Hot off the presses! Jan 01 Biomaterials

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Latest Articles Include:

• Editorial board
  - Biomaterials 33(1):IFC (2012)
  
• Porcine vena cava as an alternative to bovine pericardium in bioprosthetic percutaneous heart valves
  - Biomaterials 33(1):1-8 (2012)
  Percutaneous heart valves are revolutionizing valve replacement surgery by offering a less invasive treatment option for high-risk patient populations who have previously been denied the traditional open chest procedure. Percutaneous valves need to be crimped to accommodate a small-diameter catheter during deployment, and they must then open to the size of heart valve. Thus the material used must be strong and possess elastic recoil for this application. Most percutaneous valves utilize bovine pericardium as a material of choice. One possible method to reduce the device delivery diameter is to utilize a thin, highly elastic tissue. Here we investigated porcine vena cava as an alternative to bovine pericardium for percutaneous valve application. We compared the structural, mechanical, and in vivo properties of porcine vena cava to those of bovine pericardium. While the extracellular matrix fibers of pericardium are randomly oriented, the vena cava contains highly aligne! d collagen and elastin fibers that impart strength to the vessel in the circumferential direction and elasticity in the longitudinal direction. Moreover, the vena cava contains a greater proportion of elastin, whereas the pericardium matrix is mainly composed of collagen. Due to its high elastin content, the vena cava is significantly less stiff than the pericardium, even after crosslinking with glutaraldehyde. Furthermore, the vena cava's mechanical compliance is preserved after compression under forces similar to those exerted by a stent, whereas pericardium is significantly stiffened by this process. Bovine pericardium also showed surface cracks observed by scanning electron microscopy after crimping that were not seen in vena cava tissue. Additionally, the vena cava exhibited reduced calcification (46.64 ± 8.15 亮g Ca/mg tissue) as compared to the pericardium (86.79 ± 10.34 亮g/mg). These results suggest that the vena cava may provide enhanced leaflet flexibility,! tissue resilience, and tissue integrity in percutaneous heart! valves, ultimately reducing the device profile while improving the durability of these valves.
• Contact activation of blood plasma and factor XII by ion-exchange resins
  - Biomaterials 33(1):9-19 (2012)
  Sepharose ion-exchange particles bearing strong Lewis acid/base functional groups (sulfopropyl, carboxymethyl, quaternary ammonium, dimethyl aminoethyl, and iminodiacetic acid) exhibiting high plasma protein adsorbent capacities are shown to be more efficient activators of blood factor XII in neat-buffer solution than either hydrophilic clean-glass particles or hydrophobic octyl sepharose particles (; a.k.a autoactivation, where FXII is the zymogen and FXIIa is a procoagulant protease). In sharp contrast to the clean-glass standard of comparison, ion-exchange activators are shown to be inefficient activators of blood plasma coagulation. These contrasting activation properties are proposed to be due to the moderating effect of plasma-protein adsorption on plasma coagulation. Efficient adsorption of blood-plasma proteins unrelated to the coagulation cascade impedes FXII contacts with ion-exchange particles immersed in plasma, reducing autoactivation, and causing sluggish! plasma coagulation. By contrast, plasma proteins do not adsorb to hydrophilic clean glass and efficient autoactivation leads directly to efficient activation of plasma coagulation. It is also shown that competitive-protein adsorption can displace FXIIa adsorbed to the surface of ion-exchange resins. As a consequence of highly-efficient autoactivation and FXIIa displacement by plasma proteins, ion-exchange particles are slightly more efficient activators of plasma coagulation than hydrophobic octyl sepharose particles that do not bear strong Lewis acid/base surface functionalities but to which plasma proteins adsorb efficiently. Plasma proteins thus play a dual role in moderating contact activation of the plasma coagulation cascade. The principal role is impeding FXII contact with activating surfaces, but this same effect can displace FXIIa from an activating surface into solution where the protease can potentiate subsequent steps of the plasma coagulation cascade.
• Topographic control of the growth and function of cardiomyoblast H9c2 cells using nanodot arrays
  - Biomaterials 33(1):20-28 (2012)
  Cardiovascular stents require optimised control for the enhancement or inhibition epithelial and smooth muscle cell growth in close contact with the implant. Here we propose that the surface topology in contact with the living cells could be designed to control and optimise the growth and function of such cells. The cardiomyoblast H9c2 was cultured on nanodot arrays with dot diameters ranging between 10 and 200 nm. On the 50-nm nanodot arrays H9c2 showed maximum attachment and proliferation with largest cell area and extended lamellipodia. In contrast, 53.7% and 72.6% reductions of growth were observed on the 100- and 200-nm nanodot arrays after 3 days. Immunostaining indicated that nanodots smaller than 50-nm induced cell adhesion and cytoskeleton organization. Expression of genes associated with fibrosis and hypertrophy was up-regulated in cells grown on 100-nm nanodots. Western blot data showed high levels of expression for vinculin and plasminogen activator inhibit! or-1 for cells cultured on 50-nm nanodots. Nanotopography controls cell adhesion, morphology and proliferation. By adjusting the diameter of the nanodots, we could modulate the growth and expression of function-related genes and proteins of H9c2 cardiomyoblasts. The current study provides insights for improved design of artificial implants and parameters that affect biocompatibility.
• Active leukocyte detachment and apoptosis/necrosis on PEG hydrogels and the implication in the host inflammatory response
  - Biomaterials 33(1):29-37 (2012)
  Monocytes/Macrophages have long been recognized as key players in inflammation and wound healing and are often employed in vitro to gain an understanding of the inflammatory response to biomaterials. Previous work has demonstrated a drastic decrease in primary monocyte adherent density on biomaterial surfaces coupled with a change in monocyte behavior over time. However, the mechanism responsible for this decrease remains unclear. In this study, we explored active detachment and cellular death as possible regulating factors. Specifically, extracellular TNF-α and ROS production were analyzed as potential endogenous stimulators of cell death. MMPs, but not calpains, were found to play a key role in active monocyte detachment. Monocyte death was found to peak at 24 h and occur by both apoptosis and necrosis as opposed to polymorphonuclear leukocyte death which mainly occurred through apoptosis. Finally, TNF-α and ROS production were not found to have a causal relati! onship with monocyte death on TCPS or PEG surfaces. The occurrence of primary monocyte apoptosis/necrosis as well as active detachment from a material surface has implications not only in in vitro study, but also in the translation of the in vitro inflammatory response of these cells to in vivo applications.
• Long term performance of polycaprolactone vascular grafts in a rat abdominal aorta replacement model
  - Biomaterials 33(1):38-47 (2012)
  In the active field of vascular graft research, polycaprolactone is often used because of its good mechanical strength and its biocompatibility. It is easily processed into micro and nano-fibers by electrospinning to form a porous, cell-friendly scaffold. However, long term in vivo performance of polycaprolactone vascular grafts had yet to be investigated. In this study, polycaprolactone micro and nano-fiber based vascular grafts were evaluated in the rat abdominal aorta replacement model for 1.5, 3, 6, 12, and 18 months (n = 3 for each time point). The grafts were evaluated for patency, thrombosis, compliance, tissue regeneration, and material degradation. Results show excellent structural integrity throughout the study, with no aneurysmal dilation, and perfect patency with no thrombosis and limited intimal hyperplasia. Endothelialization, cell invasion, and neovascularization of the graft wall rapidly increased until 6 months, but at 12 and 18 months, a cellular regr! ession is observed. On the medium term, chondroid metaplasia takes place in the intimal hyperplasia layers, which contributes to calcification of the grafts. This study presents issues with degradable vascular grafts that cannot be identified with short implantation times or in vitro studies. Such findings should allow for better design of next generation vascular grafts.
• 3D cell entrapment in crosslinked thiolated gelatin-poly(ethylene glycol) diacrylate hydrogels
  - Biomaterials 33(1):48-58 (2012)
  The combined use of natural ECM components and synthetic materials offers an attractive alternative to fabricate hydrogel-based tissue engineering scaffolds to study cell-matrix interactions in three-dimensions (3D). A facile method was developed to modify gelatin with cysteine via a bifunctional PEG linker, thus introducing free thiol groups to gelatin chains. A covalently crosslinked gelatin hydrogel was fabricated using thiolated gelatin and poly(ethylene glycol) diacrylate (PEGdA) via thiol-ene reaction. Unmodified gelatin was physically incorporated in a PEGdA-only matrix for comparison. We sought to understand the effect of crosslinking modality on hydrogel physicochemical properties and the impact on 3D cell entrapment. Compared to physically incorporated gelatin hydrogels, covalently crosslinked gelatin hydrogels displayed higher maximum weight swelling ratio (Qmax), higher water content, significantly lower cumulative gelatin dissolution up to 7 days, and lowe! r gel stiffness. Furthermore, fibroblasts encapsulated within covalently crosslinked gelatin hydrogels showed extensive cytoplasmic spreading and the formation of cellular networks over 28 days. In contrast, fibroblasts encapsulated in the physically incorporated gelatin hydrogels remained spheroidal. Hence, crosslinking ECM protein with synthetic matrix creates a stable scaffold with tunable mechanical properties and with long-term cell anchorage points, thus supporting cell attachment and growth in the 3D environment.
• Regenerative potential of silk conduits in repair of peripheral nerve injury in adult rats
  - Biomaterials 33(1):59-71 (2012)
  Various attempts have been made to develop artificial conduits for nerve repair, but with limited success. We describe here conduits made from Bombyx mori regenerated silk protein, and containing luminal fibres of Spidrex®, a silk-based biomaterial with properties similar to those of spider silk. Assessment in vitro demonstrated that Spidrex® fibres support neurite outgrowth. For evaluation in vivo, silk conduits 10 mm in length and containing 0, 100, 200 or 300 luminal Spidrex® fibres, were implanted to bridge an 8 mm gap in the rat sciatic nerve. At 4 weeks, conduits containing 200 luminal Spidrex® fibres (PN200) supported 62% and 59% as much axon growth as autologous nerve graft controls at mid-conduit and distal nerve respectively. Furthermore, Spidrex® conduits displayed similar Schwann cell support and macrophage response to controls. At 12 weeks, animals implanted with PN200 conduits showed similar numbers of myelinated axons (81%) to controls, similar gast! rocnemius muscle innervation, and similar hindpaw stance assessed by Catwalk footprint analysis. Plantar skin innervation was 73% of that of controls. PN200 Spidrex® conduits were also effective at bridging longer (11 and 13 mm) gaps. Our results show that Spidrex® conduits promote excellent axonal regeneration and function recovery, and may have potential for clinical application.
• A prospective, randomised, controlled trial using a Mg-hydroxyapatite - demineralized bone matrix nanocomposite in tibial osteotomy
  - Biomaterials 33(1):72-79 (2012)
  We in vivo investigated the bone healing ability of a nanocomposite (DBSint®), constituted by biomimetic nano-structured Mg-hydroxyapatite (SINTlife®) and human demineralized bone matrix. Thirty-one subjects undergoing high tibial osteotomy for genu varus were randomly assigned to three groups: during surgery, DBSint® was inserted into nine patients, SINTlife® in thirteen patients and lyophilised bone chips, that is the routine surgery, in nine subjects. As outcome measures, clinical, radiographic and histomorphometry scores were calculated. The osseointegration was evaluated by imaging six weeks, three, six and twelve months after surgery. At six-week follow-up, DBSint® showed a significantly higher osseointegration rate in comparison with lyophilised bone chips (p = 0.008). At the same follow-up, CT-guided bone biopsies were obtained and analysed by histomorphometry: a good osteogenetic potential was demonstrated with DBSint®, as well as with SINTlife® and con! trols. Unresorbed material was evident with DBSint® and SINTlife®, with a significantly higher percentage in SINTlife® group. At 1-year follow-up, DBSint® was demonstrated as effective and safe as SINTlife® and lyophilized bone chips. More significant results could be obtained by continuing the clinical trial, by increasing the patient number and the study power. Eventually, the role of non-resorbed graft remnants is still unclear and requires further investigation.
• Enhancement of mesenchymal stem cell angiogenic capacity and stemness by a biomimetic hydrogel scaffold
  - Biomaterials 33(1):80-90 (2012)
  In this study, we examined the capacity of a biomimetic pullulan–collagen hydrogel to create a functional biomaterial-based stem cell niche for the delivery of mesenchymal stem cells (MSCs) into wounds. Murine bone marrow-derived MSCs were seeded into hydrogels and compared to MSCs grown in standard culture conditions. Hydrogels induced MSC secretion of angiogenic cytokines and expression of transcription factors associated with maintenance of pluripotency and self-renewal (Oct4, Sox2, Klf4) when compared to MSCs grown in standard conditions. An excisonal wound healing model was used to compare the ability of MSC-hydrogel constructs versus MSC injection alone to accelerate wound healing. Injection of MSCs did not significantly improve time to wound closure. In contrast, wounds treated with MSC-seeded hydrogels showed significantly accelerated healing and a return of skin appendages. Bioluminescence imaging and FACS analysis of luciferase+/GFP+ MSCs indicated that ste! m cells delivered within the hydrogel remained viable longer and demonstrated enhanced engraftment efficiency than those delivered via injection. Engrafted MSCs were found to differentiate into fibroblasts, pericytes and endothelial cells but did not contribute to the epidermis. Wounds treated with MSC-seeded hydrogels demonstrated significantly enhanced angiogenesis, which was associated with increased levels of VEGF and other angiogenic cytokines within the wounds. Our data suggest that biomimetic hydrogels provide a functional niche capable of augmenting MSC regenerative potential and enhancing wound healing.
• Damage associated molecular patterns within xenogeneic biologic scaffolds and their effects on host remodeling
  - Biomaterials 33(1):91-101 (2012)
  The immune response is an important determinant of the downstream remodeling of xenogeneic biologic scaffolds in vivo. Pro-inflammatory responses have been correlated with encapsulation and a foreign body reaction, while anti-inflammatory reactions are associated with constructive remodeling. However, the bioactive and bioinductive molecules within the extracellular matrix (ECM) that induce this polarization are unclear, although it is likely that cellular remnants such as damage associated molecular patterns (DAMPs) retained within the scaffold may play a role. The present study investigated the immunomodulatory effects of common ECM scaffolds. Results showed that tissue source, decellularization method and chemical crosslinking modifications affect the presence of the well characterized DAMP - HMGB1. In addition, these factors were correlated with differences in cell proliferation, death, secretion of the chemokines CCL2 and CCL4, and up regulation of the pro-inflamm! atory signaling receptor toll-like receptor 4 (TLR4). Inhibition of HMGB1 with glycyrrhizin increased the pro-inflammatory response, increasing cell death and up regulating chemokine and TLR4 mRNA expression. The present study suggests the importance of HMGB1 and other DAMPS as bioinductive molecules within the ECM scaffold. Identification and evaluation of other ECM bioactive molecules will be an area of future interest for new biomaterial development.
• Silk fibroin derived polypeptide-induced biomineralization of collagen
  - Biomaterials 33(1):102-108 (2012)
  Silk fibroin (SF) is extensively investigated in osteoregenerative therapy as it combines extraordinary mechanical properties and directs calcium-phosphate formation. However, the role of the peptidic fractions in inducing the protein mineralization has not been previously decoded. In this study, we investigated the mineralization of fibroin-derived polypeptides (FDPs), which were obtained through the chymotryptic separation of the hydrophobic crystalline (Cp) fractions and of the hydrophilic electronegative amorphous (Cs) fractions. When immersed in simulated body fluid (SBF), only Cs fragments demonstrated the formation of carbonated apatite, providing experimental evidence that the mineralization of SF is dictated exclusively by its electronegative amino-acidic sequences. The potential of Cs to conceptually mimic the role of anionic non-collagenous proteins in biomineralization processes was investigated via their incorporation (up to 10% by weight) in bulk osteoid-! like dense collagen (DC) gels. Within 6 h in SBF, apatite was formed in DC-Cs hybrid gels, and by day 7, carbonated hydroxylapatite crystals were extensively formed. This accelerated 3-D mineralization resulted in a nine-fold increase in the compressive modulus of the hydrogel. The tailoring of the mineralization and mechanical properties of hydrogels through hybridization with FDPs could potentially have a significant impact on cell delivery and bone regenerative medicine.
• Dermis isolated adult stem cells for cartilage tissue engineering
  - Biomaterials 33(1):109-119 (2012)
  Adult stem cells from the dermal layer of skin are an attractive alternative to primary cells for meniscus engineering, as they may be easily obtained and used autologously. Recently, chondroinducible dermis cells from caprine skin have shown promising characteristics for cartilage tissue engineering. In this study, their multilineage differentiation capacity is determined, and methods of expanding and tissue engineering these cells are investigated. It was found that these cells could differentiate along adipogenic, osteogenic, and chondrogenic lineages, allowing them to be termed dermis isolated adult stem cells (DIAS cells). Focusing on cartilage tissue engineering, it was found that passaging these cells in chondrogenic medium and forming them into self-assembled tissue engineered constructs caused upregulation of collagen type II and COMP gene expression. Further investigation showed that applying transforming growth factor 硫1 (TGF-硫1) or bone morphogenetic pr! otein 2 (BMP-2) to DIAS constructs caused increased sulfated glycosaminoglycan content. Additionally, TGF-硫1 treatment caused significant increases in compressive properties and construct contraction. In contrast, BMP-2 treatment resulted in the largest constructs, but did not increase compressive properties. These results show that DIAS cells can be easily manipulated for cartilage tissue engineering strategies, and may also be a useful cell source for other mesenchymal tissues.
• Cartilage regeneration in SCID mice using a highly organized three-dimensional alginate scaffold
  - Biomaterials 33(1):120-127 (2012)
  Tissue engineering for cartilage regeneration provides an alternative to surgery for degenerative osteoarthritis. Recently, a highly organized three-dimensional (3D) alginate scaffold was prepared using a microfluidic device; this scaffold is effective for chondrocyte culture in vitro. The performance of this scaffold was further demonstrated; an alginate scaffold seeded with porcine chondrocytes was implanted in the dorsal subcutaneous site of SCID mice. The recipients were sacrificed at 2, 4, and 6 weeks after transplantation. The grafted implants retrieved from the subcutaneous site were analyzed with histologic examinations. Real-time PCR was used to identify the gene expression patterns of the chondrocytes. The hematoxylin and eosin staining showed that the chondrocytes survived normally in SCID mice; cartilage-like structures were formed after 4 weeks implantation. Immunohistochemical staining revealed cells secreted type II collagen, produced glycosaminoglycans ! (proved by alcian blue stain), and maintained the expression of S-100. On the other hand, the cells were negative for type I and type X collagen staining. PCR showed that the mRNA expressions of aggrecan and type II collagen were up-regulated at weeks two and four, while type I and type X collagen were down-regulated during the study period. In summary, this highly organized 3D alginate scaffold provided a suitable environment and maintained functional phenotypes for chondrocytes in this animal study.
• Modulation of osteogenic differentiation in hMSCs cells by submicron topographically-patterned ridges and grooves
  - Biomaterials 33(1):128-136 (2012)
  Recent studies have shown that nanoscale and submicron topographic cues modulate a menu of fundamental cell behaviors, and the use of topographic cues is an expanding area of study in tissue engineering. We used topographically-patterned substrates containing anisotropically ordered ridges and grooves to investigate the effects of topographic cues on mesenchymal stem cell morphology, proliferation, and osteogenic differentiation. We found that human mesenchymal stem cells cultured on 1400 or 4000 nm pitches showed greater elongation and alignment relative to 400 nm pitch or planar control. Cells cultured on 400 nm pitch demonstrated significant increases in RUNX2 and BGLAP expression relative to cells cultured on 1400 or 4000 nm pitch or planar control. Four-hundred nanometer pitch enhanced extracellular calcium deposition. Cells cultured in osteoinductive medium revealed combinatory effects of topography and chemical cues on 400 nm pitch as well as up-regulation of ex! pression of ID1, a target of the BMP pathway. Our data demonstrate that a specific size scale of topographic cue promotes osteogenic differentiation with or without osteogenic agents. These data demonstrate that the integration of topographic cues may be useful for the fabrication of orthopedic implants.
• Tissue engineering bone-ligament complexes using fiber-guiding scaffolds
  - Biomaterials 33(1):137-145 (2012)
  Regeneration of bone-ligament complexes destroyed due to disease or injury is a clinical challenge due to complex topologies and tissue integration required for functional restoration. Attempts to reconstruct soft-hard tissue interfaces have met with limited clinical success. In this investigation, we manufactured biomimetic fiber-guiding scaffolds using solid free-form fabrication methods that custom fit complex anatomical defects to guide functionally-oriented ligamentous fibers in vivo. Compared to traditional, amorphous or random-porous polymeric scaffolds, the use of perpendicularly oriented micro-channels provides better guidance for cellular processes anchoring ligaments between two distinct mineralized structures. These structures withstood biomechanical loading to restore large osseous defects. Cell transplantation using hybrid scaffolding constructs with guidance channels resulted in predictable oriented fiber architecture, greater control of tissue infiltrat! ion, and better organization of ligament interface than random scaffold architectures. These findings demonstrate that fiber-guiding scaffolds drive neogenesis of triphasic bone-ligament integration for a variety of clinical scenarios.
• A paclitaxel-conjugated adenovirus vector for targeted drug delivery for tumor therapy
  - Biomaterials 33(1):146-162 (2012)
  Tumor-targeted drug delivery is an attractive strategy in cancer treatment. Our previous study demonstrated that modified adenovirus has strong tumor targeting ability and less toxicity to surrounding normal tissue. In this study, Paclitaxel (PTX), a widely used clinical anticancer drug, was conjugated to folate-modified adenovirus (Ad) nanoparticles by using succinic anhydride and Fmoc-Glu(OtBu)–OH linkers to form two prodrugs, FA-Ad-Suc-PTX and FA-Ad-ICG02-Glu-PTX. Near-infrared (NIR) fluorescent dye ICG-Der-02 was attached to –NH2–Glu(OtBu)-PTX for in vivo optical imaging. In vitro and acute toxicity study demonstrates the low toxicity of the prodrug FA-Ad-Suc-PTX and FA-Ad-ICG02-Glu-PTX compared to the free drug. The dynamic behaviors and targeting ability of FA-Ad-ICG02-Glu-PTX on MDA-MB-231 tumor-bearing mice were investigated by NIR fluorescence imaging. The result show that PTX-conjugated Ad vector could enhance the targeting and residence time in tumor s! ite. In vitro and in vivo studies demonstrate that Coxsackie adenovirus receptor (CAR) or foliate receptor (FR)-mediated uptake of FA-Ad-loaded PTX induced highly anti-tumor activity. The results support the potential of using chemically modified Ad vector as drug-loaded tumor-targeting delivery system.
• The role of iron redox state in the genotoxicity of ultrafine superparamagnetic iron oxide nanoparticles
  - Biomaterials 33(1):163-170 (2012)
  Ultrafine superparamagnetic iron oxide nanoparticles (USPION) hold great potential for revolutionising biomedical applications such as MRI, localised hyperthermia, and targeted drug delivery. Though evidence is increasing regarding the influence of nanoparticle physico-chemical features on toxicity, data however, is lacking that assesses a range of such characteristics in parallel. We show that iron redox state, a subtle though important physico-chemical feature of USPION, dramatically modifies the cellular uptake of these nanoparticles and influences their induction of DNA damage. Surface chemistry was also found to have an impact and evidence to support a potential mechanism of oxidative DNA damage behind the observed responses has been demonstrated. As human exposure to ferrofluids is predicted to increase through nanomedicine based therapeutics, these findings are important in guiding the fabrication of USPION to ensure they have characteristics that support biocom! patibility.
• An FRET-based ratiometric chemosensor for in vitro cellular fluorescence analyses of pH
  - Biomaterials 33(1):171-180 (2012)
  Ratiometric fluorescence sensing is an important technique for precise and quantitative analysis of biological events occurring under complex conditions by simultaneously recording fluorescence intensities at two wavelengths and calculating their ratios. Herein, we design a ratiometric chemosensor for pH that is based on photo-induced electron transfer (PET) and binding-induced modulation of fluorescence resonance energy transfer (FRET) mechanisms. This ratiometric chemosensor was constructed by introduction of a pH-insensitive coumarin fluorophore as an FRET donor into a pH-sensitive amino-naphthalimide derivative as the FRET acceptor. The sensor exhibited clear dual-mission signal changes in blue and green spectral windows upon pH changes. The pH sensor was applied for not only measuring cellular pH, but also for visualizing stimulus-responsive changes of intracellular pH values.
• Automated microinjection of cell-polymer suspensions in 3D ECM scaffolds for high-throughput quantitative cancer invasion screens
  - Biomaterials 33(1):181-188 (2012)
  Cell spheroids (CS) embedded in 3D extracellular matrix (ECM) serve as in vitro mimics for multicellular structures in vivo. Such cultures, started either from spontaneous cell aggregates or single cells dispersed in a gel are time consuming, applicable to restricted cell types only, prone to high variation, and do not allow CS formation with defined spatial distribution required for high-throughput imaging. Here, we describe a method where cell-polymer suspensions are microinjected as droplets into collagen gels and CS formation occurs within hours for a broad range of cell types. We have automated this method to produce CS arrays in fixed patterns with defined x-y-z spatial coordinates in 96 well plates and applied automated imaging and image analysis algorithms. Low intra- and inter-well variation of initial CS size and CS expansion indicates excellent reproducibility. Distinct cell migration patterns, including cohesive strand-like – and individual cell migration! can be visualized and manipulated. A proof-of-principle chemical screen is performed identifying compounds that affect cancer cell invasion/migration. Finally, we demonstrate applicability to freshly isolated mouse breast and human sarcoma biopsy material – indicating potential for development of personalized cancer treatment strategies.
• Nanoprobes for in vitro diagnostics of cancer and infectious diseases
  - Biomaterials 33(1):189-206 (2012)
  The successful and explosive development of nanotechnology is significantly impacting the fields of biology and medicine. Among the spectacular developments of nanobiotechnology, interest has grown in the use of nanomaterials as nanoprobes for bioanalysis and diagnosis. Herein, we review state-of-the-art nanomaterial-based probes and discuss their applications in in vitro diagnostics (IVD) and challenges in bringing these fields together. Major classes of nanoprobes include quantum dots (QDs), plasmonic nanoparticles, magnetic nanoparticles, nanotubes, nanowires, and multifunctional nanomaterials. With the advantages of high volume/surface ratio, surface tailorability, multifunctionality, and intrinsic properties, nanoprobes have tremendous applications in the areas of biomarker discovery, diagnostics of infectious diseases, and cancer detection. The distinguishing features of nanoprobes for in vitro use, such as harmlessness, ultrasensitivity, multiplicity, and point-! of-care use, will bring a bright future of nanodiagnosis.
• Molecular imaging of a cancer-targeting theragnostics probe using a nucleolin aptamer- and microRNA-221 molecular beacon-conjugated nanoparticle
  - Biomaterials 33(1):207-217 (2012)
  MicroRNAs (miRNA, miR) have been reported as cancer biomarkers that regulate tumor suppressor genes. Hence, simultaneous detecting and inhibiting of miRNA function will be useful as a cancer theragnostics probe to minimize side effects and invasiveness. In this study, we developed a cancer-targeting therangostics probe in a single system using an AS1411 aptamer - and miRNA-221 molecular beacon (miR-221 MB)-conjugated magnetic fluorescence (MF) nanoparticle (MFAS miR-221 MB) to simultaneously target to cancer tissue, image intracellularly expressed miRNA-221 and treat miRNA-221-involved carcinogenesis. AS1411 aptamer-conjugated MF (MFAS) nanoparticles displayed a great selectivity and delivery into various cancer cell lines. The miR-221 MB detached from the MFAS miR-221 MB in the cytoplasm of C6 cells clearly imaged miRNA-221 biogenesis and simultaneously resulted in antitumor therapeutic effects by inhibiting miRNA function, indicating a successful astrocytoma-targetin! g theragnostics. MFAS miRNA MB can be easily applied to other cancers by simply changing a targeted miRNA highly expressed in cancers.
• Thermosensitive/magnetic poly(organophosphazene) hydrogel as a long-term magnetic resonance contrast platform
  - Biomaterials 33(1):218-224 (2012)
  A thermosensitive/magnetic poly(organophosphazene) hydrogel (a magnetic hydrogel) was designed and synthesized for long-term magnetic resonance (MR) imaging. To turn a thermosensitive poly(organophosphazene) hydrogel (an original hydrogel) into a long-term MR contrast platform, cobalt ferrite (CoFe2O4) nanoparticles, which have hydrophobic surfaces, were bound to the original hydrogel via interactions between the hydrophobic surfaces of the nanoparticles and the L-isoleucine ethyl esters of the polymer. The magnetic hydrogel showed extremely low cytotoxicity and adequate magnetic properties for use in long-term MR imaging, in addition to possessing the same properties of the original hydrogel, such as viscosity, thermosensitivity, biodegradability, biocompatibility, a reversible sol-to-gel phase transition near body temperature, and injectability. The magnetic hydrogel was injected into a rat brain using stereotactic surgery. After the injection, the applicable potenti! ality as a long-term MR contrast platform was successfully estimated over 4–5 weeks. Consequently, it was shown that a magnetic hydrogel as a long-term MR contrast platform has the potential to be applied in a long-term theranostic hydrogel system. Furthermore, it is expected that this platform can be useful in the clinical field of incurable diseases due to either surgical difficulties or lethality, such as with brain tumors, when the platform is combined with therapeutic drugs for long-term MR theragnosis in further studies.
• Whispering gallery mode biosensor quantification of fibronectin adsorption kinetics onto alkylsilane monolayers and interpretation of resultant cellular response
  - Biomaterials 33(1):225-236 (2012)
  A Whispering Gallery Mode (WGM) biosensor was constructed to measure the adsorption of protein onto alkysilane self-assembled monolayers (SAMs) at solution concentrations unattainable with other techniques. The high sensitivity was provided by a WGM resonance excited in a silica microsphere that was functionalized with alkylsilane SAMs and integrated in a microfluidic flow cell under laminar flow conditions. It was found that FN adsorbed at biologically relevant surface densities, however, the adsorption kinetics and concentration dependent saturation values varied significantly from work published utilizing alkanethiol SAMs. Mathematical models were applied to the experimental results to interpret the observed kinetics of FN adsorption. Embryonic hippocampal neurons and skeletal myoblasts were cultured on the modified surfaces, a live–dead assay was used to determine the viability of the FN surfaces for cell culture, and major differences were noted in the biologica! l response to the different SAMs. The high sensitivity and simplicity of the WGM biosensor, combined with its ability to quantify the adsorption of any dilute protein in a label-free assay, establishes the importance of this technology for the study of surface accretion and its effect on cellular function, which can affect biomaterials for both in vivo and in vitro applications.
• Silica-shell cross-linked micelles encapsulating fluorescent conjugated polymers for targeted cellular imaging
  - Biomaterials 33(1):237-246 (2012)
  A bioinspired silification approach was successfully used to encapsulate fluorescent conjugated polymers inside silica-shell cross-linked polymeric micelles (CP-SSCL) in the highly benign synthesis environment of room temperature and near-neutral aqueous environment. Four different conjugated polymers were employed to demonstrate the versatility of the bioinspired silification, resulting in the formation of CP-SSCL with different emission wavelengths across the visible spectrum. The CP-SSCL are characterized by a large absorption coefficient and high quantum yield, indicating that they exhibit the required high fluorescence brightness for cellular imaging application. In addition, the CP-SSCL also exhibit a high colloidal stability and low cytotoxicity. The in vitro studies of using MDA-MB-231 breast cancer cells show that the CP-SSCL are successfully uptaken by the cancer cells and located at the cytoplasm of the cells. Furthermore, by conjugating folic acid on their ! surfaces, the uptake of CP-SSCL by MDA-MB-231 cells was enhanced significantly, suggesting their great potential for targeted imaging and early detection of cancer cells.
• Theranostic Gd(III)-lipid microbubbles for MRI-guided focused ultrasound surgery
  - Biomaterials 33(1):247-255 (2012)
  We have synthesized a biomaterial consisting of Gd(III) ions chelated to lipid-coated, size-selected microbubbles for utility in both magnetic resonance and ultrasound imaging. The macrocyclic ligand DOTA-NHS was bound to PE headgroups on the lipid shell of pre-synthesized microbubbles. Gd(III) was then chelated to DOTA on the microbubble shell. The reaction temperature was optimized to increase the rate of Gd(III) chelation while maintaining microbubble stability. ICP-OES analysis of the microbubbles determined a surface density of 7.5 × 105 ± 3.0 × 105 Gd(III)/μm2 after chelation at 50 °C. The Gd(III)-bound microbubbles were found to be echogenic in vivo during high-frequency ultrasound imaging of the mouse kidney. The Gd(III)-bound microbubbles also were characterized by magnetic resonance imaging (MRI) at 9.4 T by a spin-echo technique and, surprisingly, both the longitudinal and transverse proton relaxation rates were found to be roughly equal to that of no! -Gd(III) control microbubbles and saline. However, the relaxation rates increased significantly, and in a dose-dependent manner, after sonication was used to fragment the Gd(III)-bound microbubbles into non-gas-containing lipid bilayer remnants. The longitudinal (r1) and transverse (r2) molar relaxivities were 4.0 ± 0.4 and 120 ± 18 mM−1s−1, respectively, based on Gd(III) content. The Gd(III)-bound microbubbles may find application in the measurement of cavitation events during MRI-guided focused ultrasound therapy and to track the biodistribution of shell remnants.
• Effects of nanoparticle coatings on the activity of oncolytic adenovirusâ€"magnetic nanoparticle complexes
  - Biomaterials 33(1):256-269 (2012)
  Limitations to adenovirus infectivity can be overcome by association with magnetic nanoparticles and enforced infection by magnetic field influence. Here we examined three core-shell-type iron oxide magnetic nanoparticles differing in their surface coatings, particle sizes and magnetic properties for their ability to enhance the oncolytic potency of adenovirus Ad520 and to stabilize it against the inhibitory effects of serum or a neutralizing antibody. It was found that the physicochemical properties of magnetic nanoparticles are critical determinants of the properties which govern the oncolytic productivities of their complexes with Ad520. Although high serum concentration during infection or a neutralizing antibody had strong inhibitory influence on the uptake or oncolytic productivity of the naked virus, one particle type was identified which conferred high protection against both inhibitory factors while enhancing the oncolytic productivity of the internalized viru! s. This particle type equipped with a silica coating and adsorbed polyethylenimine, displaying a high magnetic moment and high saturation magnetization, mediated a 50% reduction of tumor growth rate versus control upon intratumoral injection of its complex with Ad520 and magnetic field influence, whereas Ad520 alone was inefficient. The correlations between physical properties of the magnetic particles or virus complexes and oncolytic potency are described herein.
• EGFR-specific PEGylated immunoliposomes for active siRNA delivery in hepatocellular carcinoma
  - Biomaterials 33(1):270-282 (2012)
  The development of immunoliposomes for systemic siRNA (small interfering RNA) delivery is highly desired. We reported previously the development of targeted LPD (liposome–polycation–DNA complex) conjugated with anti-EGFR (epidermal growth factor receptor) Fab′ (TLPD-FCC) for siRNA delivery, which showed superior gene silencing activity in EGFR-overexpressing breast cancers. However, TLPD-FCC did not achieve satisfactory gene silencing activity in EGFR-overexpressing hepatocellular carcinoma (HCC). In this study, some modifications including increased antibody conjugation efficiency and reduced PEGylation degree were made to TLPD-FCC to increase gene silencing activity in HCC. The resultant optimized liposomes denoted as TLPD-FP75 efficiently bound and delivered to EGFR-overexpressing HCC, resulting in enhanced gene silencing activity compared to untargeted LPD (NTLPD-FP75). Tissue distribution in vivo revealed that the accumulation of TLPD-FP75 was higher than NT! LPD-FP75 in orthotopic HCC model of mice. The promoted uptake of TLPD-FP75 in HCC cells was confirmed by confocal microscopy. To investigate the in vivo gene silencing activity, we administered TLPD-FP75 by intravenous injections into mice bearing orthotopic HCC. The results showed TLPD-FP75 potently suppressed luciferase expression, while little silencing was observed in NTLPD-FP75. TLPD-FP75 was demonstrated to possess potent gene silencing activity in HCC and will potentially increase the feasibility of HCC gene therapy.
• Effects of osteogenic growth factors on bone marrow stromal cell differentiation in a mineral-based delivery system
  - Biomaterials 33(1):283-294 (2012)
  Delivering growth factors from bone-like mineral combines osteoinductivity with osteoconductivity. The effects of individual and sequential exposure of BMP-2 and FGF-2 on osteogenic differentiation, and their release from apatite were studied to design a dual delivery system. Bone marrow stromal cells were seeded on TCPS with the addition of FGF-2 (2.5, 10, 40 ng/ml) or BMP-2 (50, 150, 450 ng/ml) for 6 days. DNA content and osteogenic response were examined weekly for 3 weeks. FGF-2 increased DNA content; however, high concentrations of FGF-2 inhibited/delayed osteogenic differentiation, while a threshold concentration of BMP-2 was required for significant osteogenic enhancement. The sequence of delivery of BMP-2 (300 ng/ml) and FGF-2 (2.5 ng/ml) also had a significant impact on osteogenic differentiation. Delivery of FGF-2 followed by BMP-2 or delivery of BMP-2 followed by BMP-2 and FGF-2 enhanced osteogenic differentiation compared to the simultaneous delivery of bot! h factors. Release of BMP-2 and FGF-2 from bone-like mineral was significantly affected by the concentration used during coprecipitation. BMP-2 also demonstrated a higher "burst" release compared to FGF-2. By integrating the results of the sequential delivery of BMP-2 and FGF-2 in solution, with the release of individual growth factors from mineral, an organic/inorganic delivery system based on coprecipitation can be designed for multiple biomolecules.
• Surface modification of pancreatic islets using heparin-DOPA conjugate and anti-CD154 mAb for the prolonged survival of intrahepatic transplanted islets in a xenograft model
  - Biomaterials 33(1):295-303 (2012)
  This study proposes a combination method of using 3,4-dihydorxy-l-phenylalanine (DOPA) conjugated heparin (heparin-DOPA) and a low dose of anti-CD154 monoclonal antibody (MR-1) treatment to improve the survival time of intrahepatic islet xenograft. To inhibit instant blood mediated inflammatory reactions, heparin-DOPA was directly grafted to the pancreatic islet surface. The surface coverage of heparin-DOPA, the viability and functionality of heparin-DOPA grafted islets were evaluated. In addition, the combined effect of grafted heparin-DOPA and a low dose of MR-1 (a T-cell targeting immunosuppressive drug) on the survival of islet was evaluated in a xenograft model. Both unmodified islets and heparin-DOPA grafted islets were completely rejected within 2 weeks after intraportal transplantation. However, when 0.1 mg/mouse of MR-1 was administered (at day 0, 2, 4, 6 of transplantation) to 11 mice that had heparin-DOPA grafted islets transplanted to, seven out of the reci! pients maintained normoglycemia over 60 days. Therefore, we propose that a developed combinatory immunoprotection protocol of surface modification of pancreatic islets using heparin-DOPA with a low dose of MR-1 can be effective in prolonging the survival rate of transplanted islets in a xenograft model.
• Local suppression of pro-inflammatory cytokines and the effects in BMP-2-induced bone regeneration
  - Biomaterials 33(1):304-316 (2012)
  The objective of this study is to investigate the effect of local inflammation suppression on the bone regeneration. Gelatin hydrogels incorporating mixed immunosuppressive triptolide-micelles and bone morphogenic protein-2 (BMP-2) were prepared. The controlled release of both the triptolide and BMP-2 from the hydrogels was observed under in vitro and in vivo conditions. When either J774.1 macrophage-like or MC3T3-E1 osteoblastic cells were cultured in the hydrogels incorporating mixed 2.5, 5 or 10 mg of triptolide-micelles and BMP-2, the expression level of pro- and anti-inflammatory cytokines including interleukin (IL)-6 and IL-10 was down-regulated, but the alkaline phosphatase (ALP) activity was promoted compared with those of hydrogels incorporating BMP-2 without triptolide-micelles. When implanted into a critical-sized bone defect of rats, the hydrogels incorporating mixed 2.5 or 5 mg of triptolide-micelles and BMP-2 showed significantly lower number of neutrophi! ls, lymphocytes, macrophages or dendritic and mast cells infiltrated into the defect, and lower expression level of IL-6, TNF-α, and IL-10 than those incorporating BMP-2 without triptolide-micelles. The reduced local inflammation responses at the defects implanted with the hydrogels incorporating mixed 2.5 or 5 mg of triptolide-micelles and BMP-2 subsequently enhanced the bone regeneration thereat. It is concluded that the proper local modulation of inflammation responses is a promising way to achieve the enhanced bone regeneration.
• The application of an alanine-substituted mutant of the C-terminal fragment of Clostridium perfringens enterotoxin as a mucosal vaccine in mice
  - Biomaterials 33(1):317-324 (2012)
  Efficient delivery of antigen to mucosal immune tissues is an essential part of mucosal vaccination. Claudin-4 is expressed on the epithelial cells that cover the mucosal immune tissues. We previously found that claudin-4-targeting is a promising strategy for mucosal vaccination by using a claudin-4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE). Substitution of Asn and Ser at positions 309 and 313, respectively, with alanine increased the affinity of C-CPE for claudin-4. However, application of the C-CPE mutant as a mucosal vaccine has never been tried. Here, we investigated whether the C-CPE mutant could serve as a mucosal vaccine. We used ovalbumin (OVA) as a model antigen and fused the C-CPE mutant to it. The resultant fusion protein was bound to claudin-4. When mice were immunized with the C-CPE mutant-fused OVA, OVA-specific serum IgG and nasal IgA increased relative to levels in mice immunized with a C-CPE-fused antigen. Immunizat! ion with the C-CPE mutant-fused OVA activated Th1- and Th2-type responses and led to increased anti-tumor activity against OVA-expressing thymoma cells relative to that of mice immunized with the C-CPE-fused antigen. These findings suggest that the alanine-substituted C-CPE mutant shows promise as a claudin-targeted mucosal vaccine.
• Enhanced gene expression promoted by the quantized folding of pDNA within polyplex micelles
  - Biomaterials 33(1):325-332 (2012)
  Selective packaging of plasmid DNA (pDNA) into folded rod or collapsed sphere structures in polyplex micelles was demonstrated by modulating the PLys segment length of poly(ethylene glycol)-block- poly(l-lysine) (PEG-PLys) block catiomers used for micelle formation. The two basic packaging structures correlated well to the integrity of double-stranded DNA contained within the micelles. Rod structures formed by the quantized folding mechanism, which results in dissociation of double-stranded DNA only at each fold. Collapsed sphere structures formed by substantial random disruption of the double-stranded DNA structure. Analysis of gene expression in a cell-free transcription/translation system, cultured cells and also skeletal muscle of mice showed that micelles containing pDNA packaged by quantized folding exhibited higher gene expression than naked pDNA and micelles containing collapsed pDNA. These results indicate that controlled packaging of pDNA into an appropriate ! structure is critical for achieving effective gene expression. Improved gene transfection and expression resulting from the quantized folding of pDNA within polyplex micelles is promising for application in therapeutic gene delivery systems.
• The phagocytosis of gas-filled microbubbles by human and murine antigen-presenting cells
  - Biomaterials 33(1):333-342 (2012)
  This study was designed to evaluate the potential of gas-filled microbubbles (MB) to be internalized by antigen-presenting cells (APC). Fluorescently labeled MB were prepared, thus permitting to track binding to, and internalization in, APC. Both human and mouse cells, including monocytes and dendritic cells (DC), prove capable to phagocyte MB in vitro. Observation by confocal laser scanning microscopy showed that interaction between MB and target cells resulted in a rapid internalization in cellular compartments and to a lesser extent in the cytoplasm. Capture of MB by APC resulted in phagolysosomal targeting as verified by double staining with anti-lysosome-associated membrane protein-1 monoclonal antibody and decrease of internalization by phagocytosis inhibitors. Fluorescent MB injected subcutaneously (s.c.) in mice were found to be associated with CD11c+DC in lymph nodes draining the injection sites 24 h after administration. Altogether, our study demonstrates tha! t MB can successfully target APC both in vitro and in vivo, and thus may serve as a potent Ag delivery system without requirement for ultrasound-based sonoporation. This adds to the potential of applications of MB already extensively used for diagnostic imaging in humans.
• The suppression of IgE-mediated histamine release from mast cells following exocytic exclusion of biodegradable polymeric nanoparticles
  - Biomaterials 33(1):343-351 (2012)
  The objective of this study is to evaluate the effect of polymeric nanoparticles (NPs) on the allergic response of mast cells that release inflammatory mediators such as histamine through exocytosis. Submicron-sized biodegradable poly(dl-lactide-co-glycolide) (PLGA) NPs were prepared by the emulsion solvent diffusion method. Here, we examined the interactions of the mast cells with two types of PLGA NPs, unmodified NPs and NPs modified with chitosan (CS), a biodegradable cationic polymer. The cellular uptake of NPs increased by CS modification due to electrostatic interactions with the plasma membrane. NPs were taken up by mast cells through an endocytic pathway (endocytic phase) and then the cellular uptake was saturated and maintained plateau level by the exclusion of NPs through exocytosis (exocytic phase). Antigen-induced histamine release from mast cells was inhibited during the exocytic phase. The extent of histamine release inhibition was related to the amount o! f excluded NPs. Exocytic exclusion of NPs competitively antagonize the antigen-induced exocytotic release of histamine by highjacking exocytosis machinery such as SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, since histamine release was recovered in mast cells that overexpress SNAP-23. The inhibitory effect of the allergic response by PLGA NPs was also evaluated in vivo using the mouse model for systemic anaphylaxis. The administration of NPs suppressed the antigen-induced systemic allergic response in vivo. In conclusion, PLGA NP itself has actions to inhibit the allergic responses mediated by mast cells.