Biomaterials (v.31, #10)

Electroconductive hydrogels (ECHs) are composite biomaterials that bring together the redox switching and electrical properties of inherently conductive electroactive polymers (CEPs) with the facile small molecule transport, high hydration levels and biocompatibility of cross-linked hydrogels. General methods for the synthesis of electroconductive hydrogels as polymer blends and as polymer co-networks via chemical oxidative, electrochemical and/or a combination of chemical oxidation followed by electrochemical polymerization techniques are reviewed. Specific examples are introduced to illustrate the preparation of electroconductive hydrogels that were synthesized from poly(HEMA)-based hydrogels with polyaniline and from poly(HEMA)-based hydrogels with polypyrrole. The key applications of electroconductive hydrogels; as biorecognition membranes for implantable biosensors, as electro-stimulated drug release devices for programmed delivery, and as the low interfacial impedance layers on neuronal prostheses are highlighted. These applications provide great new horizons for these stimuli responsive, biomimetic polymeric materials.
Keywords: Hydrogels; Polypyrrole; Polyaniline; Blends; Co-networks; Electrically conductive hydrogel;

Enhancement of osteoblast adhesion to UV-photofunctionalized titanium via an electrostatic mechanism by Fuminori Iwasa; Norio Hori; Takeshi Ueno; Hajime Minamikawa; Masahiro Yamada; Takahiro Ogawa (2717-2727).
The mechanism underlying the recently found photofunctionalization of titanium is unknown. We focused on how the initial interaction between the cells and photofunctionalized titanium is enhanced at a molecular-level and the role played by the electrostatic status of the titanium surfaces in the possible regulatory mechanism for determining their bioactivity. Rat bone marrow-derived osteoblasts were cultured on untreated and ultraviolet (UV)-treated titanium surfaces. UV treatment converted the titanium surfaces from hydrophobic to superhydrophilic. The number of osteoblasts attached to UV-treated titanium surfaces was substantially greater than that attached to untreated surfaces (5-fold and 2-fold after 3 and 24 h of incubation, respectively). Osteoblasts cultured for 3 and 24 h on these titanium surfaces were detached mechanically by vibrational force and enzymatically by trypsin treatment. Cell adhesion evaluated by the percentage of remaining cells after these detachments was substantially greater for cells on UV-treated titanium surfaces compared to untreated titanium surfaces (110–120% greater for cells incubated for 3 h and 50–60% greater for cells incubated for 24 h). Osteoblasts on UV-treated surfaces expressed more vinculin. UV-enhancing effect in cell adhesion was also demonstrated under a serum-free condition. UV-enhanced cell adhesion was abrogated when the UV-treated titanium surfaces were electrostatically neutralized by either removing the electric charge or masking with monovalent anions, while the surfaces maintained superhydrophilicity. In conclusion, the establishment of osteoblast adhesion is accelerated and augmented remarkably on UV-treated titanium surfaces, associated with upregulated expression of vinculin. This study has identified an electrostatic property of UV-treated titanium surfaces playing a regulatory role in determining their bioactivity, superseding the effect of the hydrophilic nature of these surfaces. A mechanism underlying the UV-induced conversion of titanium from bioinert to bioactive, in which direct cell–titanium interaction is exclusively enabled, is proposed.
Keywords: Bone–titanium integration; Vinculin; Osseointegration; Total hip replacement; Dental implant; Bioactive;

Direct and indirect effects of microstructured titanium substrates on the induction of mesenchymal stem cell differentiation towards the osteoblast lineage by Rene Olivares-Navarrete; Sharon L. Hyzy; Daphne L. Hutton; Christopher P. Erdman; Marco Wieland; Barbara D. Boyan; Zvi Schwartz (2728-2735).
Microstructured and high surface energy titanium substrates increase osseointegration in vivo. In vitro, osteoblast differentiation is increased, but effects of the surface directly on multipotent mesenchymal stem cells (MSCs) and consequences for MSCs in the peri-implant environment are not known. We evaluated responses of human MSCs to substrate surface properties and examined the underlying mechanisms involved. MSCs exhibited osteoblast characteristics (alkaline phosphatase, RUNX2, and osteocalcin) when grown on microstructured Ti; this effect was more robust with increased hydrophilicity. Factors produced by osteoblasts grown on microstructured Ti were sufficient to induce co-cultured MSC differentiation to osteoblasts. Silencing studies showed that this was due to signaling via α2β1 integrins in osteoblasts on the substrate surface and paracrine action of secreted Dkk2. Thus, human MSCs are sensitive to substrate properties that induce osteoblastic differentiation; osteoblasts interact with these surface properties via α2β1 and secrete Dkk2, which acts on distal MSCs.
Keywords: Titanium; Human mesenchymal stem cells; Osteoblasts; Microstructured surfaces; Hydrophilic surfaces; Co-culture;

The attenuation of platelet and monocyte activation in a rabbit model of extracorporeal circulation by a nitric oxide releasing polymer by Terry C. Major; David O. Brant; Melissa M. Reynolds; Robert H. Bartlett; Mark E. Meyerhoff; Hitesh Handa; Gail M. Annich (2736-2745).
Nitric oxide (NO) has been shown to reduce thrombogenicity by decreasing platelet and monocyte activation by the surface glycoprotein, P-selectin and the integrin, CD11b, respectively. In order to prevent platelet and monocyte activation with exposure to an extracorporeal circulation (ECC), a nitric oxide releasing (NORel) polymeric coating composed of plasticized polyvinyl chloride (PVC) blended with a lipophilic N-diazeniumdiolate was evaluated in a 4 h rabbit thrombogenicity model using flow cytometry. The NORel polymer significantly reduced ECC thrombus formation compared to polymer control after 4 h blood exposure (2.8 ± 0.7 NORel vs 6.7 ± 0.4 pixels/cm2 control). Platelet count (3.4 ± 0.3 NORel vs 2.3 ± 0.3 × 108/ml control) and function as measured by aggregometry (71 ± 3 NORel vs 17 ± 6% control) were preserved after 4 h exposure in NORel versus control ECC. Plasma fibrinogen levels significantly decreased in both NORel and control groups. Platelet P-selectin mean fluorescence intensity (MFI) as measured by flow cytometry was attenuated after 4 h on ECC to ex vivo collagen stimulation (27 ± 1 NORel vs 40 ± 2 MFI control). Monocyte CD11b expression was reduced after 4 h on ECC with NORel polymer (87 ± 14 NORel vs 162 ± 30 MFI control). These results suggest that the NORel polymer coatings attenuate the increase in both platelet P-selectin and monocytic CD11b integrin expression in blood exposure to ECCs. These NO-mediated platelet and monocytic changes were shown to improve thromboresistance of these NORel-polymer-coated ECCs for biomedical devices.
Keywords: Nitric oxide; Monocyte CD11b; Platelet P-selectin; Extracorporeal circulation; Hemocompatible polymer coating; Diazeniumdiolates;

A hybrid, localized and release-controlled delivery system for bone growth factors consisting of a liposomal core incorporated into a shell of alternating layer-by-layer self-assembled natural polyelectrolytes has been formulated. Hydrophilic, monodisperse, spherical and stable cationic nanoparticles (≤350 nm) with an extended shelf-life resulted. Cytocompatibility was previously assayed with MC3T3-E1.4 mouse preosteoblasts showing no adverse effects on cell viability. In this study, the in vivo biocompatibility of unloaded and loaded nanoparticles with osteogenic protein-1 or OP-1 was investigated. Young male Wistar rats were injected intramuscularly and monitored over a period of 10 weeks for signs of inflammation and/or adverse reactions. Blood samples (600 μL/collection) were withdrawn followed by hematological and biochemical analysis. Body weight changes over the treatment period were noted. Major organs were harvested, weighed and examined histologically for any pathological changes. Finally, the injection site was identified and examined immunohistochemically. Overall, all animals showed no obvious toxic health effects, immune responses and/or change in organ functions. This hybrid core–shell nanoparticulate delivery system localizes the effect of the released bioactive load within the site of injection in muscle with no significant tissue distress. Hence, a safe and promising carrier for therapeutic growth factors and possibly other biomolecules is presented.
Keywords: Biocompatibility; BMP; Drug delivery; Foreign body response; Haemocompatibility; Nanoparticle;

A Streptococcus pyogenes derived collagen-like protein as a non-cytotoxic and non-immunogenic cross-linkable biomaterial by Yong Y. Peng; Ayumi Yoshizumi; Stephen J. Danon; Veronica Glattauer; Olga Prokopenko; Oleg Mirochnitchenko; Zhuoxin Yu; Masayori Inouye; Jerome A. Werkmeister; Barbara Brodsky; John A.M. Ramshaw (2755-2761).
A range of bacteria have been shown to contain collagen-like sequences that form triple-helical structures. Some of these proteins have been shown to form triple-helical motifs that are stable around body temperature without the inclusion of hydroxyproline or other secondary modifications to the protein sequence. This makes these collagen-like proteins particularly suitable for recombinant production as only a single gene product and no additional enzyme needs to be expressed. In the present study, we have examined the cytotoxicity and immunogenicity of the collagen-like domain from Streptococcus pyogenes Scl2 protein. These data show that the purified, recombinant collagen-like protein is not cytotoxic to fibroblasts and does not elicit an immune response in SJL/J and Arc mice. The freeze dried protein can be stabilised by glutaraldehyde cross-linking giving a material that is stable at >37 °C and which supports cell attachment while not causing loss of viability. These data suggest that bacterial collagen-like proteins, which can be modified to include specific functional domains, could be a useful material for medical applications and as a scaffold for tissue engineering.
Keywords: Recombinant protein; Cell viability; Fibroblast; Immune response; Cross-linking;

Resorption of monetite granules in alveolar bone defects in human patients by Faleh Tamimi; Jesus Torres; David Bassett; Jake Barralet; Enrique L. Cabarcos (2762-2769).
Bone grafting is often required to restore mandibular or maxillary bone volume prior to prosthetic tooth root implantation. Preclinical animal models are often used to study the in vivo properties of new bone graft products designed for human use. Although animal studies may offer valuable data regarding bioperformance, materials do not necessarily perform the same in human patients. In this study we implanted bovine hydroxyapatite (BH), a widely used porous apatite granule, and dicalcium phosphate anhydrous (monetite) granules, bilaterally in human patients post extraction alveolar sockets. After six months, histomorphometrical analysis of the biopsies revealed that the amount of bone regenerated with monetite (59.5 ± 13%) was significantly higher than that obtained with BH (33.1% ± 4.9), while the amount of unresorbed graft was higher in the sockets treated with BH (37.8 ± 6.1) than in those implanted with monetite (25.8 ± 14.3). Resorption of calcium phosphate ceramics is discussed by applying the Hixon–Crowell dissolution model.
Keywords: Monetite; Human; Resorption; Bovine hydroxyapatite; Histomorphometry; Histology;

Regeneration of functional nerves within full thickness collagen–phosphorylcholine corneal substitute implants in guinea pigs by Christopher R. McLaughlin; M. Carmen Acosta; Carolina Luna; Wenguang Liu; Carlos Belmonte; M. Griffith; Juana Gallar (2770-2778).
Our objective was to evaluate promotion of tissue and nerve regeneration by extracellular matrix (ECM) mimics, using corneal implantation as a model system. Porcine type I collagen and 2-methacryloyloxyethyl phosphorylcholine (MPC) were crosslinked using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) and moulded into appropriate corneal dimensions to serve as substitutes for natural corneal ECM. These were implanted as full thickness grafts by penetrating keratoplasty into the corneas of guinea pigs after removal of the host tissue, and tracked over eight months, by clinical examination, slit-lamp biomicroscopy, and esthesiometry. Histopathology and ex vivo nerve terminal impulse recordings were performed at three months and at eight months. The implants promoted regeneration of corneal cells, nerves and the tear film, while retaining optical clarity. After three months, electrophysiological recordings showed evidence of mechano-nociceptors, and polymodal units inside the implants, while cold-sensitive units were present only on the peripheral host cornea. Following eight months, the incidence of nerve activity and the frequency of spontaneous firing were higher than in control eyes as reported for regenerating fibers. Active cold nerve terminals also innervated the implant area. We show that ECM mimetic materials can promote regeneration of corneal cells and functional nerves. The simplicity in fabrication and demonstrated functionality shows potential for ECM substitutes in future clinical applications.
Keywords: Cornea; Nerve regeneration; Collagen structure; Scaffold; Ophthalmology;

Transient in vitro epigenetic reprogramming of skin fibroblasts into multipotent cells by Xiang-Qing Zhu; Xing-Hua Pan; Weibo Wang; Qiang Chen; Rong-Qing Pang; Xue-Min Cai; Andrew R. Hoffman; Ji-Fan Hu (2779-2787).
Multipotent stem cells have the potential to establish a new field of promising regenerative medicine to treat tissue damage, genetic disorders, and degenerative diseases. However, limited resource of stem cells has turned to be an evitable obstacle in clinical applications. We utilized a simple in vitro epigenetic reprogramming approach to convert skin fibroblasts into multipotent cells. After transient reprogramming, stem cell markers, including Oct4 and Nanog, became activated in the treated cells. The reprogrammed cells were multipotent as demonstrated by their ability to differentiate into a variety of cells and to form teratomas. Genomic imprinting of insulin-like growth factor II (Igf2) and H19 was not affected by this short period of cell reprogramming. This study may provide an alternative strategy to efficiently generate patient-specific stem cells for basic and clinical research, solving major hurdles of virally-induced pluripotent stem (iPS) cells that entail the potential risks of mutation, gene instability, and malignancy.
Keywords: Induced multipotent stem cells; iMS cells; Epigenetic reprogramming; Multipotency; Fish oocyte extracts; Differentiation;

A versatile pH sensitive chondroitin sulfate–PEG tissue adhesive and hydrogel by Iossif Strehin; Zayna Nahas; Karun Arora; Thao Nguyen; Jennifer Elisseeff (2788-2797).
We developed a chondroitin sulfate–polyethylene glycol (CS–PEG) adhesive hydrogel with numerous potential biomedical applications. The carboxyl groups on chondroitin sulfate (CS) chains were functionalized with N-hydroxysuccinimide (NHS) to yield chondroitin sulfate succinimidyl succinate (CS–NHS). Following purification, the CS–NHS molecule can react with primary amines to form amide bonds. Hence, using six arm polyethylene glycol amine PEG–(NH2)6 as a crosslinker we formed a hydrogel which was covalently bound to proteins in tissue via amide bonds. By varying the initial pH of the precursor solutions, the hydrogel stiffness, swelling properties, and kinetics of gelation could be controlled. The sealing/adhesive strength could also be modified by varying the damping and storage modulus properties of the material. The adhesive strength of the material with cartilage tissue was shown to be ten times higher than that of fibrin glue. Cells encapsulated or in direct contact with the material remained viable and metabolically active. Furthermore, CS–PEG material produced minimal inflammatory response when implanted subcutaneously in a rat model and enzymatic degradation was demonstrated in vitro. This work establishes an adhesive hydrogel derived from biological and synthetic components with potential application in wound healing and regenerative medicine.
Keywords: Bioactivity; Chondroitin sulfate; DMA (dynamic mechanical analysis); Hydrogel; Polyethylene oxide; Tissue adhesive;

The effect of scaffold composition on the early structural characteristics of chondrocytes and expression of adhesion molecules by Jan C. Schagemann; Haymo Kurz; Michelle E. Casper; James S. Stone; Mahrokh Dadsetan; Sun Yu-Long; Eike H. Mrosek; James S. Fitzsimmons; Shawn W. O'Driscoll; Gregory G. Reinholz (2798-2805).
Previously we demonstrated that chondrocyte ECM synthesis and mitotic activity was dependent on scaffold composition when cultured on uncoated PCL scaffolds (pPCL) or PCL composites containing hyaluronan (PCL/HA), chitosan (PCL/CS), fibrin (PCL/F), or collagen type I (PCL/COL1). We hypothesized that initial cell contact with these biomaterials results in ultrastructural changes and alters CD44 and integrin beta1 expression. The current study was designed to investigate the early ultrastructural responses of chondrocytes on these scaffolds and expression of CD44 and integrin beta1. A common observation 1 h after seeding was the abundance of cell processes. Different types of cell processes occurred in different areas of the same cell and on different cells within the same composite. Chondrocytes seeded onto PCL/CS had the greatest cell surface enhancement. PCL/HA promoted CD44 expression and almost spherical cells with a low degree of surface enhancement. PCL/COL1 enabled continuing expression of integrin beta1 and CD44. In contrast, cells in PCL/CS, PCL/F and pPCL promoted elliptic cells with a higher degree of surface enhancement and no prolonged CD44 and integrin beta1 expression. A strong variability of cell surface processes indicated either reparative or degenerative adaptation to the artificial environment. Interestingly, we found initial integrin beta1 expression in all composite scaffolds, but not in pPCL although this promoted strong adhesiveness as indicated by the formation of stress fibers. In conclusion, chondrocytes respond to biomaterials early after implantation by altering ultrastructural characteristics and expression of CD44 and integrin beta1.
Keywords: Scaffolds; Polycaprolactone; Cell adhesion; CD44; Integrins; Chondrocytes;

Poly(3-hydroxybutyrate) multifunctional composite scaffolds for tissue engineering applications by Superb K. Misra; Tahera I. Ansari; Sabeel P. Valappil; Dirk Mohn; Sheryl E. Philip; Wendelin J. Stark; Ipsita Roy; Jonathan C. Knowles; Vehid Salih; Aldo R. Boccaccini (2806-2815).
Poly(3-hydroxybutyrate) (P(3HB)) foams exhibiting highly interconnected porosity (85% porosity) were prepared using a unique combination of solvent casting and particulate leaching techniques by employing commercially available sugar cubes as porogen. Bioactive glass (BG) particles of 45S5 Bioglass® grade were introduced in the scaffold microstructure, both in micrometer ((m-BG), <5 μm) and nanometer ((n-BG), 30 nm) sizes. The in vitro bioactivity of the P(3HB)/BG foams was confirmed within 10 days of immersion in simulated body fluid and the foams showed high level of protein adsorption. The foams interconnected porous microstructure proved to be suitable for MG-63 osteoblast cell attachment and proliferation. The foams implanted in rats as subcutaneous implants resulted in a non-toxic and foreign body response after one week of implantation. In addition to showing bioactivity and biocompatibility, the P(3HB)/BG composite foams also exhibited bactericidal properties, which was tested on the growth of Staphylococcus aureus. An attempt was made at developing multifunctional scaffolds by incorporating, in addition to BG, selected concentrations of Vitamin E or/and carbon nanotubes. P(3HB) scaffolds with multifunctionalities (viz. bactericidal, bioactive, electrically conductive, antioxidative behaviour) were thus produced, which paves the way for next generation of advanced scaffolds for bone tissue engineering.
Keywords: Bone tissue engineering; Scaffolds; Composite materials; Bioactive glass; Poly(3-hydroxybutyrate); Carbon nanotubes;

The effect of FGF-1 loaded alginate microbeads on neovascularization and adipogenesis in a vascular pedicle model of adipose tissue engineering by Monica L. Moya; Ming-Huei Cheng; Jung-Ju Huang; Megan E. Francis-Sedlak; Shu-wei Kao; Emmanuel C. Opara; Eric M. Brey (2816-2826).
Engineered vascularized adipose tissue could serve as an alternative to traditional tissue reconstruction procedures. Adipose formation occurs in a coordinated fashion with neovascularization. Previous studies have shown that extracellular matrix-based materials supplemented with factors that stimulate neovascularization promote adipogenesis in a number of animal models. The present study examines the ability of fibroblast growth factor (FGF-1) delivered from alginate microbeads to induce neovascularization and adipogenesis in type I collagen gels in a vascular pedicle model of adipose tissue engineering. FGF-1 loaded microbeads stimulated greater vascular network formation in an in vitro 3D co-culture model than a single bolus of FGF-1. In in vivo studies, FGF-1 loaded beads suspended in collagen and implanted in a chamber surrounding the exposed femoral pedicle of a rat resulted in a significant increase in vascular density at 1 and 6 weeks in comparison to bolus administration of FGF-1. Staining for smooth muscle actin showed that over 48% of vessels had associated mural cells. While an increase in neovascularization was achieved, there was less than 3% adipose under any condition. These results show that delivery of FGF-1 from alginate beads stimulated a more persistent neovascularization response than bolus FGF-1 both in vitro and in vivo. However, unlike previous studies, this increased neovascularization did not result in adipogenesis. Future studies need to provide a better understanding of the relationship between neovascularization and adipogenesis in order to design advanced tissue engineering therapies.
Keywords: Alginate; Fibroblast growth factor; Adipose tissue engineering; Angiogenesis;

The effect of unlocking RGD-motifs in collagen I on pre-osteoblast adhesion and differentiation by Anna V. Taubenberger; Maria A. Woodruff; Huifen Bai; Daniel J. Muller; Dietmar W. Hutmacher (2827-2835).
Denaturation of extracellular matrix proteins exposes cryptic binding sites. It is hypothesized that binding of cell adhesion receptors to these cryptic binding sites regulates cellular behaviour during tissue repair and regeneration. To test this hypothesis, we quantify the adhesion of pre-osteoblastic cells to native (Col) and partially-denatured (pdCol) collagen I using single-cell force spectroscopy. During early stages of cell attachment (≤180 s) pre-osteoblasts (MC3T3-E1) adhered significantly stronger to pdCol compared to Col. RGD (Arg-Gly-Asp)-containing peptides suppressed this elevated cell adhesion. We show that the RGD-binding α5β1- and αv-integrins mediated pre-osteoblast adhesion to pdCol, but not to Col. On pdCol pre-osteoblasts had a higher focal adhesion kinase tyrosine-phosphorylation level that correlated with enhanced spreading and motility. Moreover, pre-osteoblasts cultured on pdCol showed a pronounced matrix mineralization activity. Our data suggest that partially-denatured collagen exposes RGD-motifs that trigger binding of α5β1- and αv-integrins. These integrins initiate cellular processes that stimulate osteoblast adhesion, spreading, motility and differentiation. Taken together, these quantitative insights reveal an approach for the development of alternative collagen I- based surfaces for tissue engineering applications.
Keywords: Collagen I; Gelatin; Cell adhesion; AFM (atomic force microscopy); Biomineralisation; Osteogenesis;

The biocompatibility of Pluronic®F127 fibrinogen-based hydrogels by Yonatan Shachaf; Maya Gonen-Wadmany; Dror Seliktar (2836-2847).
Our research is focused on the design of hydrogel biomaterials that can be used for 3-D cell encapsulation and tissue engineering. In this study, our goal was to engineer a temperature-responsive biomaterial to possess bioactive properties using polymer and protein chemistry, and at the same time provide the biomaterial with susceptibility to cell-mediated remodeling. Toward this goal, we developed a biomimetic material that can harness the bioactive properties of fibrinogen and the unique structural properties of Pluronic®F127. Pluronic®F127 is a synthetic block copolymer that exhibits reverse thermal gelation (RTG) in response to small changes in ambient temperature. We conjugated fibrinogen to Pluronic®F127 to create a biosynthetic precursor with tunable physicochemical properties based on the relationship between the two constituents. A hydrogel matrix was formed from the fibrinogen-F127 adducts by free-radical polymerization using light activation (photo-polymerization). These materials displayed a reversible temperature-induced physical sol–gel transition and an irreversible light-activated chemical cross-linking. The susceptibility of this hydrogel biomaterial to protease degradation and consequent cell-mediated remodeling was controlled by the Pluronic®F127 constituent. The protein-based material also conveyed inductive signals to cells through bioactive sites on the fibrinogen backbone, as well as through structural properties such as the matrix modulus. We apply these materials as a tissue engineering hydrogel scaffold for 3-D in vitro culture of dermal fibroblasts in order to gain a better understanding of how the material bioactivity and matrix properties can independently affect cell morphology and remodeling.
Keywords: Fibrinogen; Fibroblast; Hydrogel; Polyethylene oxide; Pluronic; Scaffold;

The osteogenic properties of CaP/silk composite scaffolds by Yufeng Zhang; Chengtie Wu; Thor Friis; Yin Xiao (2848-2856).
The rationale for the present study was to develop porous CaP/silk composite scaffolds with a CaP-phase distribution and pore architecture better suited to facilitate osteogenic properties of human bone mesenchymal stromal cells (BMSCs) and in vivo bone formation abilities. This was achieved by first preparing CaP/silk hybrid powders which were then incorporated into silk to obtain uniform CaP/silk composite scaffolds, by means of a freeze-drying method. The composition, microstructure and mechanical properties of the CaP/silk composite scaffolds were ascertained by X-ray diffraction (XRD), Fourier transform infrared spectra (FTIR), scanning electron microscope (SEM) and a universal mechanical testing machine. BMSCs were cultured in these scaffolds and cell proliferation analyzed by confocal microscopy and MTS assay. Alkaline phosphatase (ALP) activity and osteogenic gene expression were assayed to determine if osteogenic differentiation had taken place. A calvarial defect model in SCID mice was used to determine the in vivo bone forming ability of the hybrid CaP/silk scaffolds. Our results showed that incorporating the hybrid CaP/silk powders into silk scaffolds improved both pore structure architecture and distribution of CaP powders in the composite scaffolds. By incorporating the CaP phase into silk scaffolds in vitro osteogenic differentiation of BMSCs was enhanced and there was increased in vivo cancellous bone formation. Here we report a method with which to prepare Ca/P composite scaffolds with a pore structure and Ca/P distribution better suited to facilitate BMSC differentiation and bone formation.
Keywords: Silk fibroin; Scaffold; Bone mesenchymal stromal cells; Bone;

Evaluation of polymer matrices for an adsorptive approach to plasma detoxification by Joseph A. Costanzo; Courtney A. Ober; Richard Black; Giorgio Carta; Erik J. Fernandez (2857-2865).
Acute liver failure arises when potentially toxic metabolites accumulate in the bloodstream because of a breakdown in liver function. New extracorporeal systems combining membrane and adsorbent technologies are being developed to replace critical liver detoxification functions between diagnosis and transplantation. This study addresses the adsorption of representative plasma components on four different hydrophobic, polymeric adsorbents for possible use in an extracorporeal hemodialysis device. The adsorbents considered span a range of pore sizes and include both strongly hydrophobic divinylbenzene (DVB) matrices as well as a less hydrophobic acrylate matrix. Adsorption equilibrium and rate measurements were made for these matrices using human serum albumin (HSA), polyclonal human immunoglobulin G (IgG), and bilirubin (BR), as representative plasma components. Pore size was found to contribute significantly to selectivity. Results demonstrated that strongly hydrophobic materials with pore sizes that allow free access to protein-bound BR are most effective for BR removal whether they are initially clean or pre-saturated with HSA.
Keywords: Plasma proteins; Protein adsorption; Bilirubin; Hemodialysis; Liver Failure;

Cu2+-labeled, SPION loaded porous silica nanoparticles for cell labeling and multifunctional imaging probes by Daksha Patel; Arnold Kell; Benoit Simard; Jixian Deng; Bo Xiang; Hung-Yu Lin; Marco Gruwel; Ganghong Tian (2866-2873).
We have developed an ion-sensing nanoparticle that is comprised of a superparamagnetic iron oxide (SPIO) core encapsulated with a porous silica shell. The latter can be readily anchored with ligands capable of coordinating with positron-emitting metal. Evidently, this nanoparticle has a great potential for use in cell tracking with magnetic resonance (MR) imaging and positron emission tomography (PET). Herein we report the synthesis, surface functionalization and characterization of the magnetic nanoparticle-based probes and evaluate their cell-labeling efficacy, cytoxicity and relaxivity in comparison to one of the most commonly utilized MRI contrast agents, Feridex®.
Keywords: Cell viability; Copper; Cytotoxicity; In vitro test; MRI (magnetic resonance imaging); Surface modification;

The efficacy of self-assembled cationic antimicrobial peptide nanoparticles against Cryptococcus neoformans for the treatment of meningitis by Huaying Wang; Kaijin Xu; Lihong Liu; Jeremy P.K. Tan; Yunbo Chen; Yongtao Li; Weimin Fan; Zeqing Wei; Jifang Sheng; Yi-Yan Yang; Lanjuan Li (2874-2881).
Cationic antimicrobial peptides have received considerable interest as new therapeutics with the potential for treatment of multiple-drug resistant infections. We recently reported that cholesterol-conjugated G3R6TAT (CG3R6TAT) formed cationic nanoparticles via self-assembly, which demonstrated strong antimicrobial activities against various types of microbes in vitro. In this study, the possibility of using these nanoparticles for treatment of Cryptococcus neoformans (yeast)-induced brain infections was studied. The antimicrobial activity of the nanoparticles was tested against 12 clinical isolates of C. neoformans in comparison with conventional antifungal agents amphotericin B and fluconazole. Minimum inhibitory concentrations (MICs) of the nanoparticles were determined to be much lower than those of fluconazole in all the isolates, but slightly higher than those of amphotericin B in some isolates. At a concentration three times higher than the MIC, the nanoparticles completely sterilized C. neoformans after 3.5 h. Cell wall disruption and release of cytoplasmic content were observed under TEM. The biodistribution studies of FITC-loaded nanoparticles in rabbits revealed that the nanoparticles were able to cross the blood-brain barrier (BBB). The efficacy of nanoparticles was further evaluated in a C. neoformans meningitis rabbit model. The nanoparticles crossed the BBB and suppressed the yeast growth in the brain tissues with similar efficiency as amphotericin B did. In addition, unlike amphotericin B, they neither caused significant damage to the liver and kidney functions nor interfered with the balance of electrolytes in the blood. CG3R6TAT nanoparticles can be a promising antimicrobial agent for treatment of brain infections caused by C. neoformans.
Keywords: Antimicrobial peptide; Cationic nanoparticles; Cryptococcus neoformans; Meningitis; Blood-brain barrier (BBB);

The intracellular drug delivery and anti tumor activity of doxorubicin loaded poly(γ-benzyl l-glutamate)-b-hyaluronan polymersomes by Kamal K. Upadhyay; Anant N. Bhatt; Anil K. Mishra; Bilikere S. Dwarakanath; Sanyog Jain; Christophe Schatz; Jean-François Le Meins; Abdullah Farooque; Godugu Chandraiah; Amit K. Jain; Ambikanandan Misra; Sébastien Lecommandoux (2882-2892).
We have investigated the intracellular delivery of doxorubicin (DOX) loaded poly(γ-benzyl l-glutamate)-block-hyaluronan (PBLG-b-HYA) based polymersomes (PolyDOX) in high (MCF-7) and low (U87) CD44 expressing cancer cell models. DOX was successfully loaded into polymersomes using nanoprecipitation method and in vitro drug release pattern were achieved at pH 5.5 and 7.4 up to 10 days. Block copolymer vesicles without loaded DOX were non cytotoxic in both cells at concentration 150–650 μg/mL. Flow cytometry data suggested successful uptake of PolyDOX in cells and high accumulation was found in MCF-7 than U87 cells. Microscopy imagings revealed that in MCF-7 cells PolyDOX was more in cytoplasm and free DOX in nuclei, whereas in U87 cells free DOX was also found in the cytoplasm. Cytotoxicity of the drug was concentration and exposure time dependent. In addition, PolyDOX significantly enhanced reactive oxygen species (ROS) level in both cells. PolyDOX also suppressed growth of breast tumor on female Sprague–Dawley (SD) rats as compared to phosphate buffer saline pH 7.4 (PBS) control group. In addition reduced level of serum enzymes (LDH and CPK) by PolyDOX formulation indicated less cardiotoxicity of DOX after loading in polymersomes. Results suggest that intracellular delivery of PolyDOX was depended on the CD44 expression level in cells due to presence of hyaluronic acid on the surface of polymersomes, and could be used as a self-targeting drug delivery cargo in over-expressed CD44 glycoprotein cells of breast cancer.
Keywords: Polymersomes; Hyaluronan; Doxorubicin; Cell uptake; Cytotoxicity; Self-assembly;

The ability of a collagen/calcium phosphate scaffold to act as its own vector for gene delivery and to promote bone formation via transfection with VEGF165 by Michael Keeney; Jeroen J.J.P. van den Beucken; Peter M. van der Kraan; John A. Jansen; Abhay Pandit (2893-2902).
Collagen/calcium phosphate scaffolds have been used for bone reconstruction due to their inherent similarities to the bone extracellular matrix. Calcium phosphate alone has also been used as a non-viral vector for gene delivery. The aim of this study was to determine the capability of a collagen/calcium phosphate scaffold to deliver naked plasmid DNA and mediate transfection in vivo. The second goal of the study was to deliver a plasmid encoding vascular endothelial growth factor165 (pVEGF165) to promote angiogenesis, and hence bone formation, in a mouse intra-femoral model. The delivery of naked plasmid DNA resulted in a 7.6-fold increase in mRNA levels of β-Galactosidase compared to the delivery of plasmid DNA complexed with a partially degraded PAMAM dendrimer (dPAMAM) in a subcutaneous murine model. When implanted in a muirne intra-femoral model, the delivery of pVEGF165 resulted in a 2-fold increase in bone volume at the defect site relative to control scaffolds without pVEGF165. It was concluded that a collagen/calcium phosphate scaffold can mediate transfection without the use of additional transfection vectors and can promote bone formation in a mouse model via the delivery of pVEGF165.
Keywords: Bone tissue engineering; Gene therapy; Calcium phosphate; Collagen;

Targeting of the prostacyclin specific IP1 receptor in lungs with molecular conjugates comprising prostaglandin I2 analogues by Johannes Geiger; Manish K. Aneja; Günther Hasenpusch; Gülnihal Yüksekdag; Grit Kummerlöwe; Burkhard Luy; Tina Romer; Ulrich Rothbauer; Carsten Rudolph (2903-2911).
Molecular conjugates comprising targeting ligands hold great promise for site-specific gene delivery to distant tumors and individual organs including the lung. Here we show that prostaglandin I2 analogues can be used to improve gene transfer efficiency of polyethylenimine (PEI) gene vectors on bronchial and alveolar epithelial cells in vitro and lungs of mice in vivo. Prostacyclin (IP1) receptor expression was confirmed in pulmonary epithelial cell lines by western blot. Iloprost (ILO) and treprostinil (TRP), two prostaglandin I2 analogues, were conjugated to fluorescein-labeled BSA (FLUO-BSA) and compared for IP1 receptor binding/uptake in different lung cell lines. Binding of FLUO-BSA-ILO was 2–4-fold higher than for FLUO-BSA-TRP and could be specifically inhibited by free ILO and IP1 receptor antagonist CAY10449. Internalization of FLUO-BSA-ILO was confirmed by confocal microscopy. Molecular conjugates of PEI and ILO (PEI-g-ILO) were synthesized with increasing coupling degree (F ILO (ILO:PEI) = 2, 5, 8, 16) and analyzed for DNA binding, particle formation and transfection efficiency. At optimized conditions (N/P 4, F ILO = 5), gene expression using PEI-g-ILO was significantly up to 46-fold higher than for PEI gene vectors and specifically inhibited by CAY10449. Gene expression in the lungs of mice after aerosol delivery was 14-fold higher with PEI-g-ILO F ILO = 5 than for PEI. We suggest that targeting of IP1 receptor using ILO represents a promising approach to improve pulmonary gene transfer.
Keywords: Gene transfer; Gene expression; Lung; Iloprost; Polyethylenimine; Aerosol;

DNA/polyethyleneimine/hyaluronic acid small complex particles and tumor suppression in mice by Tomoko Ito; Chieko Yoshihara; Katsuyuki Hamada; Yoshiyuki Koyama (2912-2918).
The highest barriers for non-viral vectors to an efficient in vivo gene transfection would be (1) non-specific interaction with biological molecules, and (2) large size of the DNA complex particles. Protective coating of the DNA/polyethyleneimine (PEI) complexes by hyaluronic acid (HA) effectively diminished the adverse interactions with biological molecules. Here we found HA also protected the DNA/PEI complexes against aggregation and inactivation through lyophilization-and-rehydration procedures. It allows us to prepare the concentrated very small DNA complex particles (<70 nm) suspension by preparing the complexes at highly diluted conditions, followed by lyophilized-and-rehydrated to a small volume. In vivo gene expression efficiency of the small complex was examined with mice subcutaneously inoculated with B16 melanoma cells. These formulations showed high reporter-gene expression level in tumor after intravenous injection into tumor-bearing mice. Small complex was then made of the plasmid encoding GM-CSF gene, and injected into the mice bearing subcutaneous solid B16 tumor. After intravenous injection, it induced apparent tumor growth suppression in 50% of the mice. Notably, significant therapeutic effect was detected in the mice that received intratumoral injection, and 75% of the mice were completely cured with disappearance of tumor.
Keywords: Gene delivery; Hyaluronic acid; Ternary complex; Lyophilization; Tumor therapy; GM-CSF;

pH responsive properties of non-fouling mixed-charge polymer brushes based on quaternary amine and carboxylic acid monomers by Luo Mi; Matthew T. Bernards; Gang Cheng; Qiuming Yu; Shaoyi Jiang (2919-2925).
In this work, we report a tunable mixed-charge copolymer surface containing positively charged quaternary amine monomers ([2-(Acryloyloxy)ethyl] trimethyl ammonium chloride, TMA) and negatively-charged carboxylic acid monomers (2-carboxy ethyl acrylate, CAA). The non-fouling properties of this copolymer coating depend on environmental pH. The surface has charge neutrality under neutral and basic conditions, and is positively charged under acidic conditions due to the protonation of the carboxylic acid group. This transition in surface charge with respect to pH allows the surface to be switched from bacteria-adhesive to bacteria-resistant. We demonstrate that the bacteria adhered to the surface under acidic conditions can be easily released as bulk pH increases. This tunable surface can be used to collect a contaminant and then be externally stimulated to release the contaminant, to allow for analysis of its composition. Its bacteria attraction and release property makes it very promising for decontamination and biomedical applications.
Keywords: Bacterial adhesion; Mixed-charge; Ultra low fouling; pH responsive; Zwitterionic materials;

Mechanism of enzymatic degradation of beta-sheet crystals by Keiji Numata; Peggy Cebe; David L. Kaplan (2926-2933).
The anti-parallel beta pleated sheet is a fundamental secondary structure in proteins and a major component in silk fibers generated by silkworms and spiders, with a key role to stabilize these proteins via physical cross-links. Importantly, these beta-sheets are fully degradable and nontoxic structures in biology, in contrast for example to beta-amyloid structures formed in disease states. Thus, insight into mechanism of enzymatic degradation would be instructive as a route to elucidating differences among these stable yet different structural features in biological systems. We report on the mechanism of enzymatic degradation of anti-parallel beta pleated sheets with Bombyx mori silk structures, leading to fibrils and subsequently to nanofilaments (2 nm thickness and 160 nm length). These nanofilaments play a role as nucleators of the crystalline regions, an important feature of the system that can be exploited to design silk-based biomaterials with predictable biodegradability and mechanical properties. The potential toxicity of degradation products from these proteolytic enzymes was also assessed in vitro and no cell toxicity found in vitro for the protease found in vivo in the human body. The degradation mechanism of beta-sheet silk crystals provides additional insight into the significant differences in biological impact between the anti-parallel beta-sheet silk biomaterials reported in this work vs. amyloid structures in disease states, adding to prior descriptions of chemical and structural differences that are more extensively documented.
Keywords: Biocompatibility; Biodegradation; Cell viability; Silk;

In this study, valved photooxidatively crosslinked acellular bovine jugular vein conduits (BJVCs) were implanted in young dogs to reconstruct the connections of pulmonary arteries and right ventricles, with acellular conduits used as controls. All acellular conduits had moderate to severe valvular dysfunction and were explanted at 1-month implantation (n = 5). Histological examination showed inflammatory cell infiltration and intimal hyperplasia in the walls, and severe inflammatory cell infiltration and thrombosis in the valves. The photooxidatively crosslinked acellular conduits were retrieved at 1-month (n = 5) and 6-month (n = 5) implantations respectively. These conduits had excellent valvular function at retrieval. Their walls and valves were still soft and smooth without calcification and hemangioma. Endothelialization in valves and luminal walls was unsatisfied at 1-month retrieval, and was improved at 6-month retrieval. Host cells infiltrated and migrated from outer layer to the middle layer, with tissue remolding and regeneration found in these recellular regions. Histological examination and tissue content assay demonstrated that degeneration and regeneration of collagens and glycosaminoglycans were comparable, but elastic fibers gradually degraded. Photooxidatively crosslinked acellular BJVCs resist calcification and thrombosis and have regeneration patterns, with excellent hemodynamic performance.
Keywords: Cross-linking; Extracellular matrix; Heart valve; Scaffold;