Hot off the presses! Jan 01 Biomater

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Latest Articles Include:

• Editorial board
  - Biomater 32(1):IFC (2011)
  
• The continuing evolution of biomaterials
  - Biomater 32(1):1-2 (2011)
  
• Acute phase proteins as biomarkers for predicting the exposure and toxicity of nanomaterials
  - Biomater 32(1):3-9 (2011)
  Recently, nanomaterials have become an integral part of our daily lives. However, there is increasing concern about the potential risk to human health. Here, we attempted to identify biomarkers for predicting the exposure and toxicity of nanomaterials by using a proteomics based approach. We evaluated the changes of protein expression in plasma after treatment with silica nanoparticles. Our analyses identified haptoglobin, one of the acute phase proteins, as a candidate biomarker. The results of ELISA showed that the level of haptoglobin was significantly elevated in plasma of mice exposed to silica nanoparticles with a diameter of 70 nm (nSP70) compared to normal mice and those exposed to silica particles with a diameter of 1000 nm. Furthermore, the other acute phase proteins, C-reactive protein (CRP) and serum amyloid A (SAA) were also elevated in plasma of nSP70 treated mice. In addition, the level of these acute phase proteins was elevated in the plasma of mice aft! er intranasal treatment with nSP30. Our results suggest that haptoglobin, CRP and SAA are highly sensitive biomarkers for assessing the risk of exposure to silica nanoparticles. We believe this study will contribute to the development of global risk assessment techniques for nanomaterials.
• The biocompatibility of titanium cardiovascular devices seeded with autologous blood-derived endothelial progenitor cells: EPC-seeded antithrombotic Ti Implants
  - Biomater 32(1):10-18 (2011)
  Implantable and extracorporeal cardiovascular devices are commonly made from titanium (Ti) (e.g. Ti-coated Nitinol stents and mechanical circulatory assist devices). Endothelializing the blood-contacting Ti surfaces of these devices would provide them with an antithrombogenic coating that mimics the native lining of blood vessels and the heart. We evaluated the viability and adherence of peripheral blood-derived porcine endothelial progenitor cells (EPCs), seeded onto thin Ti layers on glass slides under static conditions and after exposure to fluid shear stresses. EPCs attached and grew to confluence on Ti in serum-free medium, without preadsorption of proteins. After attachment to Ti for 15 min, less than 5% of the cells detached at a shear stress of 100 dyne / cm2. Confluent monolayers of EPCs on smooth Ti surfaces (Rq of 10 nm), exposed to 15 or 100 dyne / cm2 for 48 h, aligned and elongated in the direction of flow and produced nitric oxide dependent on the level ! of shear stress. EPC-coated Ti surfaces had dramatically reduced platelet adhesion when compared to uncoated Ti surfaces. These results indicate that peripheral blood-derived EPCs adhere and function normally on Ti surfaces. Therefore EPCs may be used to seed cardiovascular devices prior to implantation to ameliorate platelet activation and thrombus formation.
• The control of neural cell-to-cell interactions through non-contact electrical field stimulation using graphene electrodes
  - Biomater 32(1):19-27 (2011)
  Electric field stimulation has become one of the most promising therapies for a variety of neurological diseases. However, the safety and effectiveness of the stimulator are critical in determining the outcome. Because there are few safe and effective in vivo and/or in vitro stimulator devices, we demonstrate a method that allows for non-contact electric field stimulation with a specific strength that is able to control cell-to-cell interaction in vitro. Graphene, a form of graphite, and polyethylene terephthalate (PET) was used to create a non-cytotoxic in vitro graphene/PET film stimulator. A transient non-contact electric field was produced by charge-balanced biphasic stimuli through the graphene/PET film electrodes and applied to cultured neural cells. We found that weak electric field stimulation (pulse duration of 10 s) as low as 4.5 mV/mm for 32 min was particularly effective in shaping cell-to-cell interaction. Under weak electric field stimulation, we observed! a significant increase in the number of cells forming new cell-to-cell couplings and in the number of cells strengthening existing cell-to-cell couplings. The underlying mechanism of the altered cellular interactions may be related to an altered regulation of the endogenous cytoskeletal proteins fibronectin, actin, and vinculin. In conclusion, this technique may open a new therapeutic approach for augmenting cell-to-cell coupling in cell transplantation therapy in the central nervous system.
• The promotion of chondrogenesis, osteogenesis, and adipogenesis of human mesenchymal stem cells by multiple growth factors incorporated into nanosphere-coated microspheres
  - Biomater 32(1):28-38 (2011)
  The induction of stem cell differentiation by drugs and growth factors has been the objective of many studies designed to develop methods for the formation of new tissues or the repair of degenerated tissues via transplantation. In this study, drugs and growth factors with high potential for use in tissue repair were embedded in human mesenchymal stem cells (hMSCs), which were then induced to differentiate into chondrogenic, osteogenic, and adipogenic lineages. Additionally, microspheres coated and loaded with the drugs and growth factors successfully proliferated and, as expected, induced the differentiation of transplanted hMSCs into the desired specific cell types. Furthermore, hMSCs transplanted in micro-typed scaffolds prevented changes in differentiation. RT-PCR and western blot analysis of the resultant cartilage, bone, and adipose tissues showed that a combination of drugs and different growth factor types induced the differentiation of transplanted hMSCs. Addi! tionally, histology and immunohistochemistry showed that specific ECMs and proteins released from transplanted hMSCs were present within the nanosphere-coated microspheres. The results of this study show that the regulation of stem cell differentiation by drugs and growth factors might enable the fabrication of therapeutic materials for the delivery of stem cells that are simpler to use, less expensive, and more easily controlled than the delivery systems currently available.
• The spreading, migration and proliferation of mouse mesenchymal stem cells cultured inside hyaluronic acid hydrogels
  - Biomater 32(1):39-47 (2011)
  Synthetic hydrogel scaffolds that can be used as culture systems that mimic the natural stem cell niche are of increased importance for stem cell biology and regenerative medicine. These artificial niches can be utilized to control the stem cell fate and will have potential applications for expanding/differentiating stem cells in vitro, delivering stem cells in vivo, as well as making tissue constructs. In this study, we synthesized hyaluronic acid (HA) hydrogels that could be degraded through a combination of cell-released enzymes and used them to culture mouse mesenchymal stem cells (mMSC). To form the hydrogels, HA was modified to contain acrylate groups and crosslinked through Michael addition chemistry using non-degradable, plasmin degradable or matrix metalloproteinase (MMP) degradable crosslinkers. Using this hydrogel we found that mMSC proliferation occurred in the absence of cell spreading, that mMSCs could only spread when both RGD and MMP degradation sites w! ere present in the hydrogel and that mMSCs in hydrogels with high density of RGD (1000 μm) spread and migrated faster and more extensively than in hydrogels with low density of RGD (100 μm).
• Incorporation of biomaterials in multicellular aggregates modulates pluripotent stem cell differentiation
  - Biomater 32(1):48-56 (2011)
  Biomaterials are increasingly being used to engineer the biochemical and biophysical properties of the extracellular stem cell microenvironment in order to tailor niche characteristics and direct cell phenotype. To date, stem cell–biomaterial interactions have largely been studied by introducing stem cells into artificial environments, such as 2D cell culture on biomaterial surfaces, encapsulation of cell suspensions within hydrogel materials, or cell seeding on 3D polymeric scaffolds. In this study, microparticles fabricated from different materials, such as agarose, PLGA and gelatin, were stably integrated, in a dose-dependent manner, within aggregates of pluripotent stem cells (PSCs) prior to differentiation as a means to directly examine stem cell–biomaterial interactions in 3D. Interestingly, the presence of the materials within the stem cell aggregates differentially modulated the gene and protein expression patterns of several differentiation markers without! adversely affecting cell viability. Microparticle incorporation within 3D stem cell aggregates can control the spatial presentation of extracellular environmental cues (i.e. soluble factors, extracellular matrix and intercellular adhesion molecules) as a means to direct the differentiation of stem cells for tissue engineering and regenerative medicine applications. In addition, these results suggest that the physical presence of microparticles within stem cell aggregates does not compromise PSC differentiation, but in fact the choice of biomaterials can impact the propensity of stem cells to adopt particular differentiated cell phenotypes.
• Differentiation of neural stem cells in three-dimensional growth factor-immobilized chitosan hydrogel scaffolds
  - Biomater 32(1):57-64 (2011)
  The adult central nervous system (CNS) contains adult neural stem/progenitor cells (NSPCs) that possess the ability to differentiate into the primary cell types found in the CNS and to regenerate lost or damaged tissue. The ability to specifically and spatially control differentiation is vital to enable cell-based CNS regenerative strategies. Here we describe the development of a protein-biomaterial system that allows rapid, stable and homogenous linking of a growth factor to a photocrosslinkable material. A bioactive recombinant fusion protein incorporating pro-neural rat interferon-γ (rIFN-γ) and the AviTag for biotinylation was successfully expressed in Escherichia coli and purified. The photocrosslinkable biopolymer, methacrylamide chitosan (MAC), was thiolated, allowing conjugation of maleimide–strepatavidin via Michael-type addition. We demonstrated that biotin–rIFN-γ binds specifically to MAC-streptavidin in stoichiometric yields at 100 and 200 ng/mL in p! hotocrosslinked hydrogels. For cell studies, NSPCs were photo-encapsulated in 100 ng/mL biotin–rIFN-γ immobilized MAC based scaffolds and compared to similar NSPC-seeded scaffolds combining 100 ng/mL soluble biotin–rIFN-γ vs. no growth factor. Cells were cultured for 8 days after which differentiation was assayed using immunohistochemistry for lineage specific markers. Quantification showed that immobilized biotin-rIFN-γ promoted neuronal differentiation (72.8 ± 16.0%) similar to soluble biotin–rIFN-γ (71.8 ± 13.2%). The percentage of nestin-positive (stem/progenitor) cells as well as RIP-positive (oligodendrocyte) cells were significantly higher in scaffolds with soluble vs. immobilized biotin–rIFN-γ suggesting that 3-D immobilization results in a more committed lineage specification.
• An alginate-based hybrid system for growth factor delivery in the functional repair of large bone defects
  - Biomater 32(1):65-74 (2011)
  The treatment of challenging fractures and large osseous defects presents a formidable problem for orthopaedic surgeons. Tissue engineering/regenerative medicine approaches seek to solve this problem by delivering osteogenic signals within scaffolding biomaterials. In this study, we introduce a hybrid growth factor delivery system that consists of an electrospun nanofiber mesh tube for guiding bone regeneration combined with peptide-modified alginate hydrogel injected inside the tube for sustained growth factor release. We tested the ability of this system to deliver recombinant bone morphogenetic protein-2 (rhBMP-2) for the repair of critically-sized segmental bone defects in a rat model. Longitudinal μ-CT analysis and torsional testing provided quantitative assessment of bone regeneration. Our results indicate that the hybrid delivery system resulted in consistent bony bridging of the challenging bone defects. However, in the absence of rhBMP-2, the use of nanofiber! mesh tube and alginate did not result in substantial bone formation. Perforations in the nanofiber mesh accelerated the rhBMP-2 mediated bone repair, and resulted in functional restoration of the regenerated bone. μ-CT based angiography indicated that perforations did not significantly affect the revascularization of defects, suggesting that some other interaction with the tissue surrounding the defect such as improved infiltration of osteoprogenitor cells contributed to the observed differences in repair. Overall, our results indicate that the hybrid alginate/nanofiber mesh system is a promising growth factor delivery strategy for the repair of challenging bone injuries.
• The enhancement of endothelial cell therapy for angiogenesis in hindlimb ischemia using hyaluronan
  - Biomater 32(1):75-86 (2011)
  Growing evidence shows that injection of hyaluronan (HA) benefits ischemic injury in animals. On the other hand, cell therapy is an emerging approach to treat occlusive arterial diseases, although the low retention rate of cells after direct injection remains a major concern. Here, we tested whether injection of HA along with endothelial cells promotes the retention and growth of transplanted cells, thus improving therapeutic angiogenesis in a mouse model of hindlimb ischemia (HI). In culture, HA improved human umbilical vein endothelial cell (HUVEC) proliferation proportional to HA concentration and protected HUVECs from apoptosis. Subsequently, in immunocompromised mice HI was induced by femoral artery ligation and treatments were given 24 h later. At 4 weeks, injection of HA along with HUVECs had a greater effect for restoring blood perfusion and salvaging the ischemic limb compared to injection of HA or HUVECs alone. In addition, angiogenesis and arteriogenesis wer! e significantly increased by HA + HUVECs injection. Lastly, HA + HUVECs injection resulted in the retention of more cells than HUVECs alone, and allowed their engraftment into the vasculature of the ischemic limb. These results suggest that this combined approach can be translated into a clinical therapy for peripheral artery occlusive disease.
• Fluorescent PLLA-nanodiamond composites for bone tissue engineering
  - Biomater 32(1):87-94 (2011)
  Superior mechanical properties, rich surface chemistry, and good biocompatibility of diamond nanoparticles make them attractive in biomaterial applications. A multifunctional fluorescent composite bone scaffold material has been produced utilizing a biodegradable polymer, poly(l-lactic acid) (PLLA), and octadecylamine-functionalized nanodiamond (ND-ODA). The uniform dispersion of nanoparticles in the polymer led to significant increase in hardness and Young's modulus of the composites. Addition of 10%wt of ND-ODA resulted in more than 200% increase in Young's modulus and 800% increase in hardness, bringing the nanocomposite properties close to that of the human cortical bone. Testing of ND-ODA/PLLA as a matrix supporting murine osteoblast (7F2) cell growth for up to 1 week showed that the addition of ND-ODA had no negative effects on cell proliferation. ND-ODA serves as a multifunctional additive providing improved mechanical properties, bright fluorescence, and op! tions for drug loading and delivery via surface modification. Thus ND-ODA/PLLA composites open up numerous avenues for their use as components of bone scaffolds and smart surgical tools such as fixation devices in musculoskeletal tissue engineering and regenerative medicine. Intense fluorescence of ND-ODA/PLLA scaffolds can be used to monitor bone re-growth replacing the implant in vivo.
• Functional neovascularization of biodegradable dextran hydrogels with multiple angiogenic growth factors
  - Biomater 32(1):95-106 (2011)
  Slow vascularization of functional blood limits the transplantation of tissue constructs and the recovery of ischemic and wounded tissues. Despite the widespread investigation of polysaccharide-based hydrogel scaffolds for their therapeutic applications, blood vessel ingrowth into these hydrogel scaffolds remains a challenge. We hypothesized that modifying the properties of biodegradable hydrogel scaffolds with immobilization of multiple angiogenic growth factors (GFs) would induce a rapid proliferation of functional vasculature into the scaffolds. To this end, we remodeled the hydrogel structure by decreasing crosslinking density via reduced degree of substitution of crosslinking groups, which resulted in improved hydrogel properties including reduced rigidity, increased swelling, increased vascular endothelial GF (VEGF) release capability, and facilitated rapid hydrogel disintegration and tissue ingrowth. Immobilizing VEGF in the scaffolds promoted tissue ingrowth an! d expedited biodegradation. Furthermore, a synergistic effect of multiple angiogenic GFs was established; the coimmobilization of VEGF+ angiopoietin-1, and VEGF+ insulin-like GF+ stromal cell-derived factor-1 induced more and larger blood vessels than any individual GF, while the combination of all GFs dramatically increased the size and number of newly formed functional vessels. Altogether, our data demonstrate that rapid, efficient, and functional neovascularization can be achieved by precisely manipulating hydrogel scaffold properties and immobilizing defined angiogenic GFs.
• Towards a quantitative understanding of oxygen tension and cell density evolution in fibrin hydrogels
  - Biomater 32(1):107-118 (2011)
  The in vitro culture of hydrogel-based constructs above a critical size is accompanied by problems of unequal cell distribution when diffusion is the primary mode of oxygen transfer. In this study, an experimentally-informed mathematical model was developed to relate cell proliferation and death inside fibrin hydrogels to the local oxygen tension in a quantitative manner. The predictive capacity of the resulting model was tested by comparing its outcomes to the density, distribution and viability of human periosteum derived cells (hPDCs) that were cultured inside fibrin hydrogels in vitro. The model was able to reproduce important experimental findings, such as the formation of a multilayered cell sheet at the hydrogel periphery and the occurrence of a cell density gradient throughout the hydrogel. In addition, the model demonstrated that cell culture in fibrin hydrogels can lead to complete anoxia in the centre of the hydrogel for realistic values of oxygen diffusion ! and consumption. A sensitivity analysis also identified these two parameters, together with the proliferation parameters of the encapsulated cells, as the governing parameters for the occurrence of anoxia. In conclusion, this study indicates that mathematical models can help to better understand oxygen transport limitations and its influence on cell behaviour during the in vitro culture of cell-seeded hydrogels.
• Extracellular matrix production by adipose-derived stem cells: Implications for heart valve tissue engineering
  - Biomater 32(1):119-127 (2011)
  A key challenge in tissue engineering a heart valve is to reproduce the major tissue structures responsible for native valve function. Here we evaluated human adipose-derived stem cells (ADSCs) as a source of cells for heart valve tissue engineering investigating their ability to synthesize and process collagen and elastin. ADSCs were compared with human bone marrow mesenchymal stem cells (BmMSCs) and human aortic valve interstitial cells (hVICs). ADSCs and BmMSCs were stretched at 14% for 3 days and collagen synthesis determined by [3H]-proline incorporation. Collagen and elastin crosslinking was assessed by measuring pyridinoline and desmosine respectively, using liquid chromatography/mass spectrometry. Three-dimensional culture was obtained by seeding cells onto bovine collagen type I scaffolds for 2–20 days. Expression of matrix proteins and processing enzymes was assessed by Real Time-PCR, immunofluorescence and transmission electron microscopy. Stretch increase! d the incorporation of [3H]-proline in ADSCs and BmMSCs, however only ADSCs and hVICs upregulated COL3A1 gene. ADSCs produced collagen and elastin crosslinks. ADSCs uniformly populated collagen scaffolds after 2 days, and fibrillar-like collagen was detected after 20 days. ADSCs sense mechanical stimulation and produce and process collagen and elastin. These novel findings have important implications for the use of these cells in tissue engineering.
• The effect of source animal age upon extracellular matrix scaffold properties
  - Biomater 32(1):128-136 (2011)
  Biologic scaffold materials composed of mammalian extracellular matrix (ECM) are commonly used for the repair and reconstruction of injured tissues. An important, but unexplored variable of biologic scaffolds is the age of the animal from which the ECM is prepared. The objective of the present study was to compare the structural, mechanical, and compositional properties of small intestinal submucosa (SIS)-ECM harvested from pigs that differed only in age. Degradation product bioactivity of these ECM materials was also examined. Results showed that there are distinct differences in each of these variables among the various age source ECM scaffolds. The strength and growth factors content of ECM from 3-week-old animals is less than that of ECM harvested from 12, 26 or >52-week-old animals. The elastic modulus of SIS-ECM for 3 week and >52-week-old source was less than that of the 12 and 26 week source. Degradation products from all age source ECMs were chemotactic for pe! rivascular stem cells, with the 12 week source the most potent, while the oldest source caused the greatest increase in proliferation. In summary, distinct differences exist in the mechanical, structural, and biologic properties of SIS-ECM harvested from different aged animals.
• The surface molecular functionality of decellularized extracellular matrices
  - Biomater 32(1):137-143 (2011)
  Decellularization of tissues and organs is a successful platform technology for creating scaffolding materials for tissue engineering and regenerative medicine. It has been suggested that the success of these materials upon implantation is due to the molecular signals provided by the remaining scaffold extracellular matrix (ECM) components presented to probing cells in vivo as they repopulate the surface. For this study, decellularized matrices were created from esophagus, bladder, and small intestine harvested from adult male Fischer 344 rats. The three decellularized matrices (each originating from source tissues which included an epithelial lining on their luminal surfaces) were immunostained for collagen IV and laminin to determine basement membrane retention. Scanning electron micrographs of the surfaces were used to provide insight into the surface topography of each of the decellularized tissues. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used! to generate high-resolution mass spectra for the surfaces of each scaffold. This surface-sensitive technique allows for detailed molecular analysis of the outermost 1–2 nm of a material and has been applied previously to thin protein films and secreted ECM proteins on poly(N-isopropyl acrylamide) (polyNIPAAM) surfaces. To extract trends from within the complex ToF-SIMS dataset, a multivariate analysis technique, principal component analysis (PCA), was employed. Using this method, a molecular fingerprint of each surface was created and separation was seen in the PCA scores between the decellularized esophagus and the decellularized small intestine samples. The PCA scores for the decellularized bladder sample fell between the previous two decellularized samples. Protein films of common extracellular matrix constituents (collagen IV, collagen I, laminin, and Matrigel) were also investigated. The PCA results from these protein films were used to develop qualitative hypothese! s for the relationship of the key fragments identified from th! e PCA of the decellularized ECMs.
• Optimization of surface chemistry on single-walled carbon nanotubes for in vivo photothermal ablation of tumors
  - Biomater 32(1):144-151 (2011)
  Carbon nanotubes have shown great potential in various areas of biomedicine. Herein, we synthesize a series of amphiphilic polymers by anchoring polyethylene glycol (PEG) of different lengths at various densities on poly(maleic anhydride-alt-1-octadecene) (PMHC18). The blood circulation and biodistribution of those PEG-PMHC18-coated SWNTs in mice after intravenous injection are measured by an established Raman spectroscopy method. It is found that heavily PEGylated SWNTs with ultra-long blood circulation half-lives, although shows high uptake in the tumor, tend to accumulate in the skin dermis. A surface coating which affords SWNTs a blood half-life of 12–13 h appears to be optimal to balance the tumor-to-normal organ (T/N) uptake ratios of nanotubes in major organs. Using the selected SWNT conjugate, we then carry out a pilot in vivo photothermal therapy study and observe a promising cancer treatment efficacy. Our results highlight the importance of surface coating ! to the in vivo behaviors of nanomaterials in general and could provide guidelines to the future design of SWNT bioconjugates for various in vivo applications.
• Targeting of polymeric nanoparticles to lung metastases by surface-attachment of YIGSR peptide from laminin
  - Biomater 32(1):152-161 (2011)
  Effective therapy for disseminated metastatic cancer is currently impossible because of low drug accumulation in target sites. Here, we aimed to enhance nanoparticle (NP) targeting to lung melanoma metastases via interactions with the laminin receptor, whose expression is upregulated in metastatic cells. To enable NP follow-up and a framework for targeting ligand binding, Estapor® fluorescent NPs (299 ± 6 nm in diameter) with surface carboxylic groups were employed and the laminin receptor binding peptide (YIGSR) was attached to their surface to facilitate targeting. In vitro uptake studies performed under medium flow conditions revealed that the uptake of YIGSR-attached NPs by monolayers of B16 melanoma cells was 2-fold higher compared to the uptake of scrambled peptide-NPs. In cultures of healthy lung cells, the uptake of YIGSR-NPs was low and similar to the uptake of scrambled peptide-NPs. Competition assays using cultured B16 melanoma cells pre-incubated with sol! uble laminin confirmed that the entry of the YIGSR-modified NPs was mediated via interaction with the laminin receptor. Following intravenous (i.v.) administration into B16 melanoma tumor-bearing mice, targeting of the tumor by the YIGSR-NPs was up to five-fold higher than the scrambled peptide-NPs, with no heart, liver or lung tropism. In an experimental lung metastases model, following i.v. administration the YIGSR-NPs targeted the cancerous metastatic cells in lungs, with nearly no targeting to the healthy lung cells. Collectively, the data indicate that YIGSR-targeted NPs have a potential to be used for systemic delivery of chemotherapeutic drugs for the treatment of metastatic lung cancer.
• Development of an antibody proteomics system using a phage antibody library for efficient screening of biomarker proteins
  - Biomater 32(1):162-169 (2011)
  Proteomics-based analysis is currently the most promising approach for identifying biomarker proteins for use in drug development. However, many candidate biomarker proteins that are over- or under-expressed in diseased tissues are found by such a procedure. Thus, establishment of an efficient method for screening and validating the more valuable targets is urgently required. Here, we describe the development of an "antibody proteomics system" that facilitates the screening of biomarker proteins from many candidates by rapid preparation of cross-reacting antibodies using phage antibody library technology. Using two-dimensional differential in-gel electrophoresis analysis, 16 over-expressed proteins from breast cancer cells were identified. Specifically, proteins were recovered from the gel pieces and a portion of each sample was used for mass spectrometry analysis. The remainder was immobilized onto a nitrocellulose membrane for antibody-expressing phage enrichment! and selection. Using this procedure, antibody-expressing phages against each protein were successfully isolated within two weeks. The expression profiles of the identified proteins were then acquired by immunostaining of breast tumor tissue microarrays with the antibody-expressing phages. Using this approach, expression of Eph receptor A10, TRAIL-R2 and Cytokeratin 8 in breast tumor tissues were successfully validated. These results demonstrate the antibody proteomics system is an efficient method for screening tumor-related biomarker proteins.
• Development and in vivo evaluation of an oral drug delivery system for paclitaxel
  - Biomater 32(1):170-175 (2011)
  The aim of the present study was to investigate the effect of poly(acrylic acid)-cysteine (PAA-cysteine) exhibiting a molecular mass of 100 and 250 kDa and reduced glutathione (GSH) on the absorption of the P-glycoprotein (P-gp) and cytochrome P450 (CYP450) substrate paclitaxel in vitro and in vivo. In vitro transport studies were performed with Caco-2 monolayers. Furthermore, the delivery system based on PAA-cysteine, GSH and paclitaxel was evaluated in vivo in rats. In vitro, the formulation comprising 0.5% (m/v) PAA-cysteine (100 kDa)/0.5% (m/v) GSH improved the transport of paclitaxel 6.7-fold (Papp = 8.7 ± 1.3 × 10−6 cm/s) in comparison to paclitaxel itself serving as buffer only control (Papp = 1.3 ± 0.4 × 10−6 cm/s). Moreover, in the presence of the formulation containing 0.5% (m/v) PAA-cysteine (250 kDa)/0.5% (m/v) GSH paclitaxel absorption was even 7.4-fold (Papp = 9.7 ± 0.3 × 10−6 cm/s) improved in comparison to the buffer only control. In vivo, t! he oral administration of formulations containing 1 mg of paclitaxel, 1 mg of GSH and 8 mg of PAA-cysteine (100 kDa or 250 kDa) resulted in an improved paclitaxel plasma concentration and bioavailability. The area under the plasma concentration-time curve (AUC0–8) of paclitaxel was 4.7-fold and 5.7-fold improved in comparison to the oral formulation containing paclitaxel alone, respectively. Moreover, cmax was improved by 6.3-fold and even 7.3-fold in comparison to the oral formulation containing paclitaxel alone, respectively. Thus, according to the achieved results it is suggested that PAA-cysteine in combination with GSH would be a potentially valuable tool for improving the oral bioavailability of P-gp and CYP450 substrates such as paclitaxel.
• Surface functionalized hollow manganese oxide nanoparticles for cancer targeted siRNA delivery and magnetic resonance imaging
  - Biomater 32(1):176-184 (2011)
  Multifunctional hollow manganese oxide nanoparticles (HMON) were produced by a bio-inspired surface functionalization approach, using 3,4-dihydroxy-l-phenylalanine (DOPA) as an adhesive moiety, for cancer targeted delivery of therapeutic siRNA and simultaneous diagnosis via magnetic resonance imaging (MRI). Cationic polyethylenimine-DOPA conjugates were stably immobilized onto the surface of HMON due to the strong binding affinity of DOPA to metal oxides, as examined by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. These nanoparticles were subsequently functionalized with a therapeutic monoclonal antibody, Herceptin, to selectively target cancer cells. Confocal microscopy and MR imaging studies revealed that the surface functionalized HMON enabled the targeted detection of cancer cells in T1-weighted MRI as well as the efficient intracellular delivery of siRNA for cell-specific gene silencing. These nanomaterials are expected to be widel! y exploited as multifunctional delivery vehicles for cancer therapy and imaging applications.
• Multifunctional superparamagnetic nanocarriers with folate-mediated and pH-responsive targeting properties for anticancer drug delivery
  - Biomater 32(1):185-194 (2011)
  Multifunctional nanocarriers with multilayer core-shell architecture were prepared by coating superparamagnetic Fe3O4 nanoparticle cores with a mixture of the triblock copolymer methoxy poly(ethylene glycol)-b-poly(methacrylic acid-co-n-butyl methacrylate)-b-poly(glycerol monomethacrylate) and the folate-conjugated block copolymer folate-poly(ethylene glycol)-b-poly(glycerol monomethacrylate). The model anticancer agent adriamycin (ADR), containing an amine group and a hydrophobic moiety, was loaded into the nanocarrier at pH 7.4 by ionic bonding and hydrophobic interactions. The release rate of the loaded drug molecules was slow at pH 7.4 (i.e. mimicking the blood environment) but increased significantly at acidic pH (i.e. mimicking endosome/lysosome conditions). Acid-triggered drug release resulted from the polycarboxylate protonation of poly(methacrylic acid), which broke the ionic bond between the carrier and ADR. Cellular uptake by folate receptor-overexpressing H! eLa cells of the folate-conjugated ADR-loaded nanoparticles was higher than that of non-folated-conjugated nanoparticles. Thus, folate conjugation significantly increased nanoparticle cytotoxicity. These findings show the potential viability of a folate-targeting, pH-responsive nanocarrier for amine-containing anticancer drugs.
• Cytotoxic effects of iron oxide nanoparticles and implications for safety in cell labelling
  - Biomater 32(1):195-205 (2011)
  The in vitro labelling of cultured cells with iron oxide nanoparticles (NPs) is a frequent practice in biomedical research. To date, the potential cytotoxicity of these particles remains an issue of debate. In the present study, 4 different NP types (dextran-coated Endorem, carboxydextran-coated Resovist, lipid-coated magnetoliposomes (MLs) and citrate-coated very small iron oxide particles (VSOP)) are tested on a variety of cell types, being C17.2 neural progenitor cells, PC12 rat pheochromocytoma cells and human blood outgrowth endothelial cells. Using different NP concentrations, the effect of the NPs on cell morphology, cytoskeleton, proliferation, reactive oxygen species, functionality, viability and cellular homeostasis is investigated. Through a systematic study, the safe concentrations for every particle type are determined, showing that MLs can lead up to 67.37 ± 5.98 pg Fe/cell whereas VSOP are the most toxic particles and only reach 18.65 ± 2.07 pg Fe/cell! . Using these concentrations, it is shown that for MRI up to 500 cells/μl labelled with VSOP are required to efficiently visualize in an agar phantom in contrast to only 50 cells/μl for MLs and 200 cells/μl for Endorem and Resovist. These results highlight the importance of in-depth cytotoxic evaluation of cell labelling studies as at non-toxic concentrations, some particles appear to be less suitable for the MR visualization of labelled cells.
• The biological properties of iron oxide core high-density lipoprotein in experimental atherosclerosis
  - Biomater 32(1):206-213 (2011)
  Lipoproteins are a family of plasma nanoparticles responsible for the transportation of lipids throughout the body. High-density lipoprotein (HDL), the smallest of the lipoprotein family, measures 7–13 nm in diameter and consists of a cholesteryl ester and triglyceride core that is covered with a monolayer of phospholipids and apolipoproteins. We have developed an iron oxide core HDL nanoparticle (FeO-HDL), which has a lipid based fluorophore incorporated in the phospholipid layer. This nanoparticle provides contrast for optical imaging, magnetic resonance imaging (MRI) and transmission electron microscopy (TEM). Consequently, FeO-HDL can be visualized on the anatomical, cellular and sub-cellular level. In the current study we show that the biophysical features of FeO-HDL closely resemble those of native HDL and that FeO-HDL possess the ability to mimic HDL characteristics both in vitro as well as in vivo. We demonstrate that FeO-HDL can be applied to image HDL inter! actions and to investigate disease settings where HDL plays a key function. More generally, we have demonstrated a multimodal approach to study the behavior of biomaterials in vitro as well as in vivo. The approach allowed us to study nanoparticle dynamics in circulation, as well as nanoparticle targeting and uptake by tissues and cells of interest. Moreover, we were able to qualitatively assess nanoparticle excretion, critical for translating nanotechnologies to the clinic.
• Eradication of drug resistant Staphylococcus aureus by liposomal oleic acids
  - Biomater 32(1):214-221 (2011)
  Staphylococcus aureus (S. aureus) represents a major threat to a broad range of healthcare and community associated infections. This bacterium has rapidly evolved resistance to multiple drugs throughout its antibiotic history and thus it is imperative to develop novel antimicrobial strategies to enrich the currently shrinking therapeutic options against S. aureus. This study evaluated the antimicrobial activity and therapeutic efficacy of oleic acid (OA) in a liposomal formulation as an innate bactericide against methicillin-resistant S. aureus (MRSA). In vitro studies showed that these OA-loaded liposomes (LipoOA) could rapidly fuse into the bacterial membranes, thereby significantly improving the potency of OA to kill MRSA compared with the use of free OA. Further in vivo tests demonstrated that LipoOA were highly effective in curing skin infections caused by MRSA bacteria and preserving the integrity of the infected skin using a mouse skin model. Moreover, a prelimi! nary skin toxicity study proved high biocompatibility of LipoOA to normal skin tissues. These findings suggest that LipoOA hold great potential to become a new, effective, and safe antimicrobial agent for the treatment of MRSA infections.
• A protein transduction domain located at the NH2-terminus of human translationally controlled tumor protein for delivery of active molecules to cells
  - Biomater 32(1):222-230 (2011)
  Protein transduction domains (PTDs) are small peptides, able to penetrate biological membranes and deliver various types of cargo both in vitro and in vivo. Because use of PTDs originating from viral origins resulted in undesired effects, PTDs originating from non-viral origins are needed. Here, we report that a 10-amino acid peptide (MIIYRDLISH) derived from the NH2-terminus of human translationally controlled tumor protein (TCTP) functions as a PTD. This peptide was internalized through lipid raft-dependent endocytosis and partial macropinocytosis, and did not enter lysosome and nucleus. Beta-galactosidase fused to TCTP-PTD, when injected into mice, was efficiently delivered to liver, kidney, spleen, heart, and lungs of the animals. Preincubation of TCTP-PTD with adenovirus increased adenoviral mediated-gene expression in cells and also improved immune response to intranasally administered adenovirus expressing the triple repeat of G glycoprotein of respiratory syncy! tial virus (RSV), rAd/3×G. These findings suggest that TCTP-PTD might overcome the limitations of polycation-mediated transduction and serve as an efficient vehicle for drug delivery.
• Post-formulation peptide drug loading of nanostructures for metered control of NF-κB signaling
  - Biomater 32(1):231-238 (2011)
  The NF-κB signaling pathway is an attractive therapeutic target for cancer and chronic inflammatory diseases. In this study, we report the first strategy to achieve NF-κB inhibition with a peptide inhibitor loaded into perfluorocarbon nanoparticles with the use of a simple post-formulation mixing approach that utilizes an amphipathic cationic fusion peptide linker strategy for cargo insertion. A stable peptide-nanoparticle complex is formed (dissociation constant 0.14 μM) and metered inhibition of both NF-κB signaling and downstream gene expression (ICAM-1) is demonstrated in leukemia/lymphoma cells. This post-formulation cargo loading strategy enables the use of a generic synthetic or biologic lipidic nanostructure for drug conjugation that permits flexible specification of types and doses of peptides and/or other materials as diagnostic or therapeutic agents for metered incorporation and cellular delivery.
• Mechanisms of cellular uptake and intracellular trafficking with chitosan/DNA/poly(γ-glutamic acid) complexes as a gene delivery vector
  - Biomater 32(1):239-248 (2011)
  Chitosan (CS)-based complexes have been considered as a vector for DNA delivery; nonetheless, their transfection efficiency is relatively low. An approach by incorporating poly(γ-glutamic acid) (γ-PGA) in CS/DNA complexes was developed in our previous study to enhance their gene expression level; however, the detailed mechanisms remain to be understood. The study was designed to investigate the mechanisms in cellular uptake and intracellular trafficking of CS/DNA/γ-PGA complexes. The results of our molecular dynamic simulations suggest that after forming complexes with CS, γ-PGA displays a free γ-glutamic acid in its N-terminal end and thus may be recognized by γ-glutamyl transpeptidase in the cell membrane, resulting in a significant increase in their cellular uptake. In the endocytosis inhibition study, we found that the internalization of CS/DNA complexes took place via macropinocytosis and caveolae-mediated pathway; by incorporating γ-PGA in complexes, both ! uptake pathways were further enhanced but the caveolae-mediated pathway played a major role. TEM was used to gain directly understanding of the internalization mechanism of test complexes and confirmed our findings obtained in the inhibition experiments. After internalization, a less percentage of co-localization of CS/DNA/γ-PGA complexes with lysosomes was observed when compared with their CS/DNA counterparts. A greater cellular uptake together with a less entry into lysosomes might thus explain the promotion of transfection efficiency of CS/DNA/γ-PGA complexes. Knowledge of these mechanisms involving CS-based complexes containing γ-PGA is critical for the development of an efficient vector for DNA transfection.
• A peptide-based material platform for displaying antibodies to engage T cells
  - Biomater 32(1):249-257 (2011)
  This study investigated a strategy by which antibodies are displayed to engage T cells. A peptide composite containing binding sites was characterized in vitro and explored as an injectable system in vivo. The composite consists of two amphiphilic peptides, AEAEAKAKAEAEAKAK (referred to as "EAK") and EAK appended with six consecutive histidines at the C-terminus ("EAKH6"). Spectroscopic analysis showed the two peptides integrated into a single structure. Prior to combination, conformational analysis revealed that EAKH6 adopts a mixed α-helix/β-strand conformation. In the presence of EAK, EAKH6 exists predominately in β-strand conformation. The composite of EAK-EAKH6 was found to display His-tags, using nickel-bound horseradish peroxidase as a probe. T cell-specific antibodies were found stably displayed on the EAK-EAKH6 assembly using recombinant protein A/G and anti-histidines antibody as an adaptor. When mounted with anti-CD4 antibody, the system was shown! to capture CD4 T cells in a mixed population of lymphocytes. Antibodies were concentrated in the subcutaneous space in mice when co-administered with EAK and EAKH6 along with protein A/G and anti-histidines antibody as a solution. We report here the use of amphiphilic peptides to display Ab in vivo, the results indicating that the design can be used as a platform for engaging specific subsets of leukocytes for the purpose of immune modulation.
• Hyperbranched poly(NIPAM) polymers modified with antibiotics for the reduction of bacterial burden in infected human tissue engineered skin
  - Biomater 32(1):258-267 (2011)
  The escalating global incidence of bacterial infection, particularly in chronic wounds, is a problem that requires significant improvements to existing therapies. We have developed hyperbranched poly(NIPAM) polymers functionalized with the antibiotics Vancomycin and Polymyxin-B that are sensitive to the presence of bacteria in solution. Binding of bacteria to the polymers causes a conformational change, resulting in collapse of the polymers and the formation of insoluble polymer/bacteria complexes. We have applied these novel polymers to our tissue engineered human skin model of a burn wound infected with Pseudomonas aeruginosa and Staphylococcus aureus. When the polymers were removed from the infected skin, either in a polymer gel solution or in the form of hydrogel membranes, they removed bound bacteria, thus reducing the bacterial load in the infected skin model. These bacteria-binding polymers have many potential uses, including coatings for wound dressings.
• The use of biodegradable PLGA nanoparticles to mediate SOX9 gene delivery in human mesenchymal stem cells (hMSCs) and induce chondrogenesis
  - Biomater 32(1):268-278 (2011)
  In stem cell therapy, transfection of specific genes into stem cells is an important technique to induce cell differentiation. To perform gene transfection in human mesenchymal stem cells (hMSCs), we designed and fabricated a non-viral vector system for specific stem cell differentiation. Several kinds of gene carriers were evaluated with regard to their transfection efficiency and their ability to enhance hMSCs differentiation. Of these delivery vehicles, biodegradable poly (dl-lactic-co-glycolic acid) (PLGA) nanoparticles yielded the best results, as they complexed with high levels of plasmid DNA (pDNA), allowed robust gene expression in hMSCs, and induced chondrogenesis. Polyplexing with polyethylenimine (PEI) enhanced the cellular uptake of SOX9 DNA complexed with PLGA nanoparticles both in vitro and in vivo. The expression of enhanced green fluorescent protein (EGFP) and SOX9 increased up to 75% in hMSCs transfected with PEI/SOX9 complexed PLGA nanoparticles 2 day! s after transfection. SOX9 gene expression was evaluated by RT-PCR, real time-qPCR, glycosaminoglycan (GAG)/DNA levels, immunoblotting, histology, and immunofluorescence.
• Polycaprolactone-based fused deposition modeled mesh for delivery of antibacterial agents to infected wounds
  - Biomater 32(1):279-287 (2011)
  Infections represent a significant source of site morbidity following tissue trauma. Scarring and tissue adhesion remain the challenging issues yet to be solved. Prolonged inflammation and morphology of the re-epithelisated layer are important considerations. We hypothesized that the solution lies not only in the biochemistry of biomaterial but also the micro-architecture of the scaffold used as the matrix for wound healing. Targeted delivery of antibiotics may provide an efficacious means of infection control through adequate release. Here, we study the use of 3-dimensional polycaprolactone–tricalcium phosphate (PCL–TCP) mesh for the delivery of gentamicin sulphate (GS) fabricated using a solvent-free method. PCL–TCP meshes incorporated with varying loads of GS were evaluated in vitro for elution profile, antimicrobial efficacy and cytotoxicity. Results showed that PCL–TCP meshes incorporated with 15wt% GS (PT15) efficiently eliminate bacteria within 2 h and d! emonstrate low cytotoxicity. Subsequently, PT15 meshes were evaluated using an infected full thickness wound mice model, and observed to eliminate bacteria in the wounds effectively. Additionally, mice from the PT15 treatment group (TG) showed no observable signs of overall infection through neutrophil count by day 7 and displayed efficient wound healing (94.2% wound area reduction) by day 14. Histology also showed significantly faster healing in TG through neo-collagen deposition and wound re-epithelisation. The meshes from TG were also observed to be expelled from wounds while gauze fibers from CG were integrated into wounds during healing.
• Study of the therapeutic benefit of cationic copolymer administration to vascular endothelium under mechanical stress
  - Biomater 32(1):288-294 (2011)
  Pulmonary edema and the associated increases in vascular permeability continue to represent a significant clinical problem in the intensive care setting, with no current treatment modality other than supportive care and mechanical ventilation. Therapeutic compound(s) capable of attenuating changes in vascular barrier function would represent a significant advance in critical care medicine. We have previously reported the development of HPMA-based copolymers, targeted to endothelial glycocalyx that are able to enhance barrier function. In this work, we report the refinement of copolymer design and extend our physiological studies to demonstrate that the polymers: 1) reduce both shear stress and pressure-mediated increase in hydraulic conductivity, 2) reduce nitric oxide production in response to elevated hydrostatic pressure and, 3) reduce the capillary filtration coefficient (Kfc) in an isolated perfused mouse lung model. These copolymers represent an important tool fo! r use in mechanotransduction research and a novel strategy for developing clinically useful copolymers for the treatment of vascular permeability.
• Hierarchical scaffold design for mesenchymal stem cell-based gene therapy of hemophilia B
  - Biomater 32(1):295-305 (2011)
  Gene therapy for hemophilia B and other hereditary plasma protein deficiencies showed great promise in pre-clinical and early clinical trials. However, safety concerns about in vivo delivery of viral vectors and poor post-transplant survival of ex vivo modified cells remain key hurdles for clinical translation of gene therapy. We here describe a 3D scaffold system based on porous hydroxyapatite–PLGA composites coated with biomineralized collagen 1. When combined with autologous gene-engineered factor IX (hFIX) positive mesenchymal stem cells (MSCs) and implanted in hemophilic mice, these scaffolds supported long-term engraftment and systemic protein delivery by MSCs in vivo. Optimization of the scaffolds at the macro-, micro- and nanoscales provided efficient cell delivery capacity, MSC self-renewal and osteogenesis respectively, concurrent with sustained delivery of hFIX. In conclusion, the use of gene-enhanced MSC-seeded scaffolds may be of practical use for treatm! ent of hemophilia B and other plasma protein deficiencies.
• Combinational therapy of ischemic brain stroke by delivery of heme oxygenase-1 gene and dexamethasone
  - Biomater 32(1):306-315 (2011)
  Combinational therapies using genes and drugs are promising therapeutic strategies for various diseases. In this research, a co-delivery carrier of dexamethasone and plasmid DNA (pDNA) was developed by conjugation of dexamethasone to polyethylenimine (2 kDa, PEI2k) for combinational therapy of ischemic brain. Dynamic light scattering, atomic force microscopy and flow cytometry studies showed that the pDNA/dexamethasone-conjugated PEI2k (PEI2k-Dexa) complex was 150 nm in size and was taken up by cells more easily than PEI2k-Dexa only. The tumor necrosis factor-α (TNF-α) level was decreased more efficiently by pDNA/PEI2k-Dexa complex than dexamethasone only in hypoxia activated Raw 264.7 macrophage cells, suggesting that pDNA/PEI2k-Dexa complex increased the delivery efficiency and therapeutic effect of dexamethasone. In in vitro transfection assay, PEI2k-Dexa had higher transfection efficiency than PEI2k and lipofectamine. However, the simple mixture of PEI2k and dexa! methasone did not show this effect, suggesting that the conjugation of dexamethasone to polyethylenimine increased DNA delivery efficiency of PEI2k. To evaluate the effects of combinational therapy in vivo, pDNA/PEI2k-Dexa complex was applied to a transient focal ischemia animal model. At 24 h after the injection, mean infarction volume and the TNF-α level were reduced more efficiently in the pDNA/PEI2k-Dexa injection group, compared with the control, pDNA/PEI2k, or dexamethasone injection group. The infarction volume and inflammatory cytokines were further decreased by delivery of pSV-HO-1 using PEI2k-Dexa. Magnetic resonance imaging and microPET studies confirmed the therapeutic effect of pSV-HO-1/PEI2k-Dexa complex at 10 days after the injection. Therefore, pSV-HO-1/PEI2k-Dexa complexes may be useful in combinational therapy for ischemic diseases such as stroke.
• Snapshot: Biologic Scaffolds For Constructive Tissue Remodeling
  - Biomater 32(1):316-319 (2011)