culture polystyrene (TCPS). D-PHI triggered a smaller pro-inflammatory response (acid phosphatase, esterase, tumor necrosis factor-α) at later time points (>14 d) than PLGA suggesting that monocytes may be transitioning to a more wound-healing phenotype on the D-PHI surface. When D-PHI was coated with collagen, monocyte cell attachment did not differ with the native D-PHI; however, PLGA showed significant differences between collagen coated versus uncoated surfaces. There were more VSMCs and monocytes attached in co-culture to D-PHI when compared to PLGA. Co-cultures on D-PHI released more IL-10 (anti-inflammatory) than monocytes cultured alone, while the VSMCs retained the expression of its marker protein calponin. Together the above data suggest that co-culturing monocytes with VSMCs may aid in stimulating the attachment of VSMCs to D-PHI while eliciting the desired functional phenotypes for both monocytes (i.e. low inflammation based on IL-10 values) and VSMCs (expressing calponin, a marker of contractility). Moreover, the results of this study demonstrated that D-PHI performed equally or better to PLGA in terms of the assayed biological parameters.
Keywords: Monocyte; Vascular smooth muscle cells; Surface chemistry; Polyurethane; Poly-lactic-glycolic acid;

Cells immobilized on patterns printed in DNA by an inkjet printer by Kengo Sakurai; Yuji Teramura; Hiroo Iwata (3596-3602).
The ability to two-dimensionally align various kinds of cells freely onto substrate would be a useful tool for analysis of cell–cell interactions. In this study, we aimed to establish a method for attaching cells to the substrate, in which the pattern is drawn by an inkjet printer. Poly-deoxyribonucleic acid (DNA) was immobilized onto the cell surface by use of DNA-conjugated poly(ethylene) glycol-phospholipid (DNA–PEG-lipid), which is the amphiphilic conjugate of PEG-lipid and single-stranded DNA. The surface of the substrate was then modified with the complementary DNA using an inkjet printer. Finally, DNA-immobilized cells were attached onto the substrate through DNA hybridization. The use of the inkjet printer enabled us to draw the DNA pattern accurately on the substrate with a resolution of a few hundred micrometers. DNA-immobilized cells could be attached precisely along the DNA pattern on the substrate. In addition, various kinds of cells could be attached simultaneously by using various sequences of DNA. Our technique is promising for analysis of cell–cell interactions and differentiation induction in stem cell research.
Keywords: Cell adhesion; DNA; Surface modification; Gold; Biocompatibility;

Gemcitabine (Gem) eluting metal stents were prepared for potential application as drug delivery systems for localized treatment of malignant tumors. Pullulan, a natural polysaccharide, was chemically acetylated (pullulan acetate; PA) by different degrees (1.18, 1.71, and 2.10 acetyl groups per glucose unit of pullulan), layered on polytetrafluoroethylene (PTFE), and applied as part of a Gem-loaded controlled-release membrane for drug-eluting non-vascular stents. PA with a higher degree of acetylation had greater drug-loading capacity with more extended release of Gem over 30 days. The released Gem accumulated in CT-26 colon cancer without systemic exposure inducing total regression of tumors. The long-term biological activity of the released Gem and apoptosis of tumor tissues following localized delivery were confirmed by annexin V binding assays and histology. The controlled release of Gem from PA-PTFE covered drug-eluting stents (DES) may increase the patency of these stents for the treatment of malignant gastrointestinal cancer as well as cancer-related stenosis.
Keywords: Nonvascular drug-eluting stents (DES); Gemcitabine; Covered stents; Pullulan; Gastrointestinal cancer;

High-throughput screening of microscale pitted substrate topographies for enhanced nonviral transfection efficiency in primary human fibroblasts by Andrew F. Adler; Alessondra T. Speidel; Nicolas Christoforou; Kristian Kolind; Morten Foss; Kam W. Leong (3611-3619).
Optimization of nonviral gene delivery typically focuses on the design of particulate carriers that are endowed with desirable membrane targeting, internalization, and endosomal escape properties. Topographical control of cell transfectability, however, remains a largely unexplored parameter. Emerging literature has highlighted the influence of cell–topography interactions on modulation of many cell phenotypes, including protein expression and cytoskeletal behaviors implicated in endocytosis. Using high-throughput screening of primary human dermal fibroblasts cultured on a combinatorial library of microscale topographies, we have demonstrated an improvement in nonviral transfection efficiency for cells cultured on dense micropit patterns compared to smooth substrates, as verified with flow cytometry. A 25% increase in GFP+ cells was observed independent of proliferation rate, accompanied by SEM and confocal microscopy characterization to help explain the phenomenon qualitatively. This finding encourages researchers to investigate substrate topography as a new design consideration for the optimization of nonviral transfection systems.
Keywords: Surface topography; Gene transfer; Combinatorial screening; Microstructure; Cell spreading; Fibroblast;

The effects of cross-linked thermo-responsive PNIPAAm-based hydrogel injection on retinal function by Sanja B. Turturro; Micah J. Guthrie; Alyssa A. Appel; Pawel W. Drapala; Eric M. Brey; Victor H. Pérez-Luna; William F. Mieler; Jennifer J. Kang-Mieler (3620-3626).
There is significant interest in biomaterials that provide sustained release of therapeutic molecules to the retina. Poly(N-isopropylacrylamide) (PNIPAAm)-based materials have received significant attention as injectable drug delivery platforms due to PNIPAAm’s thermo-responsive properties at approximately 32 °C. While the drug delivery properties of PNIPAAm materials have been studied extensively, there is a need to evaluate the safety effects of hydrogel injection on retinal function. The purpose of this study was to examine the effect of poly(ethylene glycol) diacrylate (PEG-DA) crosslinked PNIPAAm hydrogel injection on retinal function. Utilizing scanning laser ophthalmoscopy (SLO), optical coherent tomography (OCT), and electroretinography (ERG), retinal function was assessed following hydrogel injection. In region near the hydrogel, there was a significant decrease in arterial and venous diameters (∼4%) and an increase in venous blood velocity (∼8%) 1 week post-injection. Retinal thickness decreased (∼6%) at 1 week and the maximum a- and b-wave amplitudes of ERG decreased (∼15%). All data returned to baseline values after week 1. These data suggest that the injection of PEG-DA crosslinked PNIPAAm hydrogel results in a small transient effect on retinal function without any long-term effects. These results further support the potential of PNIPAAm-based materials as an ocular drug delivery platform.
Keywords: Blood flow; Electroretinography; Drug delivery; Retina; Thermo-responsive hydrogel;

Modulation of immune responses by the antimicrobial peptide, epinecidin (Epi)-1, and establishment of an Epi-1-based inactivated vaccine by Han-Ning Huang; Chieh-Yu Pan; Venugopal Rajanbabu; Yi-Lin Chan; Chang-Jer Wu; Jyh-Yih Chen (3627-3636).
Current efforts to improve the effectiveness of vaccines include incorporating antimicrobial peptides mixed with a virus. The antimicrobial peptide, epinecidin (Epi)-1, was reported to have an antiviral function, and an Epi-1-based inactivated vaccine was postulated as a model and discussed. In this report, we demonstrated modulation of immune responses by Epi-1 and an Epi-1-based Japanese encephalitis virus (JEV)-inactivated vaccine against JEV infection in mice. Under in vitro conditions, Epi-1 prevented JEV infection-mediated loss of cell viability in BHK-21 cells. When Epi-1 and JEV were co-injected into mice and mice were re-challenged with JEV after 14 days, all mice survived. In addition, Epi-1 modulated the expressions of immune-responsive genes like interleukin (IL)-6, IL-10, MCP-1, tumor necrosis factor-α, interferon-γ and IL-12, and elevated the levels of anti-JEV-neutralizing antibodies in the serum. The presence of Epi-1 suppressed the multiplication of JEV in brain sections at 4 days after an injection. Mice immunized with the developed vaccine showed complete survival against JEV infection, and it was superior to the traditional formalin-based JEV-inactivated vaccine. This study demonstrates the use of Epi-1 to develop an inactivated vaccine can provide guidelines for the future design of Epi-1-virus formulations for various in vivo applications.
Keywords: Epinecidin-1; Antimicrobial peptide; Japanese encephalitis virus; Vaccine; Antiviral function; Immune responses;

Pathway of programmed cell death in HeLa cells induced by polymeric anti-cancer drugs by Yan-Qing Guan; Zhibin Li; Jiamei Chen; Huimin Tao; Wenwen Wang; Zhe Zheng; Ling Li; Jun-Ming Liu (3637-3646).
Synthesis of anticancer polymeric materials plus their biological applications is one of the most charming and active research areas in biological functional materials. However, the predominant mechanisms for controlling cancer cell viability are not yet clear. In this work, cell culture polymeric materials co-immobilized with death signal proteins interferon-γ (IFN-γ)/tumor necrosis factor-α (TNF-α) on the surface were prepared by photochemical method to develop an anticancer polymeric drug model. Various characterizations on the microstructures and compositions, including the Fourier transform infrared spectroscopy, UV absorption spectroscopy, fluorescence measurement, atomic force microscopy, and electron spectroscopy for chemical analysis, were performed. For addressing the biological applications, we investigated systematically the death pathways of HeLa cells attached onto the drug model by means of a series of cell-biology techniques. It was demonstrated that the IFN-γ plus TNF-α co-immobilized on the polymeric material surface exhibited more notable inhibitive effects than the free IFN-γ plus TNF-α, and the induced HeLa cells were mainly along apoptosis-like PCD with the translocation of EndoG from the cytoplasm to the nucleus. These findings indicate that the polymeric drugs with the co-immobilized IFN-γ plus TNF-α may offer significant potentials for therapeutic manipulation of human cervical cancer.
Keywords: Co-immobilized IFN-γ plus TNF-α; Polystyrene material; HeLa; EndoG;

A single-monomer derived linear-like PEI-co-PEG for siRNA delivery and silencing by Lin-Ren Tsai; Min-Hua Chen; Chih-Te Chien; Meng-Kai Chen; Fong-Sian Lin; Kurt Ming-Chao Lin; Yeu-Kuang Hwu; Chung-Shi Yang; Shu-Yi Lin (3647-3653).
Polyethylenimines (PEIs) are commonly used as a vehicle to deliver and protect siRNA, but the strong interaction still remains to be modulated for efficient siRNA release and silencing. Herein, a single-monomer derived linear-like PEI-co-PEG (LPEI-co-PEG, P2 ) was synthesized to substantially enhance the siRNA release, but not affect the efficiency of protection. The linear-like copolymer (P2 ) was only synthesized from a single-monomer by intensive synchrotron X-ray irradiation within 5 min, randomly producing both PEI and PEG segments. The counterpart vehicle, LPEI (P1 ), was also synthesized for comparison. We found that the P1 and P2 were able to prevent siRNA against enzymatic degradation. Most importantly, efficient siRNA release (52%) was only observed in the siRNA/P2 complexes and not in the siRNA/P1 complexes (<5%), suggesting that the PEG segment may modulate the interaction between siRNA and P2 segment. Specifically, P2 as well as P1 can emit photoluminescence; cancer cells exhibited a detectable photoluminescence after treatment with P1 and P2 , indicative of their excellent transfection efficiency. Subsequently, the siGFP/P2 complexes knocked down GFP with excellent efficiency (75%) above the siGFP/P1 complexes (19%) and siGFP/Lipofectamine complexes (20%). Importantly, the siRNA with anti-VEGF function being associated with P2 have been demonstrated an excellent efficiency in the suppression of tumor growth.
Keywords: A single-monomer copolymerization; LPEI-co-PEG; Linear-like polyethylenimine-co-poly(ethylene glycol); A non-toxic vehicle; Synchrotron X-rays irradiation; Enhanced siRNA release and mRNA silencing;

Neuronal uptake and intracellular superoxide scavenging of a fullerene (C60)-poly(2-oxazoline)s nanoformulation by Jing Tong; Matthew C. Zimmerman; Shumin Li; Xiang Yi; Robert Luxenhofer; Rainer Jordan; Alexander V. Kabanov (3654-3665).
Fullerene, the third allotrope of carbon, has been referred to as a “radical sponge” because of its powerful radical scavenging activities. However, the hydrophobicity and toxicity associated with fullerene limits its application as a therapeutic antioxidant. In the present study, we sought to overcome these limitations by generating water-soluble nanoformulations of fullerene (C60). Fullerene (C60) was formulated with poly(N-vinyl pyrrolidine) (PVP) or poly(2-alkyl-2-oxazoline)s (POx) homopolymer and random copolymer to form nano-complexes. These C60-polymer complexes were characterized by UV–vis spectroscopy, infrared spectroscopy (IR), dynamic light scattering (DLS), atomic force microscopy (AFM) and transmission electron microscopy (TEM). Cellular uptake and intracellular distribution of the selected formulations in catecholaminergic (CATH.a) neurons were examined by UV–vis spectroscopy, immunofluorescence and immunogold labeling. Electron paramagnetic resonance (EPR) spectroscopy was used to determine the ability of these C60-polymer complexes to scavenge superoxide. Their cytotoxicity was evaluated in three different cell lines. C60-POx and C60-PVP complexes exhibited similar physicochemical properties and antioxidant activities. C60-poly(2-ethyl-2-oxazoline) (PEtOx) complex, but not C60-PVP complex, were efficiently taken up by CATH.a neurons and attenuated the increase in intra-neuronal superoxide induced by angiotensin II (Ang II) stimulation. These results show that C60-POx complexes are non-toxic, neuronal cell permeable, superoxide scavenging antioxidants that might be promising candidates for the treatment of brain-related diseases associated with increased levels of superoxide.
Keywords: fullerene (C60); Polyoxazoline; Antioxidant; Free radical; Neural cell;

Induction of anti-tumor cytotoxic T cell responses through PLGA-nanoparticle mediated antigen delivery by Zhiping Zhang; Songsak Tongchusak; Yo Mizukami; Yoon Joong Kang; Tetsuya Ioji; Maki Touma; Bruce Reinhold; Derin B. Keskin; Ellis L. Reinherz; Tetsuro Sasada (3666-3678).
Nanotechnology-based antigen delivery has been developing as a vaccine strategy due to its dose-sparing and prolonged antigen presentation features. In the current study, we examined the feasibility of nanoparticle (NP)-mediated delivery of antigenic peptides to efficiently induce cytotoxic T lymphocyte responses against tumor-associated self-antigens in C57BL/6 mouse models. The biodegradable poly(d,l-lactide-co-glycolide) nanoparticle (PLGA-NP) carrying murine melanoma antigenic peptides, hgp10025–33 and TRP2180–188, were prepared by double emulsion method. Efficient uptake of PLGA-NP by murine dendritic cells was shown in vitro and in vivo, using NP labeled with the fluorescent dye DiD. Intradermal injection of peptide-loaded PLGA-NP into mice induced antigen-specific T cell responses more strongly than the peptides mixed with Freund’s adjuvant. More importantly, vaccination with PLGA-NP carrying both TRP2180–188 and a toll-like receptor 4 agonist, monophosphoryl lipid A, significantly delayed growth of subcutaneously inoculated B16 melanoma cells in a prophylactic setting. Furthermore, the anti-tumor activity of NP-mediated peptide vaccination was significantly augmented by combined treatment with interferon-γ, which might prevent tumor escape through up-regulation of MHC class I expression on tumor cells. Our findings demonstrate the feasibility of NP-mediated antigen delivery for cancer immunotherapy, in particular when immune escape mechanisms of tumor cells are blocked simultaneously.
Keywords: Nanoparticle; Peptide; Toll-like receptor 4 agonist; Tumor immunity; Interferon-γ; Immune escape;

Chondrogenesis of human mesenchymal stem cells mediated by the combination of SOX trio SOX5, 6, and 9 genes complexed with PEI-modified PLGA nanoparticles by Ji Sun Park; Han Na Yang; Dae Gyun Woo; Su Yeon Jeon; Hyun-Jin Do; Hye-Young Lim; Jae-Hwan Kim; Keun-Hong Park (3679-3688).
Target gene transfection for desired cell differentiation has recently become a major issue in stem cell therapy. For the safe and stable delivery of genes into human mesenchymal stem cells (hMSCs), we employed a non-viral gene carrier system such as polycataionic polymer, poly(ethyleneimine) (PEI), polyplexed with a combination of SOX5, 6, and 9 fused to green fluorescence protein (GFP), yellow fluorescence protein (YFP), or red fluorescence protein (RFP) coated onto PLGA nanoparticles. The transfection efficiency of PEI-modified PLGA nanoparticle gene carriers was then evaluated to examine the potential for chondrogenic differentiation by carrying the exogenous SOX trio (SOX5, 6, and 9) in hMSCs. Additionally, use of PEI-modified PLGA nanoparticle gene carriers was evaluated to investigate the potential for transfection efficiency to increase the potential ability of chondrogenesis when the trio genes (SOX5, 6, and 9) polyplexed with PEI were delivered into hMSCs. SOX trio complexed with PEI-modified PLGA nanoparticles led to a dramatic increase in the chondrogenesis of hMSCs in in vitro culture systems. For the PEI/GFP and PEI/SOX5, 6, and 9 genes complexed with PLGA nanoparticles, the expressions of GFP as reporter genes and SOX9 genes with PLGA nanoparticles showed 80% and 83% of gene transfection ratios into hMSCs two days after transfection, respectively.
Keywords: Gene transfection; hMSCs; PLGA nanoparticles; Gene carrier; SOX trio;