Hot off the presses! Mar 01 Biomater

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Latest Articles Include:

• Editorial board
  - Biomater 32(7):IFC (2011)
  
• The potential role of cobalt ions released from metal prosthesis on the inhibition of Hv1 proton channels and the decrease in Staphyloccocus epidermidis killing by human neutrophils
  - Biomater 32(7):1769-1777 (2011)
  Infection by Staphylococcus epidermidis is a devastating complication of metal-on-metal (MM) total hip arthroplasty (THA). Neutrophils are the first line of defense against infection, and these innate immune cells are potentially exposed to Co2+ ions released in the peri-prosthetic tissue by the wear of MM THA. The toxicity of Co2+ is still debated, but Co2+ is a potential inhibitor of the Hv1 proton channel that sustains the production of superoxide by neutrophils. In this study, we show that the Co2+ concentration in peri-prosthetic tissue from patients with MM THA averages 53 μM and that such high concentrations of Co2+ alter the antibacterial activity of human neutrophils in vitro by inhibiting Hv1 proton channels. We show that submillimolar concentrations of Co2+ inhibit proton currents, impair the extrusion of cytosolic acid, and decrease the production of superoxide in human neutrophils. As a result, Co2+ reduces the ability of human neutrophils to kill two str! ains of Staphyloccocus epidermidis by up to 7-fold at the maximal concentration tested of 100 μM Co2+. By inhibiting proton channels, the Co2+ ions released by metal prostheses might therefore promote bacterial infections in patients with metal-on-metal total hip arthroplasty.
• Flexible, all-polymer microelectrode arrays for the capture of cardiac and neuronal signals
  - Biomater 32(7):1778-1786 (2011)
  Microelectrode electrophysiology has become a widespread technique for the extracellular recording of bioelectrical signals. To date, electrodes are made of metals or inorganic semiconductors, or hybrids thereof. We demonstrate that these traditional conductors can be completely substituted by highly flexible electroconductive polymers. Pursuing a two-level replica-forming strategy, conductive areas for electrodes, leads and contact pads are defined as microchannels in poly(dimethylsiloxane) (PDMS) as a plastic carrier and track insulation material. These channels are coated by films of organic conductors such as polystyrenesulfonate-doped poly(3,4-ethylenedioxy-thiophene) (PEDOT:PSS) or filled with a graphite-PDMS (gPDMS) composite, either alone or in combination. The bendable, somewhat stretchable, non-cytotoxic and biostable all-polymer microelectrode arrays (polyMEAs) with a thickness below 500 μm and up to 60 electrodes reliably capture action potentials (APs) an! d local field potentials (LFPs) from acute preparations of heart muscle cells and retinal whole mounts, in vivo epicortical and epidural recordings as well as during long-term in vitro recordings from cortico-hippocampal co-cultures.
• Activation of stress-regulated transcription factors by triethylene glycol dimethacrylate monomer
  - Biomater 32(7):1787-1795 (2011)
  Triethylene glycol dimethacrylate (TEGDMA) is a resin monomer available for short exposure scenarios of oral tissues due to incomplete polymerization processes of dental composite materials. The generation of reactive oxygen species (ROS) in the presence of resin monomers is discussed as a common mechanism underlying cellular reactions as diverse as disturbed responses of the innate immune system, inhibition of dentin mineralization processes, genotoxicity and a delayed cell cycle. Yet, the signaling pathway through a network of proteins that finally initiates the execution of monomer-induced specific cell responses is unknown so far. The aim of the present study was to extend the knowledge of molecular mechanisms of monomer-induced cell death as a basis for reasonable therapy strategies. Thus, the monomer-induced expression and phosphorylation of stress-related transcription factors was analyzed in various cell lines. The time-related induction of apoptosis was invest! igated as well. The expression of p53 increased in HeLa cell cultures treated with camptothecin (positive control) for 24h, and the formation of p53Ser15 and p53Ser46 was detected in cell nuclei by Western blotting. TEGDMA (3 mm) appeared to stimulate p53 expression only slightly, but increased p21 expression was found in cell nuclei and cytoplasm. Both camptothecin and TEGDMA increased p53 expression to some extent in the nuclear fraction in human transformed pulp-derived cells (tHPC), and similar effects were detected in RAW264.7 macrophages. No clear induction of c-Jun and phospho-c-Jun by TEGDMA was detected in HeLa cell nuclei, and the expression of ATF-2 and phospho-ATF-2 was inhibited in the presence of the monomer. ATF-3 expression was found only in the nuclear fraction of camptothecin-treated HeLa cultures. TEGDMA seemed to inhibit the formation of phospho-c-Jun and phospho-ATF-2 in tHPC, and the monomer acted negatively on the expression of c-Jun, ATF-2 and ATF-3 ! in RAW264.7 macrophages. These changes in the expression and a! ctivation of stress-related transcription factors were time-related to the induction of apoptosis by TEGDMA in all cell lines. The present study provides experimental evidence that TEGDMA interferes with the regulation of cellular pathways through transcription factors activated as a consequence of DNA damage like p53 or initiated downstream of MAPK (mitogen-activated protein kinases) like c-Jun, ATF-2 and ATF-3. The direct causal correlation between DNA damage, activation or inhibition of MAPKs and transcription factors, and apoptosis is under current investigation. However, the induction of apoptosis in different cell lines in the presence of monomers like TEGDMA may be subject to a higher level of complexity than currently suggested by simple linear models.
• Real-time in vivo detection of biomaterial-induced reactive oxygen species
  - Biomater 32(7):1796-1801 (2011)
  The non-specific host response to implanted biomaterials is often a key challenge of medical device design. To evaluate biocompatibility, measuring the release of reactive oxygen species (ROS) produced by inflammatory cells in response to biomaterial surfaces is a well-established method. However, the detection of ROS in response to materials implanted in vivo has not yet been demonstrated. Here, we develop a bioluminescence whole animal imaging approach to observe ROS released in response to subcutaneously-implanted materials in live animals. We compared the real-time generation of ROS in response to two representative materials, polystyrene and alginate, over the course of 28 days. High levels of ROS were observed near polystyrene, but not alginate implants, and persisted throughout the course of 28 days. Histological analysis revealed that high levels of ROS correlated not only with the presence of phagocytic cells at early timepoints, but also fibrosis at later tim! epoints, suggesting that ROS may be involved in both the acute and chronic phase of the foreign body response. These data are the first in vivo demonstration of ROS generation in response to implanted materials, and describe a novel technique to evaluate the host response.
• The maintenance of pluripotency following laser direct-write of mouse embryonic stem cells
  - Biomater 32(7):1802-1808 (2011)
  The ability to precisely pattern embryonic stem (ES) cells in vitro into predefined arrays/geometries may allow for the recreation of a stem cell niche for better understanding of how cellular microenvironmental factors govern stem cell maintenance and differentiation. In this study, a new gelatin-based laser direct-write (LDW) technique was utilized to deposit mouse ES cells into defined arrays of spots, while maintaining stem cell pluripotency. Results obtained from these studies showed that ES cells were successfully printed into specific patterns and remained viable. Furthermore, ES cells retained the expression of Oct4 in nuclei after LDW, indicating that the laser energy did not affect their maintenance of an undifferentiated state. The differentiation potential of mouse ES cells after LDW was confirmed by their ability to form embryoid bodies (EBs) and to spontaneously become cell lineages representing all three germ layers, revealed by the expression of marker ! proteins of nestin (ectoderm), Myf-5 (mesoderm) and PDX-1 (endoderm), after 7 days of cultivation. Gelatin-based LDW provides a new avenue for stem cell patterning, with precision and control of the cellular microenvironment.
• Cellular transduction gradients via vapor-deposited polymer coatings
  - Biomater 32(7):1809-1815 (2011)
  Spatiotemporal control of gene delivery, particularly signaling gradients, via biomaterials poses significant challenges because of the lack of efficient delivery systems for therapeutic proteins and genes. This challenge was addressed by using chemical vapor deposition (CVD) polymerization in a counterflow set-up to deposit copolymers bearing two reactive chemical gradients. FTIR spectroscopy verified the formation of compositional gradients. Adenovirus expressing a reporter gene was biotinylated and immobilized using the VBABM method (virus-biotin-avidin-biotin-materials). Sandwich ELISA confirmed selective attachment of biotinylated adenovirus onto copolymer gradients. When cultured on the adenovirus gradients, human gingival fibroblasts exhibited asymmetric transduction with full confluency. Importantly, gradient transduction occurred in both lateral directions, thus enabling more advanced delivery studies that involve gradients of multiple therapeutic genes.
• A strategy for the engineering of insulin producing cells with a broad spectrum of defense properties
  - Biomater 32(7):1816-1825 (2011)
  Insulin-producing pancreatic beta cells are known to be extremely susceptible to the oxidative stress and hypoxia generated following islet transplantation in diabetic patients. We hereby present a novel in vivo selection strategy based on the isolation of insulin-producing cells with enhanced protection after repeated rounds of encapsulation and xenotransplantation. Rat insulinoma INS-1 cells were encapsulated in alginate macrobeads and transplanted in the peritoneal cavity of mice. After 2 days the beads were retrieved and cells were recovered from alginate and propagated in vitro until submitted to a second round of encapsulation and transplantation. Three days later, the surviving cells, named INS-1m2, were isolated from the alginate beads and their protection and functional activity examined. Compared to parental INS-1 cells, the selected INS-1m2 cells were more resistant to hydrogen peroxide, nitric oxide, alloxan and hypoxia. This enhanced protection of the sele! cted cells correlated with the increased level of catalase and poly (ADP-ribose) polymerase expression. Although selected cells expressed more insulin than parental cells, no change in their insulin response to glucose was observed. We conclude that the in vivo selection strategy is a powerful tool for the engineering of insulin producing cells with a broad spectrum of defense properties.
• Magnesium incorporation into hydroxyapatite
  - Biomater 32(7):1826-1837 (2011)
  The incorporation of Mg in hydroxyapatite (HA) was investigated using multinuclear solid state NMR, X-ray absorption spectroscopy (XAS) and computational modeling. High magnetic field 43Ca solid state NMR and Ca K-edge XAS studies of a 10% Mg-substituted HA were performed, bringing direct evidence of the preferential substitution of Mg in the Ca(II) position. 1H and 31P solid state NMR show that the environment of the anions is disordered in this substituted apatite phase. Both Density Functional Theory (DFT) and interatomic potential computations of Mg-substituted HA structures are in agreement with these observations. Indeed, the incorporation of low levels of Mg in the Ca(II) site is found to be more favourable energetically, and the NMR parameters calculated from these optimized structures are consistent with the experimental data. Calculations provide direct insight in the structural modifications of the HA lattice, due to the strong contraction of the MO distance! s around Mg. Finally, extensive interatomic potential calculations also suggest that a local clustering of Mg within the HA lattice is likely to occur. Such structural characterizations of Mg environments in apatites will favour a better understanding of the biological role of this cation.
• Integration of multiple cell-matrix interactions into alginate scaffolds for promoting cardiac tissue regeneration
  - Biomater 32(7):1838-1847 (2011)
  Cardiac tissue engineering aims to repair damaged myocardial tissues by applying heart patches created in vitro. Herein, we explored the possible role of a combination of two matrix-attached peptides, the adhesion peptide G4RGDY and heparin-binding peptide G4SPPRRARVTY (HBP) in cardiac tissue regeneration. Neonatal rat cardiac cells were seeded into unmodified, single peptide or double peptide-attached alginate scaffolds, all having the same physical features of porosity, hydrogel forming and matrix stiffness. The cardiac tissue developed in the HBP/RGD-attached scaffolds revealed the best features of a functional muscle tissue, as judged by all studied parameters, i.e., immunostaining of cardiac cell markers, histology, western blot of protein expressions and metabolic activity. By day 7, well-developed myocardial fibers were observed in these cell constructs. At 14 days the HBP/RGD-attached constructs presented an isotropic myofiber arrangement, while no such arrange! ment was seen in the other constructs. The expression levels of α-actinin, N-cadherin and Connexin-43, showing preservation and an increase in Connexin-43 expression (Cx-43) with time, further supported the formation a contractile muscle tissue in the HBP/RGD-attached scaffolds. Collectively, the attachment of combinatorial peptides representing different signaling in ECM-cell interactions proved to play a key role, contributing to the formation of a functional cardiac muscle tissue, in vitro.
• Probing cell migration in confined environments by plasma lithography
  - Biomater 32(7):1848-1855 (2011)
  Cellular processes are regulated by various mechanical and physical factors in their local microenvironment such as geometric confinements, cell–substrate interactions, and cell–cell contact. Systematic elucidation of these regulatory mechanisms is crucial for fundamental understanding of cell biology and for rational design of biomedical devices and regenerative medicine. Here, we report a generally applicable plasma lithography technique, which performs selective surface functionalization on large substrate areas, for achieving long-term, stable confinements with length scales from 100 nm to 1 cm toward the investigation of cell–microenvironment interactions. In particular, we applied plasma lithography for cellular confinement of neuroblastomas, myoblasts, endothelial cells, and mammary gland epithelial cells, and examined the motion of mouse embryonic fibroblasts in directionality-confined environments for studying the effect of confinements on migratory beha! vior. In conjunction with live cell imaging, the distance traveled, velocity, and angular motion of individual cells and collective cell migration behaviors were measured in confined environments with dimensions comparable to a cell. A critical length scale that a cell could conceivably occupy and migrate to was also identified by investigating the behaviors of cells using confined environments with subcellular length scales.
• The significance of pore microarchitecture in a multi-layered elastomeric scaffold for contractile cardiac muscle constructs
  - Biomater 32(7):1856-1864 (2011)
  Multi-layered poly(glycerol-sebacate) (PGS) scaffolds with controlled pore microarchitectures were fabricated, combined with heart cells, and cultured with perfusion to engineer contractile cardiac muscle constructs. First, one-layered (1L) scaffolds with accordion-like honeycomb shaped pores and elastomeric mechanical properties were fabricated by laser microablation of PGS membranes. Second, two-layered (2L) scaffolds with fully interconnected three dimensional pore networks were fabricated by oxygen plasma treatment of 1L scaffolds followed by stacking with off-set laminae to produce a tightly bonded composite. Third, heart cells were cultured on scaffolds with or without interstitial perfusion for 7 days. The laser-microablated PGS scaffolds exhibited ultimate tensile strength and strain-to-failure higher than normal adult rat left ventricular myocardium, and effective stiffnesses ranging from 220 to 290 kPa. The 7-day constructs contracted in response to electrica! l field stimulation. Excitation thresholds were unaffected by scaffold scale up from 1L to 2L. The 2L constructs exhibited reduced apoptosis, increased expression of connexin-43 (Cx-43) and matrix metalloprotease-2 (MMP-2) genes, and increased Cx-43 and cardiac troponin-I proteins when cultured with perfusion as compared to static controls. Together, these findings suggest that multi-layered, microfabricated PGS scaffolds may be applicable to myocardial repair applications requiring mechanical support, cell delivery and active implant contractility.
• The inhibition of retinal neovascularization by gold nanoparticles via suppression of VEGFR-2 activation
  - Biomater 32(7):1865-1871 (2011)
  The pathological angiogenesis in the retina is the major cause of vision loss at all ages. In particular, retinopathy of prematurity (ROP) is a leading cause of blindness in children. This study investigated whether gold nanoparticle (GNP) could inhibit retinal neovascularization in the animal model of ROP. Intravitreal injection of GNP significantly inhibited retinal neovascularization in the mouse model of ROP. In addition, GNP effectively suppressed VEGF-induced in vitro angiogenesis of retinal microvascular endothelial cells including proliferation, migration and capillary-like networks formation. GNP blocked VEGF-induced auto-phosphorylation of VEGFR-2 to inhibit consequently ERK 1/2 activation. GNP never affected on the cellular viability of retinal microvascular endothelial cells and induced no retinal toxicity. Our data suggest that GNP could be a potent inhibitor to retinal neovascularization without retinal toxicity. Furthermore, GNP could be extensively appl! ied to variable vaso-proliferative retinopathies mediated by VEGF.
• Endothelial cell scaffolds generated by 3D direct writing of biodegradable polymer microfibers
  - Biomater 32(7):1872-1879 (2011)
  The engineering of large (thickness > 100 μm) tissues requires a microvascular network to supply nutrients and remove waste. To produce microvasculature in vitro, a scaffold is required to mechanically support and stimulate endothelial cell (EC) adhesion and growth. Scaffolds for ECs are currently produced by patterning polymers or other biomaterials into configurations which often possess isotropic morphologies such as porous films and fibrous mats. We propose a new "direct-write" process for fabricating scaffolds composed of suspended polymer microfibers that are precisely oriented in 3D, providing directional architecture for selectively guiding cell growth along a desired pathway. The diameters of the fibers produced with this process were predictably and repeatably controlled through modulation of the system parameters, enabling production of fibers with microvascular-scale diameters (5–20 μm) from a variety of biodegradable polymers. These scaffolds were ! successfully seeded with ECs, which conformed to the geometry of the fibers and proliferated over the course of one week.
• PEGylation of hyaluronic acid nanoparticles improves tumor targetability in vivo
  - Biomater 32(7):1880-1889 (2011)
  A major drawback of hyaluronic acid (HA)-based drug conjugates or nanoparticles for cancer therapy is their preferential accumulation in the liver after systemic administration. In an attempt to investigate the physicochemical characteristics and in vivo fates of poly(ethylene glycol) (PEG)-conjugated HA nanoparticles (HA-NPs), amphiphilic HA derivatives were prepared by varying the degree of PEGylation. The PEGylated HA-NPs formed self-assembled nanoparticles (217–269 nm in diameter) with the negatively charged surfaces in the physiological condition. Although PEGylation of HA-NPs reduced their cellular uptake in vitro, larger amounts of nanoparticles were taken up by cancer cells over-expressing CD44, an HA receptor, than by normal fibroblast cells. The ex vivo images of the organs using an optical imaging technique after the intravenous injection of Cy5.5-labeled nanoparticles into normal mice demonstrated that PEGylation could effectively reduce the liver uptake ! of HA-NPs and increase their circulation time in the blood. When the nanoparticles were systemically administered into tumor-bearing mice for in vivo real-time imaging, the strongest fluorescence signals were detected at the tumor site of the mice for the whole period of time studied, indicating their high tumor targetability. Interestingly, PEGylated HA-NPs were more effectively accumulated into the tumor tissue up to 1.6-fold higher than bare HA-NPs. The high tumor targetability of PEGylated HA-NPs was further supported by the intravital tumor imaging, in which their rapid extravasation into the tumor tissue was clearly observed. These results suggest that PEGylated HA-NPs can be useful as a means for cancer therapy and diagnosis.
• Multi-functional magnetic nanoparticles for magnetic resonance imaging and cancer therapy
  - Biomater 32(7):1890-1905 (2011)
  We have developed a multi-layer approach for the synthesis of water-dispersible superparamagnetic iron oxide nanoparticles for hyperthermia, magnetic resonance imaging (MRI) and drug delivery applications. In this approach, iron oxide core nanoparticles were obtained by precipitation of iron salts in the presence of ammonia and provided β-cyclodextrin and pluronic polymer (F127) coatings. This formulation (F127250) was highly water dispersible which allowed encapsulation of the anti-cancer drug(s) in β-cyclodextrin and pluronic polymer for sustained drug release. The F127250 formulation has exhibited superior hyperthermia effects over time under alternating magnetic field compared to pure magnetic nanoparticles (MNP) and β-cyclodextrin coated nanoparticles (CD200). Additionally, the improved MRI characteristics were also observed for the F127250 formulation in agar gel and in cisplatin resistant ovarian cancer cells (A12780CP) compared to MNP and CD200 formulations.! Furthermore, the drug-loaded formulation of F127250 exhibited many folds of imaging contrast properties. Due to the internalization capacity of the F127250 formulation, its curcumin-loaded formulation (F127250-CUR) exhibited almost equivalent inhibition effects on A2780CP (ovarian), MDA-MB-231 (breast), and PC-3 (prostate) cancer cells even though curcumin release was only 40%. The improved therapeutic effects were verified by examining molecular effects using Western blotting and transmission electron microscopic (TEM) studies. F127250-CUR also exhibited haemocompatibility, suggesting a nanochemo-therapuetic agent for cancer therapy.
• A strategy for ZnO nanorod mediated multi-mode cancer treatment
  - Biomater 32(7):1906-1914 (2011)
  Combinations of cancer therapy modalities are attracting attention to improve the outcome of treatment, since single modality has not always been sufficiently effective. The aim of this study was to investigate a new strategy of combined application of ZnO nanorods with anticancer drug daunorubicin (DNR) in photodynamic therapy (PDT). Using a simple one-step solid state reaction in air at room temperature, we were able to fabricate ZnO nanorods as the drug carrier of DNR in drug delivery system. The combination of ZnO nanorods with DNR induced the remarkable improvement in the anti-tumor activity, which has been demonstrated by the flow cytometry, MTT assay and nuclear DAPI staining. Furthermore, the possible signaling pathway was explored by immunocytochemistry. It was noted that the notable photodynamic activity of the non-cytotoxic ZnO nanorods could considerably increase cancer cell injury mediated by reactive oxygen species (ROS). For instance, in human hepatocarc! inoma cells (SMMC-7721 cells), our observations demonstrated that ZnO nanorods could obviously increase the intracellular concentration of DNR and enhance its potential anti-tumor efficiency, indicating that ZnO nanorods could act as an efficient drug delivery carrier importing DNR into target cancer cells. Furthermore, photodynamic ZnO nanorods loaded chemotherapeutic agent could induce distinguished improvement in anti-tumor activity with UV illumination. These findings revealed that such modality combinations represent a promising approach in cancer therapy.
• Molecular beacon-based bioimaging of multiple microRNAs during myogenesis
  - Biomater 32(7):1915-1922 (2011)
  MicroRNAs (miRNAs, miR) are associated with multiple cellular processes and diseases. Here, we designed fluorescent DNA probes composed of stem loop-structured DNA complementary to miRNAs and fluorophore–quencher pairs [molecular beacon (MB)] to simultaneously monitor the biogenesis of miR-206 and miR-26a, which are highly expressed during myogenic differentiation. C2C12 cells were transfected with an MB targeting miR-26a and containing a 6-FAM-BHQ1 pair (miRNA-26a MB) or an MB targeting miR-206 with a Texas Red-BHQ2 pair (miRNA-206 MB). In vitro and in vivo fluorescence analysis revealed that, only in differentiated single C2C12 cell, significantly increased fluorescence signals of miRNA-26a MB, miRNA-206 MB or simultaneous incubation of both beacons were detected due to the hybridization of miR-206 or miR-26a with their respective beacons, resulting in activation of fluorescence. Our MB-based miRNA imaging system may serve as a new imaging probe for monitoring mult! iple miRNAs during various cellular or disease processes associated with miRNAs.
• Quantum dot-antisense oligonucleotide conjugates for multifunctional gene transfection, mRNA regulation, and tracking of biological processes
  - Biomater 32(7):1923-1931 (2011)
  It was demonstrated that oligonucleotides, independent of their base sequence and length, could effectively induce the cellular uptake of mercapto acid-capped CdTe QDs after the oligonucleotides were covalently attached on the surface of the QDs. Following these experimental observations, a conjugate composed of covalently linked anti-survivin antisense oligonucleotides (ASON) and CdTe QDs was designed and synthesized. Then, the survivin mRNA down-regulation and the apoptosis of HeLa cells induced by ASON were studied. Systematic experimental results revealed that CdTe-ASON could effectively induce the apoptosis of HeLa cells, while CdTe QDs offered the possibility to visualize the specific intracellular localization of the CdTe-ASON probes strongly associated with their biological functions.
• A magnetic, reversible pH-responsive nanogated ensemble based on Fe3O4 nanoparticles-capped mesoporous silica
  - Biomater 32(7):1932-1942 (2011)
  Stimuli-sensitive mesoporous silica nanoparticles (MSNs)-based hybrid "gate-like" ensembles capable of performing specific programmed release mode represent a new generation delivery system in recent years. In this paper, a magnetic and reversible pH-responsive, MSNs-based nanogated ensemble was fabricated by anchoring superparamagnetic Fe3O4 nanoparticles on the pore outlet of MSNs via a reversible boronate esters linker. To achieve this, MSNs and Fe3O4 nanoparticles were first synthesized and functionalized by polyalcohol derivative and boronic acid, respectively. The successful incorporation of Fe3O4 nanoparticles onto the MSNs was confirmed by the results of XRD, TEM, XPS and N2 adsorption-desorption method. The pH-driven "gate-like" effect was studied by in vitro release of an entrapped model dexamethasone from the pore voids into the bulk solution at different pH values. The results indicated that at pH 5–8, the pores of the MSNs were effectively capped! with Fe3O4 nanoparticles and the drug release was strongly inhibited. While at pH 2–4, the hydrolysis of the boroester bond took place and thus resulted in a rapid release of the entrapped drug. And by alternately changing the pH from 3 to 7, these Fe3O4 cap gate could be switched "on" and "off" and thereby released the entrapped drug in a pulsinate manner (in small portions). Additionally, this nanogated release system exhibited good magnetic property, high cell biocompatibility and cellular uptake for MC3T3-E1 cells. The present data suggest that it is possible to obtain simple and very effective pH-driven pulsinate release using these Fe3O4-capped-MSNs, and this new platform represents a promising candidate in the formulation of in vivo targeted delivery of therapeutic agents to low pH tissues, such as tumors and inflammatory sites.
• Robust MeO2MA/vinyl-4,6-diamino-1,3,5-triazine copolymer hydrogels-mediated reverse gene transfection and thermo-induced cell detachment
  - Biomater 32(7):1943-1949 (2011)
  We have fabricated a robust temperature sensitive hydrogel by photoinitiated copolymerization of 2-(2-methoxyethoxy)ethyl methacrylate (MeO2MA), 2-vinyl-4,6-diamino-1,3,5-triazine (VDT) and crosslinker polyethylene glycol diacrylate (PEGDA). It was shown that self-hydrogen bondings of VDT moieties in the bulk considerably strengthened the mechanical properties of gels, which was dependent on the weight ratio of MeO2/VDT and the initial monomer concentration; the VDT motifs on the surface could efficiently bind DNA for reverse gene transfection. On this soft-wet platform, gene expression lasted 7 days and the re-treated gels could be reused for new cycle of transfection. The gene modified cells could be detached by thermo-triggered switchable surface hydrophilicity of PMEO2MA in hydrogel. MTT assay showed low cytotoxicity of hydrogels. The results suggested that this type of mechanically strong H-bonded and thermoresponsive hydrogels hold a great potential as an integra! ted functional soft-wet platform for the unharmful harvest of gene modified seed cells for tissue engineering or as implantable scaffold for gene therapy and regenerative medicine applications.
• A scalable controlled-release device for transscleral drug delivery to the retina
  - Biomater 32(7):1950-1956 (2011)
  A transscleral drug-delivery device, designed for the administration of protein-type drugs, that consists of a drug reservoir covered with a controlled-release membrane was manufactured and tested. The controlled-release membrane is made of photopolymerized polyethylene glycol dimethacrylate (PEGDM) that contains interconnected collagen microparticles (COLs), which are the routes for drug permeation. The results showed that the release of 40-kDa FITC-dextran (FD40) was dependent on the COL concentration, which indicated that FD40 travelled through the membrane-embedded COLs. Additionally, the sustained-release drug formulations, FD40-loaded COLs and FD40-loaded COLs pelletized with PEGDM, fine-tuned the release of FD40. Capsules filled with COLs that contained recombinant human brain-derived neurotrophic factor (rhBDNF) released bioactive rhBDNF in a manner dependent on the membrane COL concentration, as was found for FD40 release. When capsules were sutured onto scler! ae of rabbit eyes, FD40 was found to spread to the retinal pigment epithelium. Implantation of the device was easy, and it did not damage the eye tissues. In conclusion, our capsule is easily modified to accommodate different release rates for protein-type drugs by altering the membrane COL composition and/or drug formulation and can be implanted and removed with minor surgery. The device thus has great potential as a conduit for continuous, controlled drug release.
• Real-time monitoring of sustained drug release using the optical properties of porous silicon photonic crystal particles
  - Biomater 32(7):1957-1966 (2011)
  A controlled and observable drug delivery system that enables long-term local drug administration is reported. Biodegradable and biocompatible drug-loaded porous Si microparticles were prepared from silicon wafers, resulting in a porous 1-dimensional photonic crystal (rugate filter) approx. 12 μm thick and 35 μm across. An organic linker, 1-undecylenic acid, was attached to the Si–H terminated inner surface of the particles by hydrosilylation and the anthracycline drug daunorubicin was bound to the carboxy terminus of the linker. Degradation of the porous Si matrix in vitro was found to release the drug in a linear and sustained fashion for 30 d. The bioactivity of the released daunorubicin was verified on retinal pigment epithelial (RPE) cells. The degradation/drug delivery process was monitored in situ by digital imaging or spectroscopic measurement of the photonic resonance reflected from the nanostructured particles, and a simple linear correlation between obse! rved wavelength and drug release was observed. Changes in the optical reflectance spectrum were sufficiently large to be visible as a distinctive red to green color change.
• Mucosal irritation potential of polyelectrolyte multilayer capsules
  - Biomater 32(7):1967-1977 (2011)
  Polyelectrolyte multilayer capsules have recently gained interest as carriers for drug delivery. When envisioning mucosal administration, one is focused with potential concerns such as tissue irritation and tissue damage, induced by the carrier itself. In this paper we demonstrate the use of a slug-based (Arion lusitanicus) assay to evaluate the mucosal irritation potential of different types of polyelectrolytes, their complexes and multilayer capsules. This assay allows to assess in a simple yet efficient way mucosal tissue irritation without using large numbers of vertebrates such as mice, rabbits or non-human primates. We found that although single polyelectrolyte components do induce tissue irritation, this response is dramatically reduced upon complexation with an oppositely charged polyelectrolyte, rendering fairly inert polyelectrolyte complexes. These findings put polyelectrolyte multilayer capsules further en route towards drug delivery applications.
• Lifelong reporter gene imaging in the lungs of mice following polyethyleneimine-mediated sleeping-beauty transposon delivery
  - Biomater 32(7):1978-1985 (2011)
  Polyethyleneimine (PEI) is a cationic polymer that is effective in gene delivery in vivo. Plasmid DNA incorporating the Sleeping-Beauty (SB) transposon has been shown to induce long-term transgene expression in mouse lungs after PEI-mediated delivery. In the current report, we followed the reporter gene expression mediated by PEI/SB delivery in lungs of mice using the non-invasive bioluminescent imaging (BLI) technology. After delivery, the reporter gene signal showed a rapid decay in the first two weeks to a nearly undetectable level, but then the signal augmented gradually in the following weeks and finally reached a stable level that maintained until the natural death of animals. The stabilization of transgene expression is associated with the multiplication of a small number of PEI/SB-labeled alveolar cells, which proliferated both under normal conditions and in response to acute local injury for epithelia repair, and may play a role in long-term homeostatic mainte! nance in alveoli. The data presented here suggests that systemic delivery of PEI/SB induces stable transfection specifically in a small population of alveolar progenitor cells. The technique provides a promising platform for future research in distal lung biology and tissue regenerative therapy.
• A mesoporous silica nanoparticulate/β-TCP/BG composite drug delivery system for osteoarticular tuberculosis therapy
  - Biomater 32(7):1986-1995 (2011)
  A composite scaffold drug delivery system (CS-DDS) for osteoarticular tuberculosis therapy has been prepared by loading bi-component drugs into a mesoporous silica nanoparticles (MSNs)-coated porous β-TCP scaffold, which was followed by an additional bioactive glass coating. Such a CS-DDS showed high performances in the local and extremely sustained delivery of the bi-component antitubercular drugs and excellent biocompatibility. N2 sorption isotherms indicated greatly increased surface area of the composites compared to pure β-TCP scaffold, and the mesopores were around 2.6 nm which were large enough to encapsulate drugs such as isoniazide and rifampicin. The in vitro and in vivo release tests demonstrated extra sustained co-release profiles of rifampicin and isoniazide from such a CS-DDS, and both drug concentrations kept higher than their effective values to kill mycobacterium tuberculosis for as long as 42 days. The hepatic and renal function tests indicated that! the CS-DDS had neglectable long-term lesions to liver and kidney.
• Bioactivation of dermal scaffolds with a non-viral copolymer-protected gene vector
  - Biomater 32(7):1996-2003 (2011)
  The use of scaffolds in skin tissue engineering is accompanied with low regeneration rates and high risk of infection. In this study, we activated an FDA-approved collagen scaffold for dermal regeneration by incorporation of copolymer-protected gene vectors (COPROGs) to induce a temporary release of VEGF. In vitro results show that the presence of COPROGs did not affect the distribution, attachment, proliferation and viability of cells in the scaffold. A transient release of VEGF was observed for up to 3 weeks. Moreover a high amount of VEGF was also found in the cells and associated with the scaffold. In a full skin defect model in nude mice, VEGF levels were significantly increased compared to controls in VEGF gene activated scaffolds 14 d after implantation, but not in skin from the wound edge. Results showed an increased amount of non-adherent cells, especially erythrocytes, and von Willebrandt factor (vWF) and a yellow red appearance of gene activated scaffolds in! relation to controls. This suggests the presence of leaky vessels. In this work we show that the bioactivation of collagen scaffolds with COPROGs presents a new technology that allows a local release of therapeutic proteins thus enhancing the regenerative potential in vivo.
• Tuning the non-equilibrium state of a drug-encapsulated poly(ethylene glycol) hydrogel for stem and progenitor cell mobilization
  - Biomater 32(7):2004-2012 (2011)
  Injectable and biodegradable hydrogels have been increasingly studied for sustained drug delivery in various molecular therapies. However, it remains a challenge to attain desired delivery rate at injection sites due to local tissue pressures exerted on the soft hydrogels. Furthermore, there is often limited controllability of stiffness and degradation rates, which are key factors required for achieving desired drug release rate and therapeutic efficacy. This study presents a stiff and metastable poly(ethylene glycol) diacrylate (PEGDA)-poly(ethylene imine) (PEI) hydrogel which exhibits an elastic modulus equivalent to bulk plastic materials, and controllable degradation rate independent of its initial elastic modulus. Such unique stiffness was attained from the highly branched architecture of PEI, and the decoupled controllability of degradation rate was achieved by tuning the non-equilibrium swelling of the hydrogel. Furthermore, a single intramuscular administration! of granulocyte colony stimulating factor (GCSF)-encapsulated PEGDA-PEI hydrogel extended the mobilization of mononuclear cells to four days. A larger yield of expanded CD34+ and CD31+ endothelial progenitor cells (EPCs) was also obtained as compared to the daily bolus administration. Overall, the hydrogel created in this study will be useful for the controlled and sustained delivery of a wide array of drug molecules.