Hot off the presses! Feb 01 Biomater

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Latest Articles Include:

• Editorial board
  - Biomater 32(4):OFC (2011)
  
• Differential response of Staphylococci and osteoblasts to varying titanium surface roughness
  - Biomater 32(4):951-960 (2011)
  The surface roughness of metallic orthopaedic implants has typically been used to influence osseointegration and spatially control load transfer to the surrounding bone. Because of the increasing recognition of biomaterials-associated infection as a leading implant failure mode, we are interested to know the relative importance of roughness not only on surface–osteoblast interactions but also on surface–bacteria interactions. This in vitro study thus compares the effects of surface topography on Staphylococcus epidermidis and human osteoblast behavior using four clinically relevant titanium surface finishes: polished, satin, grit-blasted and plasma-sprayed. Important differences between these surfaces are manifested not only by their vertical roughness parameters but also by the lateral length scales over which topographic fluctuations occur. We find that S. epidermidis adhesion and growth is substantially higher on the satin and grit-blasted surfaces than on the p! olished or plasma-sprayed surfaces. The former are both substantially rougher at length scales comparable to that of bacteria. In contrast, based on imaging and biochemical assays of proliferation, differentiation and matrix formation, we find that desirable osteoblast–surface interactions are maximized on plasma-sprayed surfaces and minimized on satin-finished surfaces. We attribute these differences to the fact that the plasma-sprayed surface is relatively smooth compared to the size of an individual osteoblast, while the satin surface is rough at this length scale. These findings indicate that both the vertical and lateral character of surface roughness can be modified to not only optimize implant–bone interactions but to simultaneously minimize implant–bacteria interactions.
• Functionalizable and nonfouling zwitterionic carboxybetaine hydrogels with a carboxybetaine dimethacrylate crosslinker
  - Biomater 32(4):961-968 (2011)
  We have introduced a dimethacrylate carboxybetaine-based crosslinker that has excellent compatibility with zwitterionic hydrogel systems. Poly(carboxybetaine methacrylate) (pCBMA) hydrogels prepared with the new CBMA crosslinker (CBMAX) result in considerably improved solubility, homogeneity, and mechanical properties (up to 8 MPa compressive modulus) over those prepared with the commercially available N,N'-methylenebis(acrylamide) (MBAA) crosslinker. The zwitterionic nature of the CBMAX crosslinker provides continuity of ordered hydration in the CBMA hydrogel and retains its nonfouling properties. CBMAX-crosslinked CBMA hydrogels had lower cell fouling than MBAA-crosslinked CBMA hydrogels and in fact reduced cell adhesion by about 90% relative to pHEMA hydrogels. Furthermore, unlike pHEMA, CBMA hydrogels are readily functionalizable. Cell adhesion on nonfouling CBMA hydrogels was controlled by cRGD functionalization.
• Volumetric interpretation of protein adsorption: Interfacial packing of protein adsorbed to hydrophobic surfaces from surface-saturating solution concentrations
  - Biomater 32(4):969-978 (2011)
  The maximum capacity of a hydrophobic adsorbent is interpreted in terms of square or hexagonal (cubic and face-centered-cubic, FCC) interfacial packing models of adsorbed blood proteins in a way that accommodates experimental measurements by the solution-depletion method and quartz-crystal-microbalance (QCM) for the human proteins serum albumin (HSA, 66 kDa), immunoglobulin G (IgG, 160 kDa), fibrinogen (Fib, 341 kDa), and immunoglobulin M (IgM, 1000 kDa). A simple analysis shows that adsorbent capacity is capped by a fixed mass/volume (e.g. mg/mL) surface-region (interphase) concentration and not molar concentration. Nearly analytical agreement between the packing models and experiment suggests that, at surface saturation, above-mentioned proteins assemble within the interphase in a manner that approximates a well-ordered array. HSA saturates a hydrophobic adsorbent with the equivalent of a single square or hexagonally-packed layer of hydrated molecules whereas the lar! ger proteins occupy two-or-more layers, depending on the specific protein under consideration and analytical method used to measure adsorbate mass (solution depletion or QCM). Square or hexagonal (cubic and FCC) packing models cannot be clearly distinguished by comparison to experimental data. QCM measurement of adsorbent capacity is shown to be significantly different than that measured by solution depletion for similar hydrophobic adsorbents. The underlying reason is traced to the fact that QCM measures contribution of both core protein, water of hydration, and interphase water whereas solution depletion measures only the contribution of core protein. It is further shown that thickness of the interphase directly measured by QCM systematically exceeds that inferred from solution-depletion measurements, presumably because the static model used to interpret solution depletion does not accurately capture the complexities of the viscoelastic interfacial environment probed by Q! CM.
• Competitive time- and density-dependent adhesion of staphylococci and osteoblasts on crosslinked poly(ethylene glycol)-based polymer coatings in co-culture flow chambers
  - Biomater 32(4):979-984 (2011)
  Biomaterial-associated infections (BAI) remain a serious clinical complication, often arising from an inability of host tissue-implant integration to out-compete bacterial adhesion and growth. A commercial polymer coating based on polyethylene glycol (PEG), available in both chemically inert and NHS-activated forms (OptiChem®), was compared for simultaneous growth of staphylococci and osteoblasts. In the absence of staphylococci, osteoblasts adhered and proliferated well on glass controls and on the NHS-reactive PEG-based coating over 48 h, but not on the inert PEG coating. Staphylococcal growth was low on both PEG-based coatings. When staphylococci were pre-adhered on surfaces for 1.5 h to mimic peri-operative contamination, osteoblast growth and spreading was reduced on glass but virtually absent on both reactive and inert PEG-based coatings. Thus although NHS-reactive, PEG-based coatings stimulated tissue–cell interactions in the absence of contaminating staphylo! cocci, the presence of adhering staphylococci eliminated osteoblast adhesion advantages on the PEG surface. This study demonstrates the importance of using bacterial and cellular co-cultures compared to monocultures when assessing functionalized biomaterials coatings for infectious potential.
• In vivo tissue responses to thermal-responsive shape memory polymer nanocomposites
  - Biomater 32(4):985-991 (2011)
  To explore the safe use of thermal-responsive shape memory polymers (SMPs) as minimally invasive tissue scaffolds, we recently developed a class of biodegradable POSS-SMP nanocomposites exhibiting stable temporary shape fixing and facile shape recovery within a narrow window of physiological temperatures. The materials were covalently crosslinked from star-branched building blocks consisting a bioinert polyhedral oligomeric silsesquioxane (POSS) core and 8 degradable poly(d,l-lactide) (PLA) arms. Here we examine the degradation profiles and immunogenicity of POSS-SMPs as a function of the PLA arm lengths using a rat subcutaneous implantation model. We show that POSS-SMPs elicited a mild foreign body type immune response upon implantation. The degradation rates of POSS-SMPs, both in vitro and in vivo, inversely correlated with the length of the PLA chains within the crosslinked amorphous network. Upon in vivo degradation of POSS-SMPs, a second acute inflammatory respons! e was elicited locally, and the inflammation was able to resolve over time without medical interventions. One year after the implantation of POSS-SMPs, no pathologic abnormities were detected from the vital/scavenger organs examined. These minimally immunogenic and biodegradable SMPs are promising candidates for scaffold-assisted tissue repair where both facile surgical delivery and controlled degradation of the scaffold are desired for achieving optimal short-term and long-term clinical outcomes.
• Design of culture substrates for large-scale expansion of neural stem cells
  - Biomater 32(4):992-1001 (2011)
  Neural stem cells (NSCs) have been frequently used to investigate in vitro the molecular and cellular mechanisms underlying the development of the central nervous system (CNS). In addition, NSCs are regarded as one of the potential sources for the cell replacement therapy of CNS disorders. Most of these studies have utilized NSCs prepared by neurosphere culture. However, this method normally yields a heterogeneous population containing differentiated neural cells as well as NSCs. In addition, the rate of cell expansion is not high enough for obtaining a large quantity of NSCs in a short period. Here we report the design of culture substrates that allow highly selective and rapid expansion of NSCs. We synthesize epidermal growth factor fused with a hexahistidine sequence (EGF-His) and a polystyrene-binding peptide (EGF-PSt), and these engineered growth factors were surface-anchored to a nickel-chelated glass plate and a polystyrene dish, respectively. The EGF-His-chelat! ed glass substrate was further used to assemble a culture module. Neurosphere-forming cells prepared from the fetal rat striatum were used to examine the selective expansion of NSCs using the EGF-His-chelated module and the EGF-PSt-bound polystyrene dish. Our results show that the culture module enables to selectively expand NSCs in a closed system more efficiently than the standard neurosphere culture. The EGF-PSt-bound polystyrene dish also permits efficient expansion of NSCs, providing a straightforward means to acquire a large quantity of pure NSCs in standard laboratories.
• Hydrogels with time-dependent material properties enhance cardiomyocyte differentiation in vitro
  - Biomater 32(4):1002-1009 (2011)
  Tissue-specific elastic modulus (E), or 'stiffness,' arises from developmental changes in the extracellular matrix (ECM) and suggests that progenitor cell differentiation may be optimal when physical conditions mimic tissue progression. For cardiomyocytes, maturing from mesoderm to adult myocardium results in a 9-fold stiffening originating in part from a change in collagen expression and localization. To mimic this temporal stiffness change in vitro, thiolated-hyaluronic acid (HA) hydrogels were crosslinked with poly(ethylene glycol) diacrylate, and their dynamics were modulated by changing crosslinker molecular weight. With the hydrogel appropriately tuned to stiffen as heart muscle does during development, pre-cardiac cells grown on collagen-coated HA hydrogels exhibit a 3-fold increase in mature cardiac specific markers and form up to 60% more maturing muscle fibers than they do when grown on compliant but static polyacrylamide hydrogels over 2 weeks. Though es! ter hydrolysis does not substantially alter hydrogel stiffening over 2 weeks in vitro, model predictions indicate that ester hydrolysis will eventually degrade the material with additional time, implying that this hydrogel may be appropriate for in vivo applications where temporally changing material properties enhance cell maturation prior to its replacement with host tissue.
• Spherical bioactive glass particles and their interaction with human mesenchymal stem cells in vitro
  - Biomater 32(4):1010-1018 (2011)
  Sub-micron particles of bioactive glass (SMBGs) with composition 85 mol% SiO2 and 15 mol% CaO were synthesised and characterised. Bioactivity was demonstrated by the formation of calcium apatite following 5 days immersion in simulated body fluid (SBF). The effect of a 24 h exposure of SMBGs (100 μg/ml, 150 μg/ml, 200 μg/ml) to human mesenchymal stem cells (hMSCs) on cell viability, metabolic activity and proliferation were determined using the LIVE/DEAD, MTT, total DNA and LDH assays after 1, 4 and 7 days of culture. None of the SMBG concentrations caused significant cytotoxicity at 1 and 4 days, but the doses of 150 and 200 μg/ml significantly decreased hMSC metabolic activity after 7 days of culture. Cell proliferation decreased as SMBG concentration increased; however none of the SMBGs tested had a significant effect on DNA quantity compared to the control. Confocal microscopy confirmed cellular uptake and localisation of the SMBGs in the hMSC cytoskeleton. Tran! smission electron microscopy revealed that the SMBGs localised inside the cell cytoplasm and cell endosomes. These findings are important for assessing the toxicity of sub-micron particles that may either be used as injectables for bone regeneration or generated by wear or degradation of bioactive glass scaffolds.
• The healing of full-thickness burns treated by using plasmid DNA encoding VEGF-165 activated collagen–chitosan dermal equivalents
  - Biomater 32(4):1019-1031 (2011)
  Repair of deep burn by use of the dermal equivalent relies strongly on the angiogenesis and thereby the regeneration of dermis. To enhance the dermal regeneration, in this study plasmid DNA encoding vascular endothelial growth factor-165 (VEGF-165)/N,N,N-trimethyl chitosan chloride (TMC) complexes were loaded into a bilayer porous collagen–chitosan/silicone membrane dermal equivalents (BDEs), which were applied for treatment of full-thickness burn wounds. The DNA released from the collagen–chitosan scaffold could remain its supercoiled structure but its degree was decayed along with the prolongation of incubation time. The released DNA could transfect HEK293 cells in vitro with decayed efficiency too. Human umbilical vein endothelial cells (HUVECs) in vitro cultured in the scaffold loaded with TMC/pDNA-VEGF complexes expressed a significantly higher level of VEGF and showed higher viability than those cultured in the controls, i.e. blank scaffold, and scaffolds loa! ded with naked pDNA-VEGF and TMC/pDNA-eGFP, respectively. The four different BDEs were then transplanted in porcine full-thickness burn wounds. Results showed that the TMC/pDNA-VEGF group had a significantly higher number of newly-formed and mature blood vessels, and fastest regeneration of the dermis. RT-qPCR and western blotting found that the experimental group also had the highest expression of VEGF, CD31 and α-SMA in both mRNA and protein levels. Furthermore, ultra-thin skin grafting was performed on the regenerated dermis 14 days later, leading to complete repair of the burn wounds with normal histology. Moreover, the tensile strength of the repaired tissue increased along with the time prolongation of post grafting, resulting in a value of approximately 70% of the normal skin at 105 days.
• The influence of scaffold architecture on chondrocyte distribution and behavior in matrix-associated chondrocyte transplantation grafts
  - Biomater 32(4):1032-1040 (2011)
  Scaffold architecture and composition are important parameters in cartilage tissue engineering. In this in vitro study, we compared the morphology of four different cell-graft systems applied in clinical cartilage regeneration and analyzed the cell distribution (DAPI nuclei staining) and cell–scaffold interaction (SEM, TEM). Our investigations revealed major differences in cell distribution related to scaffold density, pore size and architecture. Material composition influenced the quantity of autogenous matrix used for cellular adhesion. Cell bonding was further influenced by the geometry of the scaffold subunits. On scaffolds with widely spaced fibers and a thickness less than the cell diameter, chondrocytes surrounded the scaffold fibers with cell extensions. On those fibers, chondrocytes were spherical, suggesting a differentiated phenotype. Fiber sizes smaller than chondrocyte size, and widely spaced, are therefore beneficial in terms of improved adhesion by cel! l shape adaptation. They also support the differentiated stage of chondrocytes by preventing the fibroblast-like and polygonal cell shape, at least briefly.
• Robust cell integration from co-transplantation of biodegradable MMP2-PLGA microspheres with retinal progenitor cells
  - Biomater 32(4):1041-1050 (2011)
  The failure of the adult mammalian retina to regenerate can be partly attributed to the barrier formed by inhibitory extracellular matrix (ECM) and cell adhesion molecules, such as CD44 and neurocan, after degeneration. These molecules act to separate a sub-retinal graft from integrating into the host retina. It has been shown that matrix metalloproteinase 2 (MMP2) can promote host-donor integration by degrading these molecules. In order to enhance cellular integration and promote retinal repopulation, we co-transplanted biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres that have the ability to deliver active MMP2 with retinal progenitor cells (RPCs) to the sub-retinal space of adult retinal degenerative Rho-/- mice. Following delivery, significant degradation of CD44 and neurocan at the outer surface of the degenerative retina without disruption of the host retinal architecture was observed. Coincident with this, we observed a significant increase in the! number of cells migrating beyond the barrier into the degenerative retina. No changes in the differentiation characteristics of RPCs were observed. Cells in the outer nuclear layer (ONL) could express the mature photoreceptor markers recoverin, make contacts with residual protein kinase C (PKC)-positive cells and express the ribbon synapse protein bassoon. Thus, co-transplantation of MMP2-PLGA microspheres with RPCs provides controlled release of active MMP2 to the site of retinal degeneration, stimulating inhibitory barrier removal and enhancing cell integration. This suggests a practical and effective strategy for retinal repair.
• Biocompatibility and osteogenesis of biomimetic Bioglass-Collagen-Phosphatidylserine composite scaffolds for bone tissue engineering
  - Biomater 32(4):1051-1058 (2011)
  A novel biomimetic composite scaffold Bioglass-Collagen-Phosphatidylserine (BG-COL-PS) was fabricated with a freeze-drying technique. The macrostructure and morphology as well as mechanical strength of the scaffolds were characterized. Scanning electronic microscopy (SEM) showed that the BG-COL-PS scaffolds exhibited interconnected porous structures with pore sizes of several microns up to about 300 μm. The scaffolds have a porosity of 75.40% and the corresponding compressive strength of 1.5469 Mpa. Rat mesenchymal stem cells (rMSCs) were seeded on BG-COL-PS or BG-COL scaffolds and cultured for 21 days in vitro. Based on the results of SEM, dsDNA content, alkaline phosphatase (ALP) activity, osteogenic gene expression analysis and alizarin red staining, the responses of MSCs to the scaffold exhibited a higher degree of attachment, growth as well as osteogenic differentiation than those on BG-COL scaffolds in vitro. To investigate the in vivo biocompatibility and osteo! genesis of the composite scaffolds, both pure BG-COL-PS scaffolds and MSC/scaffold constructs were implanted in rat femurs defects for 6 weeks and studied histologically and radiographically. The in vivo results showed that BG-COL-PS composite scaffolds exhibited good biocompatibility and extensive osteoconductivity with host bone. Moreover, the BG-COL-PS/MSC constructs dramatically enhanced the efficiency of new bone formation than pure BG-COL-PS scaffolds or BG-COL/MSC constructs. All these results demonstrate the usefulness of PS composited BG-COL-PS scaffolds for inducing enhanced bone formation. The BG-COL-PS scaffolds fulfill the basic requirements of bone tissue engineering scaffold and have the potential to be applied in orthopedic and reconstructive surgery.
• Nanofibrous architecture of silk fibroin scaffolds prepared with a mild self-assembly process
  - Biomater 32(4):1059-1067 (2011)
  Besides excellent biocompatibility and biodegradability, a useful tissue engineering scaffold should provide suitable macropores and nanofibrous structure, similar to extracellular matrix (ECM), to induce desired cellular activities and to guide tissue regeneration. In the present study, a mild process to prepare porous and nanofibrous silk-based scaffolds from aqueous solution is described. Using collagen to control the self-assembly of silk, nanofibrous silk scaffolds were firstly achieved through lyophilization. Water annealing was used to generate insolubility in the silk-based scaffolds, thereby avoiding the use of organic solvents. The nano-fibrils formed in the silk-collagen scaffolds had diameters of 20–100 nm, similar with native collagen in ECM. The silk-collagen scaffolds dissolved slowly in PBS solution, with about a 28% mass lost after 4 weeks. Following the dissolution or degradation, the nanofibrous structure inside the macropore walls emerged and inte! racted with cells directly. During in vitro cell culture, the nanofibrous silk-collagen scaffolds containing 7.4% collagen demonstrated significantly improved cell compatibility when compared with salt-leached silk scaffolds and silk-collagen scaffolds containing 20% collagen that emerged less nano-fibrils. Therefore, this new process provides useful scaffolds for tissue engineering applications. Furthermore, the process involves all-aqueous, room temperature and pressure processing without the use of toxic chemicals or solvents, offering new green chemistry approaches, as well as options to load bioactive drugs or growth factors into process.
• Chitosan/Poly(-caprolactone) blend scaffolds for cartilage repair
  - Biomater 32(4):1068-1079 (2011)
  Chitosan (CHT)/poly(-caprolactone) (PCL) blend 3D fiber-mesh scaffolds were studied as possible support structures for articular cartilage tissue (ACT) repair. Micro-fibers were obtained by wet-spinning of three different polymeric solutions: 100:0 (100CHT), 75:25 (75CHT) and 50:50 (50CHT) wt.% CHT/PCL, using a common solvent solution of 100 vol.% of formic acid. Scanning electron microscopy (SEM) analysis showed a homogeneous surface distribution of PCL. PCL was well dispersed throughout the CHT phase as analyzed by differential scanning calorimetry and Fourier transform infrared spectroscopy. The fibers were folded into cylindrical moulds and underwent a thermal treatment to obtain the scaffolds. μCT analysis revealed an adequate porosity, pore size and interconnectivity for tissue engineering applications. The PCL component led to a higher fiber surface roughness, decreased the scaffolds swelling ratio and increased their compressive mechanical properties. Biologic! al assays were performed after culturing bovine articular chondrocytes up to 21 days. SEM analysis, live-dead and metabolic activity assays showed that cells attached, proliferated, and were metabolically active over all scaffolds formulations. Cartilaginous extracellular matrix (ECM) formation was observed in all formulations. The 75CHT scaffolds supported the most neo-cartilage formation, as demonstrated by an increase in glycosaminoglycan production. In contrast to 100CHT scaffolds, ECM was homogenously deposited on the 75CHT and 50CHT scaffolds. Although mechanical properties of the 50CHT scaffold were better, the 75CHT scaffold facilitated better neo-cartilage formation.
• Irreversible optical clearing of sclera by dehydration and cross-linking
  - Biomater 32(4):1080-1090 (2011)
  This study manipulates both clear cornea and opaque sclera by two dehydration processes for revealing the relationship between altered tissue structures and change in optical functions. In contrast to the high levels of light scattering in dehydrated tissues by critical point dry, a simple dehydration at 4–8 °C effectively and significantly improved their visible-light transmission, even in the sclera, with accompanying dense fiber packing. Further improvement in visible-light transmission, from 40–50% to 80–90%, has been achieved by flatting tissue surface with cover glasses during dehydration at low temperature. Such optical clearing of sclera by dehydration is reversible. However, chemical cross-linking effectively stabilizes their densely packed microscopic structures and visible-light transmission at over 50% irreversibly, even at wet conditions. Interestingly, the repetition of both low temperature dehydration/cross-linking treatments effectively reduced t! he required amounts of cross-linking reagents to keep a high transparency. Wet transparent cross-linked sclera can also show a characteristic strong tensile strength. Furthermore, rabbit corneal epithelium has regenerated on the transparent sclera with cross-linking in vitro.
• Regeneration of the cavernous nerve by Sonic hedgehog using aligned peptide amphiphile nanofibers
  - Biomater 32(4):1091-1101 (2011)
  SHH plays a significant role in peripheral nerve regeneration and has clinical potential to be used as a regenerative therapy for the CN in prostatectomy patients and in other patients with neuropathy of peripheral nerves. Efforts to regenerate the cavernous nerve (CN), which provides innervation to the penis, have been minimally successful, with little translation into improved clinical outcomes. We propose that, Sonic hedgehog (SHH), is critical to maintain CN integrity, and that SHH delivered to the CN by novel peptide amphiphile (PA) nanofibers, will promote CN regeneration, restore physiological function, and prevent penile morphology changes that result in erectile dysfunction (ED). We performed localization studies, inhibition of SHH signaling in the CN, and treatment of crushed CNs with SHH protein via linear PA gels, which are an innovative extended release method of delivery. Morphological, functional and molecular analysis revealed that SHH protein is essential to maintain CN architecture, and that SHH treatment promoted CN regeneration, suppressed penile apoptosis and caused a 58% improvement in erectile function in less than half the time reported in the literature. These studies ! show that SHH has substantial clinical application to regenerate the CN in prostatectomy and diabetic patients, that this methodology has broad application to regenerate any peripheral nerve that SHH is necessary for maintenance of its structure, and that this nanotechnology method of protein delivery may have wide spread application as an in vivo delivery tool in many organs.
• Human embryonic stem cell-derived microvascular grafts for cardiac tissue preservation after myocardial infarction
  - Biomater 32(4):1102-1109 (2011)
  We present use of a synthetic, injectable matrix metalloproteinase (MMP)- responsive, bioactive hydrogel as an in situ forming scaffold to deliver thymosin β4 (Tβ4), a pro-angiogenic and pro-survival factor, along with vascular cells derived from human embryonic stem cells (hESC) in ischemic injuries to the heart in a rat model. The gel was found to substitute the degrading extracellular matrix in the infarcted myocardium of rats and to promote structural organization of native endothelial cells, while some of the delivered hESC-derived vascular cells formed de novo capillaries in the infarct zone. Magnetic resonance imaging (MRI) revealed that the microvascular grafts effectively preserved contractile performance 3 d and 6 wk after myocardial infarction, attenuated left ventricular dilation, and decreased infarct size as compared to infarcted rats treated with PBS injection as a control (3 d ejection fraction, + 7%, P < 0.001; 6 wk ejection faction, + 12%, P < 0.001! ). Elevation in vessel density was observed in response to treatment, which may be due in part to elevations in human (donor)-derived cytokines EGF, VEGF and HGF (1 d). Thus, a clinically relevant matrix for dual delivery of vascular cells and drugs may be useful in engineering sustained tissue preservation and potentially regenerating ischemic cardiac tissue.
• Drug delivery with upconversion nanoparticles for multi-functional targeted cancer cell imaging and therapy
  - Biomater 32(4):1110-1120 (2011)
  Upconversion nanoparticles (UCNPs) with unique multi-photon excitation photoluminescence properties have recently been intensively explored as novel contrast agents for low-backgroundbiomedical imaging. In this work, we functionalize UCNPs with a polyethylene glycol (PEG) grafted amphiphilic polymer. The PEGylated UCNPs are loaded with a commonly used chemotherapy molecule, doxorubicin (DOX), by simple physical adsorption via a supramolecular chemistry approach for intracellular drug delivery. The loading and releasing of DOX from UCNPs are controlled by varying pH, with an increased drug dissociation rate in acidic environment, favorable for controlled drug release. Upconversion luminescence (UCL) imaging by a modified laser scanning confocal microscope reveals the time course of intracellular delivery of DOX by UCNPs. It is found that DOX is shuttled into cells by the UCNP nano carrier and released inside cells after endocytosis. By conjugating nanoparticles with fol! ic acid, which targets folate receptors over expressed on various types of cancer cells, we further demonstrate targeted drug delivery and UCL cell imaging with UCNPs. Besides DOX, this non-covalent drug loading strategy can also be used for loading of photosensitizer molecules on UCNPs for potential near-infrared light induced photodynamic therapy. Our results suggest the promise of UCNPs as interesting nano carriers for multi-functional cancer therapy and imaging.
• In vitro comparison of the photothermal anticancer activity of graphene nanoparticles and carbon nanotubes
  - Biomater 32(4):1121-1129 (2011)
  The present study compared the photothermal anticancer activity of near-infrared (NIR)-excited graphene nanoparticles and carbon nanotubes (CNT). Despite lower NIR-absorbing capacity, suspension of polyvinylpyrrolidone-coated graphene sheets exposed to NIR radiation (808 nm, 2 W/cm2) generated more heat than DNA or sodium dodecylbenzenesulfonate-solubilized single-wall CNT under the same conditions. Accordingly, graphene nanoparticles performed significantly better than CNT in inducing photothermal death of U251 human glioma cells in vitro. The superior photothermal sensitivity of graphene sheets could be largely explained by their better dispersivity, which has been supported by a simple calculation taking into account thermodynamic, optical and geometrical properties of the two type of carbon nanoparticles. The mechanisms of graphene-mediated photothermal killing of cancer cells apparently involved oxidative stress and mitochondrial membrane depolarization resulting ! in mixed apoptotic and necrotic cell death characterized by caspase activation/DNA fragmentation and cell membrane damage, respectively.
• A multimodal nanoparticle-based cancer imaging probe simultaneously targeting nucleolin, integrin αvβ3 and tenascin-C proteins
  - Biomater 32(4):1130-1138 (2011)
  Molecular imaging of cancers has been characterized based on the sensitivity and selectivity of a single cancer probe targeting a cancer biomarker of a specific cancer cell line. Here, we designed a multimodal nanoparticle-based Simultaneously Multiple Aptamers and RGD Targeting (SMART) cancer probe targeting multiple cancer biomarkers to enhance the specificity and signal sensitivity for various cancers. Transmission electron microscopy revealed that the multimodal SMART cancer probe was spheric and well dispersed. Fluorescence, radioisotope, and magnetic resonance analysis demonstrated that the SMART cancer probe simultaneously targeting the nucleolin, integrin αvβ3 and Tnc proteins had dramatically enhanced specificity and signal intensity when used to target cancers from C6, NPA, DU145, HeLa and A549 cells when compared with single cancer probes conjugated with AS1411, RGD or TTA1 targeting a single cancer biomarker. The results demonstrated that the SMART cancer! probe will be useful for the diagnosis of different cancers as a cancer master probe.
• Au@Pt nanostructures as oxidase and peroxidase mimetics for use in immunoassays
  - Biomater 32(4):1139-1147 (2011)
  In this paper, we demonstrated that Au nanorods coated with a shell composed of Pt nanodots (Au@Pt nanostructures) exhibited intrinsic oxidase-like, peroxidase-like and catalase-like activity, catalyzing oxygen and hydrogen peroxide reduction and the dismutation decomposition of hydrogen peroxide to produce oxygen. Based on these findings, we established an Au@Pt nanostructures based enzyme linked immunosorbent assay (ELISA) for the detection of mouse interleukin 2 (IL-2). In comparison with natural enzymes, Au@Pt nanostructures have advantages of low cost, easy preparation, better stability, and tunable catalytic activity (compared with HRP), which make them a promising enzyme mimetic candidate and may find potential applications in biocatalysis, bioassays, and nano-biomedicine such as reactive oxygen species (ROS)-related fields (anti-aging and therapeutics for neurodegenerative diseases and cancers).
• Fluorine-18-labeled Gd3+/Yb3+/Er3+ co-doped NaYF4 nanophosphors for multimodality PET/MR/UCL imaging
  - Biomater 32(4):1148-1156 (2011)
  Molecular imaging modalities provide a wealth of information that is highly complementary and rarely redundant. To combine the advantages of molecular imaging techniques, 18F-labeled Gd3+/Yb3+/Er3+ co-doped NaYF4 nanophosphors (NPs) simultaneously possessing with radioactivity, magnetic, and upconversion luminescent properties have been fabricated for multimodality positron emission tomography (PET), magnetic resonance imaging (MRI), and laser scanning upconversion luminescence (UCL) imaging. Hydrophilic citrate-capped NaY0.2Gd0.6Yb0.18Er0.02F4 nanophosphors (cit-NPs) were obtained from hydrophobic oleic acid (OA)-coated nanoparticles (OA-NPs) through a process of ligand exchange of OA with citrate, and were found to be monodisperse with an average size of 22 × 19 nm. The obtained hexagonal cit-NPs show intense UCL emission in the visible region and paramagnetic longitudinal relaxivity (r1 = 0.405 s−1·(mM)−1). Through a facile inorganic reaction based on the stro! ng binding between Y3+ and F−, 18F-labeled NPs have been fabricated in high yield. The use of cit-NPs as a multimodal probe has been further explored for T1-weighted MR and PET imaging in vivo and UCL imaging of living cells and tissue slides. The results indicate that 18F-labeled NaY0.2Gd0.6Yb0.18Er0.02 is a potential candidate as a multimodal nanoprobe for ultra-sensitive molecular imaging from the cellular scale to whole-body evaluation.
• The use of hydrogel microparticles to sequester and concentrate bacterial antigens in a urine test for Lyme disease
  - Biomater 32(4):1157-1166 (2011)
  Hydrogel biomarker capturing microparticles were evaluated as a biomaterial to amplify the sensitivity of urine testing for infectious disease proteins. Lyme disease is a bacterial infection transmitted by ticks. Early diagnosis and prompt treatment of Lyme disease reduces complications including arthritis and cardiac involvement. While a urine test is highly desirable for Lyme disease screening, this has been difficult to accomplish because the antigen is present at extremely low concentrations, below the detection limit of clinical immunoassays. N-isopropylacrylamide (NIPAm) – acrylic acid (AAc) microparticles were covalently functionalized with amine containing dyes via amidation of carboxylic groups present in the microparticles. The dyes act as affinity baits towards protein analytes in solution. NIPAm/AAc microparticles functionalized with acid black 48 (AB48) mixed with human urine, achieved close to one hundred percent capture and 100 percent extraction yield! of the target antigen. In urine, microparticles sequestered and concentrated Lyme disease antigens 100 fold, compared to the absence of microparticles, achieving an immunoassay detection sensitivity of 700 pg/mL in 10 mL urine. Antigen present in a single infected tick could be readily detected following microparticle sequestration. Hydrogel microparticles functionalized with high affinity baits can dramatically increase the sensitivity of urinary antigen tests for infectious diseases such as Lyme disease. These findings justify controlled clinical studies evaluating the sensitivity and precision of Lyme antigen testing in urine.
• The cell labeling efficacy, cytotoxicity and relaxivity of copper-activated MRI/PET imaging contrast agents
  - Biomater 32(4):1167-1176 (2011)
  A new class of nanoparticle-based dual-modality positron emission tomography/magnetic resonance imaging (PET/MRI) contrast agents has been developed. The probe consists of a superparamagnetic iron oxide (SPIO) or manganese oxide core coated with 3,4-dihydroxy-d,l-phenylalanine (dl-DOPA). The chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to DOPA termini. The DOTA modified nanoparticles allow chelation of copper for PET imaging. These surface functionalized nanoparticle-based probes have been characterized by various analytical techniques. The cell-labeling efficacy, cytotoxicity and relaxivity of these nanoparticles have been evaluated and compared with the same properties of one of the most commonly utilized MRI contrast agents, Feridex®. Evidently, this new nanoparticle has a great potential for use in cell tracking with MRI and PET in the absence of transfecting agent. It is noteworthy that there is a sharp increase in r2 re! laxivity of these nanoparticles on coordination with Cu2+ ions. Thus these iron oxide nanoparticles can also be explored as the smart magnetic resonance (MR) sensor for the detection of micromolar changes in copper concentration for neurodegenerative diseases such as Alzheimer's disease, Menkes and Wilson's diseases, amyotrophic lateral sclerosis and prion diseases.
• A multicomponent recognition and separation system established via fluorescent, magnetic, dualencoded multifunctional bioprobes
  - Biomater 32(4):1177-1184 (2011)
  Accurate and rapid recognition and separation of multiple types of biological targets such as molecules, cells, bacteria or viruses from complex sample mixtures is of great importance for a wide range of diagnostic and therapeutic strategies. To achieve this goal, a set of fluorescent, magnetic, dual-encoded multifunctional bioprobes has been constructed by co-embedding different-sized quantum dots and varying amounts of γ-Fe2O3 magnetic nanoparticles into swollen poly(styrene/acrylamide) copolymer nanospheres. The dual-encoded bioprobes, which possessed different photoluminescent property and magnetic susceptibility, were proven to be capable of simultaneously recognizing and separating multiple components from a complex sample when three kinds of lectins were used as the targets. The lectins were separated with high efficiency and kept their bioactivity during the process. Compared to the conventional batchwise separation, this method does not require a large number! of sequential reaction steps, which is economical of time and can be very reagent-saving. By combining the multiplexing capability of quantum dots with the superparamagnetic properties of iron oxide nanoparticles, this dual-encoded technique is expected to open new opportunities in high-throughput and multiplex bioassays, such as cell sorting, proteomical and genomical applications, drug screening etc.
• Fluorescence-enhanced gadolinium-doped zinc oxide quantum dots for magnetic resonance and fluorescence imaging
  - Biomater 32(4):1185-1192 (2011)
  We report here the development of Gd-doped ZnO quantum dots (QDs) as dual modal fluorescence and magnetic resonance imaging nanoprobes. They are fabricated in a simple, versatile and environmentally friendly method, not only decreasing the difficulty and complexity, but also avoiding the increase of particle's size brought about by silica coating procedure in the synthesis of nanoprobes reported previously. These nanoprobes, with exceptionally small size and enhanced fluorescence resulting from the Gd doping, can label successfully the HeLa cells in short time and present no evidence of toxicity or adverse affect on cell growth even at the concentration up to 1 mm. These results show that such nanoprobes have low toxicity, especially in comparison with the traditional PEGylated CdSe/ZnS or CdSe/CdS QDs. In MRI studies, they exert strong positive contrast effect with a large longitudinal relaxivity (r1) of water proton of 16 mm−1 s−1. Their capability of imaging H! eLa cells with MRI implies that they have great potential as MRI contrast agents. Combining the high sensitivity of fluorescence imaging with high spatial resolution of MRI, We expect that the as-prepared Gd-doped Zno QDs can provide a better reliability of the collected data and find promising applications in biological, medical and other fields.
• A reducible polycationic gene vector derived from thiolated low molecular weight branched polyethyleneimine linked by 2-iminothiolane
  - Biomater 32(4):1193-1203 (2011)
  To improve transfection efficiency and reduce the cytotoxicity of polymeric gene vectors, reducible polycations (RPC) were synthesized from low molecular weight (MW) branched polyethyleneimine (bPEI) via thiolation and oxidation. RPC (RPC-bPEI0.8 kDa) possessed MW of 5 kDa–80 kDa, and 50%–70% of the original proton buffering capacity of bPEI0.8 kDa was preserved in the final product. The cytotoxicity of RPC-bPEI0.8 kDa was 8–19 times less than that of the gold standard of polymeric transfection reagents, bPEI25 kDa. Although bPEI0.8 kDa exhibited poor gene condensing capacities (2 μm at a weight ratio (WR) of 40), RPC-bPEI0.8 kDa effectively condensed plasmid DNA (pDNA) at a WR of 2. Moreover, RPC-bPEI0.8 kDa/pDNA (WR ≥2) formed 100–200 nm-sized particles with positively charged surfaces (20–35 mV). In addition, the results of the present study indicated that thiol/polyanions triggered the release of pDNA from RPC-bPEI0.8 kDa/pDNA via the fragmentation of ! RPC-bPEI0.8 kDa and ion-exchange. With negligible polyplex-mediated cytotoxicity, the transfection efficiencies of RPC-bPEI0.8 kDa/pDNA were approximately 1200–1500-fold greater than that of bPEI0.8 kDa/pDNA and were equivalent or superior (7-fold) to that of bPEI25 kDa/pDNA. Interestingly, the distribution of high MW RPC-bPEI0.8 kDa/pDNA in the nucleus of the cell was higher than that of low MW RPC-bPEI0.8 kDa/pDNA. Thus, the results of the present study suggest that RPC-bPEI0.8 kDa has the potential to effectively deliver genetic materials with lower levels of toxicity.
• Doxycycline hydrogels with reversible disulfide crosslinks for dermal wound healing of mustard injuries
  - Biomater 32(4):1204-1217 (2011)
  Doxycycline hydrogels containing reversible disulfide crosslinks were investigated for a dermal wound healing application. Nitrogen mustard (NM) was used as a surrogate to mimic the vesicant effects of the chemical warfare agent sulfur mustard. An 8-arm-poly(ethylene glycol) (PEG) polymer containing multiple thiol (-SH) groups was crosslinked using hydrogen peroxide (H2O2 hydrogel) or 8-arm-S-thiopyridyl (S-TP hydrogel) to form a hydrogel in situ. Formulation additives (glycerin, PVP and PEG 600) were found to promote dermal hydrogel retention for up to 24 h. Hydrogels demonstrated high mechanical strength and a low degree of swelling (< 1.5%). Doxycycline release from the hydrogels was biphasic and sustained for up to 10-days in vitro. Doxycycline (8.5 mg/cm3) permeability through NM-exposed skin was elevated as compared to non vesicant-treated controls at 24, 72 and 168 h post-exposure with peak permeability at 72 h. The decrease in doxycycline permeability at 168 h ! correlates to epidermal re-epithelialization and wound healing. Histology studies of skin showed that doxycycline loaded (0.25% w/v) hydrogels provided improved wound healing response on NM-exposed skin as compared to untreated skin and skin treated with placebo hydrogels in an SKH-1 mouse model. In conclusion, PEG-based doxycycline hydrogels are promising for dermal wound healing application of mustard injuries.
• Functional TNFα gene silencing mediated by polyethyleneimine/TNFα siRNA nanocomplexes in inflamed colon
  - Biomater 32(4):1218-1228 (2011)
  During inflammatory bowel disease, TNFα is the major pro-inflammatory cytokine mainly secreted from macrophages and dendritic cells. Here, we have demonstrated that TNFα siRNA/polyethyleneimine loaded into polylactide at an optimal concentration of 20 g/L nanoparticles covered with polyvinyl alcohol are efficiently taken up by inflamed macrophages and inhibit TNFα secretion by the macrophages. Those nanoparticles have a diameter of 380 nm and zeta potential of −8 mV at pH 7.2, and are non-cytotoxic. Complexation, interactions and protection from RNAse between TNFα siRNA and polyethyleneimine were higher than those using chitosan. Importantly, complexation between TNFα siRNA and polyethyleneimine facilitated higher rates of siRNA loading into nanoparticles, compared to Chi or free siRNA mixed with Lipofectamine. Oral administration of encapsulated TNFα siRNA-loaded nanoparticles specifically reduced the TNFα expression/secretion in colonic tissue in LPS-treated! mice. In conclusion, we have shown: (1) that proposed TNFα siRNA-loaded NPs are prepared via a non-denaturing synthetic process; (2) a high encapsulation rate of TNFα siRNA complexed to polyethyleneimine into NPs; (3) effective enzymatic protection of TNFα siRNA by polyethyleneimine; (4) non-cytotoxicity and biodegradability of nanoparticles loaded with polyethyleneimine/TNFα siRNA; and (5) in vitro and in vivo significant anti-inflammatory effects at low TNFα siRNA dose that is specific and restricted to the colonic cells. Our results collectively indicate that polyethyleneimine/TNFα siRNA nanocomplexes represent an efficient therapeutic option for diseases such as IBD.
• A robust high-throughput sandwich cell-based drug screening platform
  - Biomater 32(4):1229-1241 (2011)
  Hepatotoxicity evaluation of pharmaceutical lead compounds in early stages of drug development has drawn increasing attention. Sandwiched hepatocytes exhibiting stable functions in culture represent a standard model for hepatotoxicity testing of drugs. We have developed a robust and high-throughput hepatotoxicity testing platform based on the sandwiched hepatocytes for drug screening. The platform involves a galactosylated microfabricated membrane sandwich to support cellular function through uniform and efficient mass transfer while protecting cells from excessive shear. Perfusion bioreactor further enhances mass transfer and cellular functions over long period; and hepatoctyes are readily transferred to 96-well plate for high-throughput robotic liquid handling. The bioreactor design and perfusion flow rate are optimized by computational fluid dynamics simulation and experimentally. The cultured hepatocytes preserved 3D cell morphology, urea production and cytochrome ! p450 activity of the mature hepatocytes for 14 days. When the perfusion-cultured sandwich is transferred to 96-well plate for drug testing, the hepatocytes exhibited improved drug sensitivity and low variability in hepatotoxicity responses amongst cells transferred from different dates of perfusion culture. The platform enables robust high-throughput screening of drug candidates.
• Plasmid pORF-hTRAIL and doxorubicin co-delivery targeting to tumor using peptide-conjugated polyamidoamine dendrimer
  - Biomater 32(4):1242-1252 (2011)
  A combination cancer therapy was investigated via co-delivery of therapeutic gene encoding human tumor necrosis factor-related apoptosis-inducing ligand (pORF-hTRAIL) and doxorubicin (DOX) using a tumor-targeting carrier, peptide HAIYPRH (T7)-conjugated polyethylene glycol-modified polyamidoamine dendrimer (PAMAM-PEG-T7). T7, a transferrin receptor-specific peptide, was chosen as the ligand to target the co-delivery system to the tumor cells expressing transferrin receptors. The result of fluorescence scanning showed that about 375 DOX molecules were bound to one pORF-hTRAIL molecule. The co-delivery system was constructed based on the electrostatic interactions between pORF-hTRAIL–DOX complex and cationic PAMAM-PEG-T7. T7-modified co-delivery system showed higher efficiency in cellular uptake and gene expression than unmodified co-delivery system in human liver cancer Bel-7402 cells, and accumulated in tumor more efficiently in vivo. In comparison with single DOX or! pORF-hTRAIL delivery system, co-delivery system induced apoptosis of tumor cells in vitro and inhibited tumor growth in vivo more efficiently. In mice bearing Bel-7402 xenografts, lower doses of co-delivery system (4 μg DOX/mouse, about 0.16 mg/kg) effectively inhibited tumor growth comparable to high doses (5 mg/kg) of free doxorubicin (77% versus 69%). These results suggested that T7-mediated co-delivery system of DOX and pORF-hTRAIL was a simply prepared, combined delivery platform which can significantly improve the anti-tumor effect. This co-delivery system might widen the therapeutic window and allow for the selective destruction of cancer cells.