Biomaterials (v.30, #31)

In situ gelling properties of chitosan-thioglycolic acid conjugate in the presence of oxidizing agents by Duangkamon Sakloetsakun; Juliane M.R. Hombach; Andreas Bernkop-Schnürch (6151-6157).
The rheological behaviour of chitosan-thioglycolic acid conjugate (chitosan-TGA) in the presence of four oxidizing agents was investigated. Chitosan-TGA was synthesized via amide bond formation between the primary amino group of chitosan and the carboxylic acid group of thioglycolic acid. The sol–gel phase transition of the polymer was determined by rheological measurements. Moreover, cytotoxicity of the gel in combination with each oxidizing agent was evaluated utilizing LDH and MTT assay. The modified chitosan displayed 1053 ± 44 μmol/g thiol groups. Results of rheological studies showed that 1% (m/v) chitosan-TGA without any oxidizing agents became gel within 40 min. In contrast, when the oxidizing agents hydrogen peroxide, sodium periodate, ammonium persulfate and sodium hypochlorite were added, respectively, gelation took place within a few minutes. Within 20 min, hydrogen peroxide having been added in a final concentration of 25.2 nmol/L increased dynamic viscosity of 1% (m/v) chitosan-TGA up to 16,500-fold. This can be explained by the formation of inter- and/or intramolecular disulfide bonds which were indirectly verified via the decrease in thiol groups. Additionally, evidence of an increase in cross-linking of thiolated chitosan as a function of time was provided by frequency sweep measurements. Furthermore, viability of Caco-2 cells having been incubated with chitosan-TGA/oxidizing agent systems assessed by MTT assay was 70–85% and the percentage of LDH release was only in case of the chitosan-TGA/ammonium persulfate system significantly (p  < 0.05) raising compared to the negative control. According to these results, chitosan-TGA/oxidizing agent combinations might be a promising novel in situ gelling system for various pharmaceutical applications such as a controlled drug release carrier or for tissue engineering.
Keywords: In situ gelling; Chitosan-TGA/oxidizing agent systems; Sol–gel transition; Oxidizing agents; Thiomers;

The insulation performance of reactive parylene films in implantable electronic devices by John P. Seymour; Yaseen M. Elkasabi; Hsien-Yeh Chen; Joerg Lahann; Daryl R. Kipke (6158-6167).
Parylene-C (poly-chloro-p-xylylene) is an appropriate material for use in an implantable, microfabricated device. It is hydrophobic, conformally deposited, has a low dielectric constant, and superb biocompatibility. Yet for many bioelectrical applications, its poor wet adhesion may be an impassable shortcoming. This research contrasts parylene-C and poly(p-xylylene) functionalized with reactive group X (PPX-X) layers using long-term electrical soak and adhesion tests. The reactive parylene was made of complementary derivatives having aldehyde and aminomethyl side groups (PPX-CHO and PPX-CH2NH2 respectively). These functional groups have previously been shown to covalently react together after heating. Electrical testing was conducted in saline at 37 °C on interdigitated electrodes with either parylene-C or reactive parylene as the metal layer interface. Results showed that reactive parylene devices maintained the highest impedance. Heat-treated PPX-X device impedance was 800% greater at 10 kHz and 70% greater at 1 Hz relative to heated parylene-C controls after 60 days. Heat treatment proved to be critical for maintaining high impedance of both parylene-C and the reactive parylene. Adhesion measurements showed improved wet metal adhesion for PPX-X, which corresponds well with its excellent high frequency performance.
Keywords: Parylene; Electronic material; Micromachining; Electrochemistry; Adhesion; Neural prosthesis;

Biofunctional alendronate–Hydroxyapatite thin films deposited by Matrix Assisted Pulsed Laser Evaporation by Adriana Bigi; Elisa Boanini; Chiara Capuccini; Milena Fini; Ion N. Mihailescu; Carmen Ristoscu; Felix Sima; Paola Torricelli (6168-6177).
We applied Matrix Assisted Pulsed Laser Evaporation (MAPLE) in order to synthesize alendronate-hydroxyapatite thin films on titanium substrates. Alendronate-hydroxyapatite composite nanocrystals with increasing bisphosphonate content (0, 3.9, 7.1% wt) were synthesized in aqueous medium. Then, they were suspended in deionised water, frozen at liquid nitrogen temperature and used as targets for MAPLE experiments. The depositions were conducted with a KrF* excimer laser source (l = 248 nm, tFWHM  = 25 ns) in mild conditions of temperature and pressure. The obtained thin films had a good crystallinity, which slightly decreases with the increase of alendronate content, and exhibited a porous-like structure. Osteoblast-like MG63 cells and human osteoclasts were cultured on the thin films up to 14 days. In the presence of alendronate, MG63 cells displayed a normal morphology, increased proliferation and higher values of differentiation parameters, namely type I collagen, osteocalcin, and osteoprotegerin/TNF-related activation-induced cytokine receptor ratio. In contrast, osteoclasts showed significantly reduced proliferation, and increased level of Caspase 3. Moreover, the coatings synthesized from hydroxyapatite at relatively high bisphosphonate content (7.1% wt) displayed a reduced production of Tumour Necrosis Factor alpha (TNF-α) and Interleukin 6 (IL-6), suggesting a down-regulatory role of alendronate on the inflammatory reaction. The successful deposition of alendronate modified hydroxyapatite thin films yields coatings with enhanced bioactivity, able to promote osteoblast differentiation and to inhibit osteoclast proliferation.
Keywords: Bisphosphonate; Alendronate-doped hydroxyapatite films; MAPLE; Osteoblast; Osteoclast;

Chemically crosslinkable and thermosensitive poly(organophosphazenes) containing multiple thiol (–SH) groups along with hydrophobic isoleucine ethyl ester and hydrophilic α-amino-ω-methoxy-poly(ethylene glycol) of the molecular weight 550 have been synthesized and characterized as an injectable biomaterial. The aqueous solutions of these polymers were transformed into hydrogel with desired gel strength at body temperature via hydrophobic interactions, and the gel strength was further improved by the cross-linking of thiol groups with crosslinkers, divinyl sulfone (VS) and PEG divinyl sulfone (PEGVS) under physiological conditions. The kinetics of cross-linking behavior of polymer thiol groups with crosslinkers was studied in both in vitro and in vivo conditions. Field Emission-Scanning Electron Microscopy (FE-SEM), swelling experiments, and rheology study of present polymers revealed that the inner three-dimensional hydrogel networks depended on the degree of thiol units in the polymer network. From the in vivo (in mice) degradation studies, the dual cross-linked gels showed to have a controlled degradation. These results demonstrate that the inner network of the hydrogels can be tuned, gel strength and degradation rate can be controlled, and the chemically crosslinkable and thermosensitive poly(organophosphazenes) hold promises for uses as injectable systems for biomedical applications including tissue engineering and protein delivery.
Keywords: Injectable; Thermosensitive; Chemically crosslinkable; Biodegradable; Hydrogels; Degree of substitution;

Microarrays of over 2000 hydrogels – Identification of substrates for cellular trapping and thermally triggered release by Rong Zhang; Albert Liberski; Rosario Sanchez-Martin; Mark Bradley (6193-6201).
In this paper we describe an approach whereby over 2000 individual polymers were synthesized, in situ, on a microscope slide using inkjet printing. Subsequent biological analysis of the entire library allowed the rapid identification of specific polymers with the desired properties. Herein we demonstrate how this array of new materials could be used for the identification of polymers that allow cellular adherence, proliferation and then mild thermal release, for multiple cell lines, including mouse embryonic stem (mES) cells. The optimal, identified hydrogels were successfully scaled-up and demonstrated excellent cell viability after thermal detachment for all cell lines tested. We believe that this approach offers an avenue to the discovery of a specific thermal release polymer for every cell line.
Keywords: Hydrogel microarrays; Inkjet printing; Thermo-responsive polymers; Cellular trapping; Cellular release; Stem cells;

Dynamic in vivo biocompatibility of angiogenic peptide amphiphile nanofibers by Shahram Ghanaati; Matthew J. Webber; Ronald E. Unger; Carina Orth; James F. Hulvat; Sarah E. Kiehna; Mike Barbeck; Angela Rasic; Samuel I. Stupp; C. James Kirkpatrick (6202-6212).
Biomaterials that promote angiogenesis have great potential in regenerative medicine for rapid revascularization of damaged tissue, survival of transplanted cells, and healing of chronic wounds. Supramolecular nanofibers formed by self-assembly of a heparin-binding peptide amphiphile and heparan sulfate-like glycosaminoglycans were evaluated here using a dorsal skinfold chamber model to dynamically monitor the interaction between the nanofiber gel and the microcirculation, representing a novel application of this model. We paired this model with a conventional subcutaneous implantation model for static histological assessment of the interactions between the gel and host tissue. In the static analysis, the heparan sulfate-containing nanofiber gels were found to persist in the tissue for up to 30 days and revealed excellent biocompatibility. Strikingly, as the nanofiber gel biodegraded, we observed the formation of a de novo vascularized connective tissue. In the dynamic experiments using the dorsal skinfold chamber, the material again demonstrated good biocompatibility, with minimal dilation of the microcirculation and only a few adherent leukocytes, monitored through intravital fluorescence microscopy. The new application of the dorsal skinfold model corroborated our findings from the traditional static histology, demonstrating the potential use of this technique to dynamically evaluate the biocompatibility of materials. The observed biocompatibility and development of new vascularized tissue using both techniques demonstrates the potential of these angiogenesis-promoting materials for a host of regenerative strategies.
Keywords: Angiogenesis; Biocompatibility; Peptide amphiphile; Regenerative medicine; Self-assembly;

A molecularly defined array based on native fibrillar collagen for the assessment of skin tissue engineering biomaterials by G. Lammers; G.S. Tjabringa; J. Schalkwijk; W.F. Daamen; T.H. van Kuppevelt (6213-6220).
Large-scale in vivo evaluation of biomaterials is time-consuming and limited by ethical considerations. The availability of a library of biomaterials would allow a fast and rational in vitro selection of those biomaterials to be evaluated in vivo. For this reason, we developed an array of 48 different, molecularly-defined films based on native fibrillar collagen. The films differed in the type and amount of extracellular matrix components (type I/IV collagens, fibrous/solubilised elastin, glycosaminoglycans, heparin, chondroitin sulfate or dermatan sulfate), method of preparation (homogenisation) and method and extent of crosslinking (carbodiimide (EDC/NHS) or glutaraldehyde). The array was evaluated by studying morphology, proliferation and differentiation of primary human keratinocytes/fibroblasts. Major differences were observed. Only a small selection of films (especially those containing elastin fibres) specifically stimulated the proliferation of keratinocytes, but not fibroblasts. Such films may be the biomaterials of choice for in vivo evaluation for skin tissue engineering and regenerative medicine.
Keywords: Cell proliferation; Collagen; ECM (extracellular matrix); Elastin; In vitro test; Keratinocyte;

The current tissue engineering paradigm is that successfully engineered thick tissues must include vasculature. As biological approaches alone, such as VEGF, have fallen short of their promises, one may look for an engineering approach to build microvasculature. Layer-by-layer approaches for customized fabrication of cell/scaffold constructs have shown some potential in building complex 3D structures. With the advent of cell printing, one may be able to build precise human microvasculature with suitable bio-ink. Human microvascular endothelial cells (HMVEC) and fibrin were studied as bio-ink for microvasculature construction. Endothelial cells are the only cells to compose the human capillaries and also form the entire inner lining of cardiovascular system. Fibrin has been already widely recognized as tissue engineering scaffold for vasculature and other cells, including skeleton/smooth muscle cells and chondrocytes. In our study, we precisely fabricated micron-sized fibrin channels using a drop-on-demand polymerization. This printing technique uses aqueous processes that have been shown to induce little, if any, damage to cells. When printing HMVEC cells in conjunction with the fibrin, we found the cells aligned themselves inside the channels and proliferated to form confluent linings. The 3D tubular structure was also found in the printed patterns. We conclude that a combined simultaneous cell and scaffold printing can promote HMVEC proliferation and microvasculature formation.
Keywords: Inkjet printing; Human microvascular endothelial cell; Fibrin; Microvasculature;

Development and analysis of multi-layer scaffolds for tissue engineering by Bernke J. Papenburg; Jun Liu; Gustavo A. Higuera; Ana M.C. Barradas; Jan de Boer; Clemens A. van Blitterswijk; Matthias Wessling; Dimitrios Stamatialis (6228-6239).
The development of 3D scaffolds consisting of stacked multi-layered porous sheets featuring microchannels is proposed and investigated in this work. In this concept, the inner-porosity of the sheets allows diffusion of nutrients and signalling products between the layers whereas the microchannels facilitate nutrient supply on all layers as they provide space for the culture medium to be perfused throughout the scaffold. Besides the above, these scaffolds have excellent distribution of the cells as seeding and attaching of the cells occurs on individual layers that are subsequently stacked. In addition, these scaffolds enable gaining local data from within the scaffolds as unstacking of the stacked layers allows for determination of various parameters per layer. Here, we show the proof of this concept by culturing C2C12 pre-myoblasts and A4-4 cells on stacked Poly(l-lactic acid) (PLLA) sheets featuring microchannels. The results obtained for culturing under static conditions clearly indicate that despite inhibited cell proliferation due to nutrient limitations, diffusion between the layers takes place and cells on various layers stay viable and also affect each other. Under dynamic conditions, medium flow through the channels improves nutrient availability to the cells on the various layers, drastically increasing cell proliferation on all layers.
Keywords: 3D scaffold; Tissue engineering; Micropatterning; Porosity; Nutrient transport; Cell behaviour;

Orthotopic replacement of the aortic valve with decellularized allograft in a sheep model by Hassina Baraki; Igor Tudorache; Maike Braun; Klaus Höffler; Adelheid Görler; Artur Lichtenberg; Christopher Bara; Alex Calistru; Gudrun Brandes; Marion Hewicker-Trautwein; Andres Hilfiker; Axel Haverich; Serghei Cebotari (6240-6246).
Tissue engineered (TE) allografts have been successfully applied in pulmonary circulation. The behavior of TE valves based on decellularized scaffolds in systemic circulation remains unexplored. We investigated the function, histological changes, potential of in-vivo re-endothelialization of decellularized aortic valve allografts in orthotopic position in sheep. Ovine aortic valve conduits (n  = 12) were decellularized with detergents and implanted as an aortic root in lambs (35–45 kg). For controls, fresh native ovine aortic valve conduits (n  = 6) were implanted. The valves were explanted at 3 and 9 months. In the experimental group, the valves exhibited trivial regurgitation and normal morphology with no signs of graft dilatation, degeneration or rejection. In some animals (n  = 2), we documented minimal calcification in the area of arterial anastomosis and in one, microthrombi formation on the leaflet surface. The luminal sides of the grafts were partially covered with an endothelial cell monolayer, neovasculogenesis was observed at the adventitial side. The valves in the control group appeared thickened, shrunken with marked calcification/degeneration signs, and advanced valve insufficiency. Detergent decellularized aortic valve allografts satisfy the higher requirements of the systemic circulation in sheep. As valve conduits become repopulated by endothelial and interstitial cells, they may re-gain the potential for growth.
Keywords: Aorta; Valves; Surgery; Tissue engineering;

Combined delivery of heme oxygenase-1 gene and fibroblast growth factor-2 protein for therapeutic angiogenesis by Suk H. Bhang; Ju H. Kim; Hee S. Yang; Wan-Geun La; Tae-Jin Lee; Ah-Young Sun; Ga H. Kim; Minhyung Lee; Byung-Soo Kim (6247-6256).
Ectopic expression of heme oxygenase-1 (HO-1) in ischemic tissue protects the tissue from apoptosis and necrosis and promotes angiogenesis. However, apoptosis and necrosis will decrease HO-1 gene transfection efficacy. We hypothesized that fibroblast growth factor-2 (FGF2) would attenuate ischemic damage during the incipient period, improve HO-1 gene transfection and, in turn, enhance neovascularization. To test this hypothesis, we employed a mouse model of hindlimb ischemia and treated the mice with HO-1 gene therapy alone, FGF2 alone, or HO-1 gene therapy plus FGF2. As controls, a group of mice was left untreated. At 12 h, prior to the expression of exogenously delivered HO-1, apoptosis was significantly reduced in mice treated with FGF2, either alone or in combination with HO-1 gene therapy. At 3 days, HO-1 expression was greater in mice that also received FGF2 than in mice treated with HO-1 gene therapy alone. The expression of angiogenic growth factors and angiogenesis was greater in mice treated with HO-1 gene therapy plus FGF2 than in mice treated with HO-1 gene therapy alone. These data indicate that FGF2 rescued muscle necrosis prior to the exogenous expression of HO-1 and enhanced HO-1 gene transfection in ischemic murine limbs.
Keywords: Angiogenesis; Basic fibroblast growth factor; Heme oxygenase-1; Mouse hindlimb ischemia;

Electrochemical modulation of epithelia formation using conducting polymers by Karl Svennersten; Maria H. Bolin; Edwin W.H. Jager; Magnus Berggren; Agneta Richter-Dahlfors (6257-6264).
Conducting polymers are soft, flexible materials, exhibiting material properties that can be reversibly changed by electrochemically altering the redox state. Surface chemistry is an important determinant for the molecular events of cell adhesion. Therefore, we analyzed whether the redox state of the conducting polymer PEDOT:Tosylate can be used to control epithelial cell adhesion and proliferation. A functionalized cell culture dish comprising two adjacent electrode surfaces was developed. Upon electronic addressing, reduced and oxidized surfaces are created within the same device. Simultaneous analysis of how a homogenous epithelial MDCK cell population responded to the electrodes revealed distinct surface-specific differences. Presentation of functional fibronectin on the reduced electrode promoted focal adhesion formation, involving αvβ3 integrin, cell proliferation, and ensuing formation of polarized monolayers. In contrast, the oxidized surface harbored only few cells with deranged morphology showing no indication of proliferation. This stems from the altered fibronectin conformation, induced by the different surface chemistry of the PEDOT:Tosylate electrode in the oxidized state. Our results demonstrate a novel use of PEDOT:Tosylate as a cell-hosting material in multiple-electrode systems, where cell adhesion and proliferation can be controlled by electrochemical modulation of surface properties.
Keywords: Actin; Cell adhesion; Electroactive polymer; Epithelial cell; Fibronectin; RGD peptide;

The dynamic regulation of integrin-binding peptides is crucial for chondrogenic differentiation. Here, we revealed the feasibility for flexible modification of RGD by embedding a large molecular weight and slightly charged (isoelectric point, 6–6.25) RGD-chimeric protein (CBD–RGD) with cellulose-binding domain (CBD) in three dimensional (3D) alginate beads to evaluate the chondrogenesis of adipose-derived adult stem cells (ADAS). The binding of CBD–RGD with cells and its diffusion from alginate beads were studied on fluorescein isothiocyanate (FITC)-conjugated CBD–RGD. The increases in gene expression (Sox9, Aggrecan, fibronectin and collagen II), accumulation of chondrogenic matrices and decrease of collagen X gene expression during TGF-β3 induction were only observed for those beads containing 10 mg/g CBD–RGD initially, with 20.18 ± 0.73% of that released in a week. The contrary was observed for beads with CBD–RGD 20 mg/g initially and having higher persistence (only 8.6 ± 2.17% released in a week). The 10 mg/g CBD–RGD-mediated enhancement was demonstrated via the activation of integrin α5 and β1-dependent pathway, and especially related to the upregulation of Sox9 gene and the temporary block of fibronectin expression as well as sustained inhibition of RhoA activity in the early differentiation stage. Thus, we speculated that the dynamic mobility of CBD–RGD may account for the enhanced chondrogenesis. It was concluded that the CBD–RGD–alginate culture system promoted the chondrogenesis of mesenchymal stem cells coordinated with TGF-β3 induction in an RGD dose-dependent manner.
Keywords: RGD-chimeric protein; Alginate; Adipose-derived adult stem cells; Chondrogenesis; Fibronectin; RhoA activity;

Segmental bone regeneration using an rhBMP-2-loaded gelatin/nanohydroxyapatite/fibrin scaffold in a rabbit model by Yue Liu; Yun Lu; Xuezhong Tian; Geng Cui; Yanmei Zhao; Qiang Yang; Shunlu Yu; Guosheng Xing; Boxun Zhang (6276-6285).
We aimed to develop a hybrid scaffold with a porous structure and similar composition as natural bone for the controlled release of bone morphogenetic protein-2 (BMP-2) to enhance bone regeneration. We fabricated a gelatin/nanohydroxypatite (nHAP) scaffold by glutaraldehyde chemical cross-linking a gelatin aqueous solution with nHAP granules at a 5:1 ratio (v/w). Then, fibrin glue (FG) mixed with recombinant human BMP-2 (rhBMP-2) was infused into the gelatin/nHAP scaffold and lyophilized to develop an rhBMP-2-loaded gelatin/nHAP/FG scaffold. On scanning electron microscopy, the composite had a 3-D porous structure. The rhBMP-2 release kinetics from the hybrid scaffold was sustained and slow, and release of rhBMP-2 was complete at 40 days. Immunohistochemistry, azo-coupling and alizarin S-red staining were used to study in vitro differentiation of human bone-marrow mesenchymal cells (hBMSCs). Strong positive staining results confirmed that rhBMP-2 released from the scaffold could improve osteocalcin (OCN) and alkaline phosphatase (ALP) expression and calcium deposition formation. RT-PCR results showed significantly high mRNA expression of ALP and OCN in hBM-MSCs cultured on the gelatin/nHAP/FG scaffold with rhBMP-2. DNA assay demonstrated that the scaffold was noncytotoxic and could promote hBMSC proliferation from the components of the hybrid scaffold, not released rhBMP-2. The hybrid scaffolds were then used to repair critical-size segmental bone defects of rabbit radius. Gross specimen, X-ray, bone histomorphology and bone mineral density assay demonstrated that the rhBMP-2-loaded gelatin/nHAP/FG scaffold had good osteogenic capability and could repair the segmental bone defect completely in 12 weeks.
Keywords: Bone tissue engineering; Scaffold; Gelatin; Bone regeneration; Bone morphogenetic protein-2;

The effects of heparin releasing hydrogels on vascular smooth muscle cell phenotype by Jeffrey A. Beamish; Leah C. Geyer; Nada A. Haq-Siddiqi; Kandice Kottke-Marchant; Roger E. Marchant (6286-6294).
Poly(ethylene glycol) diacrylate (PEGDA) hydrogel scaffolds were engineered to promote contractile smooth muscle cell (SMC) phenotype via controlled release of heparin. The scaffold design was evaluated by quantifying the effects of free heparin on SMC phenotype, engineering hydrogels to provide controlled release of heparin, and synthesizing cell-adhesive, heparin releasing hydrogels to promote contractile SMC phenotype. Heparin inhibited SMC proliferation and up-regulated expression of contractile SMC phenotype markers, including smooth muscle α-actin, calponin, and SM-22α, in a dose-dependent fashion (6 μg/ml to 3.2 mg/ml). Heparin release from PEGDA hydrogels was controlled by altering PEGDA molecular weight (MW 1000–6000) and concentration at polymerization (10–30% w/w), yielding release profiles ranging from hours to weeks in duration. Heparin released from PEGDA gels, formulated for optimized heparin loading and release kinetics (30% w/w PEGDA, MW 3000), stimulated SMCs to up-regulate contractile marker mRNA. A cell-instructive scaffold construct was prepared by polymerizing a thin hydrogel film, with pendant RGD peptides for cell attachment, over the optimized hydrogel depots. SMCs seeded on these constructs had elevated levels of contractile marker mRNA after 3 d of culture compared with SMCs on control constructs. These results indicate that RGD-modified, heparin releasing PEGDA gels can act as cell-instructive scaffolds that promote contractile SMC phenotype.
Keywords: Smooth muscle cell; Arterial tissue engineering; Hydrogel; Scaffold; Vascular graft;

An injectable and thermosensitive poly(organophosphazene)–RGD conjugate to enhance functionality was synthesized by a covalent amide linkage between a cell adhesion peptide, GRGDS and carboxylic acid-terminated poly(organophosphazene). The aqueous solutions of synthesized poly(organophosphazene)–GRGDS conjugates existed in an injectable fluid state at room temperature and immediately formed a hydrogel at body temperature. The rabbit mesenchymal stem cells (rMSCs) on the polymer–GRGDS conjugate (conjugate 1-2, 0.05 mol fraction as GRGDS) hydrogel constructs using an injection method into a nude mouse were proved to express markers at mRNA level for all stages towards osteogenesis and mainly a sharp increase of osteocalcin (OCN, a typical late osteogenic differentiation marker) levels at 4th week post-induction indicated that the maturation process has started within this period. By histological and immunohistochemical evaluations, significantly high mineralization level by calcium contents was detected qualitatively and collagen type I (Col I), a major characteristic marker protein, was mainly and highly expressed by the rMSCs cultivated in the polymer–GRGDS conjugate hydrogel constructs formed into the nude mouse. The results suggest that the poly(organophosphazene)–GRGDS conjugate to enhance biofunctionality hold a promise for cell delivery material to induce osteogenic differentiation of MSC for enhancing ectopic bone formation.
Keywords: Thermosensitive; Injectable; Polyphosphazene–RGD conjugate; Mesenchymal stem cells (MSCs); Osteogenic differentiation; Hydrogel;

This paper reports on the damaging effects of different percentage porosities on the fatigue life of acrylic bone cement as used in the fixation of orthopaedic implants. Both hand-mixed (HM) and vacuum-mixed (VM) specimens containing different levels of porosity were fatigue tested to failure. A negative correlation between porosity level and fatigue life was demonstrated for both techniques. Considerable scatter was present in the data. Using the pore size distributions for HM and VM cement virtual HM and VM specimens were created containing various levels of porosity. Incorporating the effect of pore size and pore clustering quantified previously using the theory of critical distances a fatigue life prediction could be obtained for the virtual specimens. The virtual data agreed strongly with the experimental findings, predicting the correlation and more significantly the scatter in the experimental results. Using the virtual porosity failure model, it was demonstrated that given a constant porosity the fatigue life can vary by over an order of magnitude in both HM and VM cement. This suggests that not only porosity level but pore size distribution is extremely important in controlling the fatigue life of bone cement. It was verified that pore clustering and pore size are the major contributors to failure in HM and VM cement respectively. Furthermore, given the beneficial effects of porosity it has been proposed that an even distribution of small pores would provide an optimal bone cement mantle. Using the virtual model, it was determined that neither technique was capable of achieving such a distribution indicating a need for a new more reliable technique. The TCD based virtual porosity failure model should prove to be a powerful tool in the design of such a technique.
Keywords: Bone cement; Porosity; Fatigue; Scatter; Critical distance; Probability;

Craniofacial vertical bone augmentation: A comparison between 3D printed monolithic monetite blocks and autologous onlay grafts in the rabbit by Faleh Tamimi; Jesus Torres; Uwe Gbureck; Enrique Lopez-Cabarcos; David C. Bassett; Mohammad H. Alkhraisat; Jake E. Barralet (6318-6326).
Onlay autografting is amongst the most predictable techniques for craniofacial vertical bone augmentation, however, complications related to donor site surgery are common and synthetic alternatives to onlay autografts are desirable. Recent studies have shown that the acidic calcium phosphates, brushite and monetite, are osteoconductive, osteoinductive and resorb faster in vivo than hydroxyapatite. Moreover, they can be 3D printed allowing precise host bone–implant conformation. The objectives of this study were to confirm that craniofacial screw fixation of 3D printed monetite blocks was possible and to compare the resulting vertical bone augmentation with autograft. 3D printed monolithic monetite onlay implants were fixed with osteosynthesis screws on the calvarial bone surface of New Zealand rabbits. After 8 weeks, integration between the implant and the calvarial bone surface was observed in all cases. Histomorphometry revealed that 42% of the monetite was resorbed and that the new bone formed within the implant occupied 43% of its volume, sufficient for immediate dental implant placement. Bone tissue within the autologous onlay occupied 60% of the volume. We observed that patterns of regeneration within the implants differed throughout the material and propose that this was due to the anatomy and blood supply pattern in the region. Rapid prototyped monetite being resorbable osteoconductive and osteoinductive would appear to be a promising biomaterial for many bone regeneration strategies.
Keywords: Monetite; Bone; Autograft; Onlay; Monolithic; Histomorphometry;

In this work, therapeutic magnetic micro carriers (TMMC) guided in real time by a magnetic resonance imaging (MRI) system are proposed as a mean to improve drug delivery to tumor sites. MRI steering constraints and physiological parameters for the chemoembolization of liver tumors were taken into account to design magnetic iron–cobalt nanoparticles encapsulated into biodegradable poly(d,l-lactic-co-glycolic acid) (PLGA) microparticles with the appropriate saturation magnetization (M s). FeCo nanoparticles displayed a diameter of 182 nm and an M s of 209 emu g−1. They were coated with a multilayered graphite shell to minimize the reduction of M s during the encapsulation steps. FeCo–PLGA microparticles, with a mean diameter of 58 μm and an M s of 61 emu g−1, were steered in a phantom mimicking the hepatic artery and its bifurcation, with a flow in the same order of magnitude as that of the hepatic artery flow. The steering efficiency, defined as the amount of FeCo–PLGA microparticles in the targeted bifurcation channel divided by the total amount of FeCo–PLGA microparticles injected, reached 86%. The data presented in this paper confirms the feasibility of the steering of these TMMC.
Keywords: Magnetism; Nanoparticle; Microencapsulation; MRI (magnetic resonance imaging); Embolization;

Antifungal activity of silver nanoparticles against Candida spp. by Aleš Panáček; Milan Kolář; Renata Večeřová; Robert Prucek; Jana Soukupová; Vladimír Kryštof; Petr Hamal; Radek Zbořil; Libor Kvítek (6333-6340).
The antifungal activity of the silver nanoparticles (NPs) prepared by the modified Tollens process was evaluated for pathogenic Candida spp. by means of the determination of the minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), and the time-dependency of yeasts growth inhibition. Simultaneously the cytotoxicity of the silver NPs to human fibroblasts was determined. The silver NPs exhibited inhibitory effect against the tested yeasts at the concentration as low as 0.21 mg/L of Ag. The inhibitory effect of silver NPs was enhanced through their stabilization and the lowest MIC equal to 0.05 mg/L was determined for silver NPs stabilized by sodium dodecyl sulfate against Candida albicans II. The obtained MICs of the silver NPs and especially of the stabilized silver NPs were comparable and in some cases even better than MICs of the conventional antifungal agents determined by E-test. The silver NPs effectively inhibited the growth of the tested yeasts at the concentrations below their cytotoxic limit against the tested human fibroblasts determined at a concentration equal to 30 mg/L of Ag. In contrast, ionic silver inhibited the growth of the tested yeasts at the concentrations comparable to the cytotoxic level (approx. 1 mg/L) of ionic silver against the tested human fibroblasts.
Keywords: Silver; Nanoparticles; Antifungal; Fungicidal; Yeasts; Candida spp.;

Antiangiogenic properties of silver nanoparticles by Sangiliyandi Gurunathan; Kyung-Jin Lee; Kalimuthu Kalishwaralal; Sardarpasha Sheikpranbabu; Ramanathan Vaidyanathan; Soo Hyun Eom (6341-6350).
Angiogenesis is an important phenomenon involved in normal growth and wound healing processes. An imbalance of the growth factors involved in this process, however, causes the acceleration of several diseases including malignant, ocular, and inflammatory diseases. Inhibiting angiogenesis through interfering in its pathway is a promising methodology to hinder the progression of these diseases. The function and mechanism of silver nanoparticles (Ag-NPs) in angiogenesis have not been elucidated to date. PEDF is suggested to be a potent anti-angiogenic agent. In this study, we postulated that Ag-NPs might have the ability to inhibit angiogenesis, the pivotal step in tumor growth, invasiveness, and metastasis. We have demonstrated that Ag-NPs could also inhibit vascular endothelial growth factor (VEGF) induced cell proliferation, migration, and capillary-like tube formation of bovine retinal endothelial cells like PEDF. In addition, Ag-NPs effectively inhibited the formation of new blood microvessels induced by VEGF in the mouse Matrigel plug assay. To understand the underlying mechanism of Ag-NPs on the inhibitory effect of angiogenesis, we showed that Ag-NPs could inhibit the activation of PI3K/Akt. Together, our results indicate that Ag-NPs can act as an anti-angiogenic molecule by targeting the activation of PI3K/Akt signaling pathways.
Keywords: Bovine retinal endothelial cell; Vascular endothelial growth factor; Ag-NPs; PEDF; Angiogenesis;

The inhibition of neuronal calcium ion channels by trace levels of yttrium released from carbon nanotubes by Lorin M. Jakubek; Spiro Marangoudakis; Jesica Raingo; Xinyuan Liu; Diane Lipscombe; Robert H. Hurt (6351-6357).
Carbon nanotubes (CNTs) are used with increasing frequency in neuroengineering applications. CNT scaffolds are used to transmit electrical stimulation to cultured neurons and to control outgrowth and branching patterns of neurites. CNTs have been reported to disrupt normal neuronal function including alterations in endocytotic capability and inhibition of ion channels. Calcium ion channels regulate numerous neuronal and cellular functions including endo and exocytosis, neurite outgrowth, and gene expression. Strong CNT interactions with neuronal calcium ion channels would have profound biological implications. Here we show that physiological solutions containing CNTs inhibit neuronal voltage-gated calcium ion channels in a dose-dependent and CNT sample-dependent manner with IC50 as low as 1.2 μg/ml. Importantly, we demonstrate that the inhibitory activity does not involve tubular graphene as previously reported, but rather very low concentrations of soluble yttrium released from the nanotube growth catalyst. Cationic yttrium potently inhibits calcium ion channel function with an inhibitory efficacy, IC50, of 0.07 ppm w/w. Because of this potency, unpurified and even some reportedly “purified” CNT samples contain sufficient bioavailable yttrium to inhibit channel function. Our results have important implications for emerging nano-neurotechnology and highlight the critical role that trace components can play in the biological response to complex nanomaterials.
Keywords: Biocompatibility; Nanoparticle; Brain; Metal ion release; CaV2.2; Voltage-gated calcium channel;

Biodegradable micelles with sheddable poly(ethylene glycol) shells for triggered intracellular release of doxorubicin by Huanli Sun; Bingnan Guo; Ru Cheng; Fenghua Meng; Haiyan Liu; Zhiyuan Zhong (6358-6366).
Biodegradable micelles with sheddable poly(ethylene glycol) shells were developed based on disulfide-linked poly(ethylene glycol)-b-poly(ɛ-caprolactone) (PEG-SS-PCL) diblock copolymer and applied for rapid intracellular release of doxorubicin (DOX). PEG-SS-PCL was prepared with controlled block lengths via exchange reaction between PEG orthopyridyl disulfide and mercapto PCL. The micelles formed from PEG-SS-PCL, though sufficiently stable in water, were prone to fast aggregation in the presence of 10 mm dithiothreitol (DTT), due to shedding of the PEG shells through reductive cleavage of the intermediate disulfide bonds. Interestingly, the in vitro release studies revealed that these shell-sheddable micelles released DOX quantitatively within 12 h under a reductive environment analogous to that of the intracellular compartments such as cytosol and the cell nucleus. In contrast, minimal drug release (<20%) was observed within 24 h for the reduction insensitive PEG–PCL micelles under the same conditions as well as for PEG-SS-PCL micelles under the non-reductive conditions. Remarkably, cell experiments showed that these shell-sheddable micelles accomplished much faster release of DOX inside cells and higher anticancer efficacy as compared to the reduction insensitive control. These shell-sheddable biodegradable micelles are highly promising for the efficient intracellular delivery of various lipophilic anticancer drugs to achieve improved cancer therapy.
Keywords: Reduction-sensitive; Shell-sheddable; Degradation; Micelle; Doxorubicin; Drug delivery;

Nanostructured polyelectrolyte multilayer drug delivery systems for bone metastasis prevention by Florence Daubiné; Delphine Cortial; Guy Ladam; Hassan Atmani; Youssef Haïkel; Jean-Claude Voegel; Philippe Clézardin; Nadia Benkirane-Jessel (6367-6373).
Polyelectrolyte multilayers (PEM) are well established nanoarchitectures with numerous potential applications, in particular as biomaterial coatings. They may exhibit specific biological properties in terms of controlled cell activation or local drug delivery. Here, in a new approach for bone metastasis prevention, we employed poly-l-lysine covalently grafted with β-cyclodextrin as a polycationic vector (PLL–CD) for the antitumor bisphosphonate drug risedronate (RIS). Molar ratio for maximum loading of the PLL–CD vector with RIS was determined by Raman microspectroscopy. The efficacy of RIS at inhibiting cancer cell invasion in vitro was strongly enhanced upon complexation, whatever PLL–CD:RIS complexes were in solution or embedded into PEM nanoarchitectures. Complexes in solution also clearly prevented cancer-induced bone metastasis in animals. Incorporation of the complexes into PEM nanoarchitectures covering bone implants appears of interest for in situ prevention of bone metastasis after ablation.
Keywords: Bioactive coatings; Polyelectrolyte multilayers; Bone metastasis; Bisphosphonates; Cyclodextrins;

This study was conducted to reveal the chondrogenesis of mesenchymal stem cells that had been genetically modified with the green fluorescence protein (GFP) gene and then co-cultured with chondrocytes in vitro and in vivo. Subsequent mixing of chondrocytes in the hydrogel constructs induced increased chondrogenic differentiation of the transfected hMSCs. The proliferation and differentiation of MSCs that were transfected with the GFP gene and co-cultured with chondrocytes (1:1 and 1:3) or chondrocytes alone were evaluated by a live/dead assay, MTT assay, GAG & DNA assay, RT-PCR, real time-PCR, and histological and immunochemical analysis in vitro and in vivo. Real-time PCR revealed that the expression of aggrecan and COMP by genetically modified hMSCs co-cultured with chondrocytes was 2 or 3 times greater than that of genetically modified MSCs alone. Moreover, the expression of collagen type II was more than 3.5 times greater than that of genetically modified MSCs alone. 3-D hydrogel constructs co-cultured with chondrocytes and genetically modified MSCs showed a significantly higher number of specific lacunae phenotypes at the end of the 4 week study, regardless of whether they were co-cultured in the presence of chondrocytes. These findings indicate that co-culture with chondrocytes and genetically modified MSCs can be used to engineer well designed implants for the formation of neocartilage by transplanted genetically modified MSCs.
Keywords: Hydrogel; GFP; Chondrocytes; Genetically modified MSC; Co-culture;

Delivering genes to mediate functions of cells is a crucial technology for both basic science and clinical applications. Though numerous non-viral gene delivery systems have been developed, the diversity of mammalian cells poses a great challenge to the material design. Here, we demonstrate that surface-induced mineralization represents a promising approach to systematically customize DNA delivery with respect to the characteristics of cells. We initially examined gene transfer in nine cell types derived from different tissues and organisms by surface-induced DNA-doped calcium carbonate nanocomposites derived from a library of mineral solutions. Subsequently, we correlated gene transfer efficiency with cellular uptake, pH responsiveness of nanocomposites, and phagosomal pH of individual cell types. Based on the correlation, we were able to optimize the DNA delivery to the cell types of interest. Surface-induced mineralization possesses great potential for customizing gene transfer in realizing gene- and cell-based therapy and probing functions of genes.
Keywords: Nanocomposite; Tissue engineering; Biomineralization; Gene transfer;

Modeling the steady-state deformation of the solid phase of articular cartilage by J.S. Bell; C.P. Winlove; C.W. Smith; H. Dehghani (6394-6401).
The transient response of articular cartilage (AC) to compressive loads has been described by complex multicomponent models. However, the steady-state behaviour is determined by the collagen network which is heterogeneous through the depth of the tissue, a characteristic which is omitted from most theoretical models. Experimental data are now available on the local responses of the network to compressive loads and the aim of this study was to develop minimal models capable of simulating this behaviour. A series of finite element models (FEMs) of AC under load were developed of increasing complexity, assuming the AC was i) completely homogeneous, ii) layered and isotropic and iii) layered and anisotropic. The geometry of the layered cartilage model was based on the recent experimental data. It is shown that a layered transversely isotropic elastic model is required to accurately recreate the experimental data. Stress distributions within the models are analysed, and the relevance of this work to transient modeling of AC is discussed. The work presented is a fundamental step forward in the understanding of the distribution of local physiological stresses and strains in AC, and has applications in modeling chondrocyte mechanotransduction as well as the effects of pathogenesis.
Keywords: Cartilage; Collagen structure; ECM (extracellular matrix); Elasticity; Mechanical properties;