Hot off the presses! Oct 01 Biomaterials

The Oct 01 issue of the Biomaterials is now up on Pubget (About Biomaterials): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• Editorial board
  - Biomaterials 32(28):IFC (2011)
  
• The modulation of platelet adhesion and activation by chitosan through plasma and extracellular matrix proteins
  - Biomaterials 32(28):6655-6662 (2011)
  Chitosan has been shown to promote initial wound closure events to prevent blood loss. Platelet adhesion and activation are crucial early events in these processes after traumatic bleeding leading to thrombus formation. Platelet adhesion to chitosan was found to be enhanced in the presence of adsorbed plasma and extracellular matrix proteins and was found to be primarily mediated by αIIbβ3 integrins, while α2β1 integrins were found to be involved in platelet adhesion to collagen and perlecan. Platelets were found to be activated by chitosan, as shown by an increase in the expression of αIIbβ3 integrins and P-selectin, while the extent of activation was modulated by the presence of proteins including perlecan and fibrinogen. Collagen-coated chitosan was found to activate platelets to the same extent as either chitosan or collagen alone. These data support the role of plasma and extracellular matrix proteins in promoting chitosan mediated platelet adhesion and acti! vation supporting the hypothesis that chitosan promotes wound healing via these interactions.
• Spatio-temporal detachment of single cells using microarrayed transparent electrodes
  - Biomaterials 32(28):6663-6669 (2011)
  This paper describes a dynamic cell manipulation approach by which cells can be selectively detached from transparent indium tin oxide (ITO) electrodes via electrochemical desorption of a self-assembled monolayer (SAM) of alkanethiol. Changes in the surface properties of ITO following modification and electrical desorption of the SAM were characterized. By using these reactions on ITO, cells were readily attached and then detached from ITO electrodes in a very rapid manner, with greater than 90% of the cells being detached within 5 min. Furthermore, we fabricated micropatterns of ITO electrodes using photolithography. Cells on the micropatterned ITO electrodes could be detached with single-cell resolution. This approach could potentially lead to on-demand harvesting or elimination of one population from others under microscopy, for a wide range of purposes.
• The Goldilocks Surface
  - Biomaterials 32(28):6670-6675 (2011)
  A minimum in the biological response to materials that is observed to occur within a narrow surface energy range is related to the properties of water at these biology-contacting surfaces. Wetting energetics are calculated using a published theory from which it is further estimated that water molecules bind to these special surfaces through a single hydrogen bond, leaving three other hydrogen bonds to interact with proximal water molecules. It is concluded that, at this Goldilocks Surface, the local chemical environment of surface-bound water is nearly identical to that experienced in bulk water; neither deprived of hydrogen bond opportunities, as it is in contact with a more hydrophobic surface, nor excessively hydrogen bonded to a more hydrophilic surface. A minimum in the biological response occurs because water vicinal (near) to the Goldilocks Surface is not chemically different than bulk water. A more precise definition of the relative terms hydrophobic and hydrop! hilic for use in biomaterials becomes evident from calculations: >1.3 kJ/mole-of-surface-sites is expended in wetting a hydrophilic surface whereas <1.3 kJ/mole-of-surface-sites is expended in wetting hydrophobic surfaces; hydrophilic surfaces wet with >1 hydrogen bond per water molecule whereas hydrophobic surfaces wet with <1 hydrogen bond per water molecule.
• Polyamidoamine dendrimer-conjugated quantum dots for efficient labeling of primary cultured mesenchymal stem cells
  - Biomaterials 32(28):6676-6682 (2011)
  Monitoring of cells in vivo after transplantation could supply important information for determining the efficacy of stem cell therapy. The use of quantum dots (QDs) has several advantages for in vivo imaging, such as remarkable resistance to photo bleaching, high fluorescence efficiency, and size-tunable emission. After they are taken up by cells via endocytosis, QDs lose their fluorescence intensity in endosomes/lysosomes at low pH because the intensity cannot survive under acidic conditions. Moreover, the amount of QD uptake by mesenchymal stem cells (MSCs) is extremely small. Therefore, for effective labeling of MSCs and long observation of MSCs labeled by QDs in vivo, it is essential both to increase cellular uptake of QDs and to promote endosomal escape into the cytosol. The polyamidoamine (PAMAM) dendrimer had plenty of cationic charge, which promoted cellular uptake though electrostatic interactions, and a "buffering capacity," which enhanced endosomal esca! pe into the cytosol. In this study, QDs were modified with PAMAM dendrimer for the efficient labeling of MSCs by QDs. The uptake efficiency and cytosolic distribution of QDs in primary cultured MSCs were increased by the modification of the PAMAM dendrimer. The fluorescence intensity in MSCs labeled by PAMAM dendrimer-conjugated QDs lasted for a longer time in harvested culture plates or in cell-transplanted mice than that in MSCs labeled by non-conjugated QDs.
• The generation of iPS cells using non-viral magnetic nanoparticlebased transfection
  - Biomaterials 32(28):6683-6691 (2011)
  Induced pluripotent stem (iPS) cells have been generated from various somatic cells; however, a major restriction of the technology is the use of potentially harmful genome-integrating viral DNAs. Here, without a viral vector, we generated iPS cells from fibroblasts using a non-viral magnetic nanoparticle-based transfection method that employs biodegradable cationic polymer PEI-coated super paramagnetic nanoparticles (NP). Our findings support the possible use of transient expression of iPS genes in somatic cells by magnet-based nanofection for efficient generation of iPS cells. Results of dynamic light scattering (DLS) analysis and TEM analyses demonstrated efficient conjugation of NP with iPS genes. After transfection, nanofection-mediated iPS cells showed ES cell-like characteristics, including expression of endogenous pluripotency genes, differentiation of three germ layer lineages, and formation of teratomas. Our results demonstrate that magnet-based nanofection m! ay provide a safe method for use in generation of virus-free and exogenous DNA-free iPS cells, which will be crucial for future clinical applications in the field of regenerative medicine.
• Immune responses to implants – A review of the implications for the design of immunomodulatory biomaterials
  - Biomaterials 32(28):6692-6709 (2011)
  A key for long-term survival and function of biomaterials is that they do not elicit a detrimental immune response. As biomaterials can have profound impacts on the host immune response the concept emerged to design biomaterials that are able to trigger desired immunological outcomes and thus support the healing process. However, engineering such biomaterials requires an in-depth understanding of the host inflammatory and wound healing response to implanted materials. One focus of this review is to outline the up-to-date knowledge on immune responses to biomaterials. Understanding the complex interactions of host response and material implants reveals the need for and also the potential of "immunomodulating" biomaterials. Based on this knowledge, we discuss strategies of triggering appropriate immune responses by functional biomaterials and highlight recent approaches of biomaterials that mimic the physiological extracellular matrix and modify cellular immune responses.
• Directed cell attachment by tropoelastin on masked plasma immersion ion implantation treated PTFE
  - Biomaterials 32(28):6710-6718 (2011)
  The ability to generate cell patterns on polymer surfaces is critical for the detailed study of cellular biology, the fabrication of cell-based biosensors, cell separation techniques and for tissue engineering. In this study contact tape masking and steel shadow masks were used to exclude plasma immersion ion implantation (PIII) treatment from defined areas of polytetrafluoroethylene (PTFE) surfaces. This process enabled patterned covalent binding of the cell adhesive protein, tropoelastin, without employing chemical linking molecules. Tropoelastin coating rendered the untreated regions cell adhesive and the PIII-treated area non-adhesive, allowing very fine patterning of cell adhesion to PTFE surfaces. A blocking step, such as with BSA or PEG, was not required to prevent cell binding to the underlying PIII-treated regions as tropoelastin coating alone performed this blocking function. Although tropoelastin coated the entire PTFE surface, the cell binding C-terminus of! tropoelastin was markedly less solvent exposed on the PIII-treated, hydrophilic regions. The differential exposure of the C-terminus correlated with the patterned distribution of tropoelastin-mediated cell adhesion. This new methodology specifically enables directed cell behavior on a polymer surface using a simple one-step treatment process, by modulating the adhesive activity of a single extracellular matrix protein.
• Selective immobilization of Sonic hedgehog on benzylguanine terminated patterned self-assembled monolayers
  - Biomaterials 32(28):6719-6728 (2011)
  Patterned two-component, self-assembled monolayers on gold were produced by UV lithography. An oligo(ethylene glycol) terminated disulfide served as inert matrix reducing unspecific protein adsorption and cell adhesion. The second component of the self-assembled monolayer (SAM) presented a benzylguanine moiety for the immobilization of Sonic hedgehog (Shh) fused to a mutant O6-alkylguanine-DNA alkyltransferase (SNAP-tag™). The enzymatic activity of the SNAP-tag allows selective and covalent immobilization of the linked Shh. Time-of-flight secondary ion mass spectrometry verified the correct lateral distribution of the benzylguanine head groups in the patterned SAM. The quantification of unspecific and specific protein binding to mixed SAMs showed increased adsorption of albumin with increasing benzylguanine/(ethylene glycol) ratios. However, the immobilization of SNAP-tagged Shh was not blocked by pre-adsorbed albumin. Furthermore, the obtained micro-patterned substr! ates permitted direct immobilization of SNAP-tagged Shh even in the presence of many competing proteins from conditioned media of transfected HEK293 cells. Therefore, the presented system is suited for the controlled immobilization of fusion proteins from complex mixtures avoiding purification steps.
• Tailoring the porosity and pore size of electrospun synthetic human elastin scaffolds for dermal tissue engineering
  - Biomaterials 32(28):6729-6736 (2011)
  We obtained low and high porosity synthetic human elastin scaffolds by adapting low (1 mL/h) and high (3 mL/h) flow rates respectively during electrospinning. Physical, mechanical and biological properties of these scaffolds were screened to identify the best candidates for the bioengineering of dermal tissue. SHE scaffolds that were electrospun at the higher flow rate presented increased fiber diameter and greater average pore size and over doubling of overall scaffold porosity. Both types of scaffold displayed Young's moduli comparable to that of native elastin, but the high porosity scaffolds possessed higher tensile strength. Low and high porosity scaffolds supported early attachment, spreading and proliferation of primary dermal fibroblasts, but only high porosity scaffolds supported active cell migration and infiltration into the scaffold. High porosity SHE scaffolds promoted cell persistence and scaffold remodeling in vitro with only moderate scaffold contract! ion. The scaffolds persisted for at least 6 weeks in a mouse subcutaneous implantation study with fibroblasts on the exterior and infiltrating, evidence of scaffold remodeling including de novo collagen synthesis and early stage angiogenesis.
• The promotion of neural progenitor cells proliferation by aligned and randomly oriented collagen nanofibers through β1 integrin/MAPK signaling pathway
  - Biomaterials 32(28):6737-6744 (2011)
  In regenerative medicine, accumulating evidence demonstrates that the property of substrates monitors neural stem cells behavior. However, how stem cells sense and interpret biochemical and topographical cues remains elusive. This study aimed to explore the mechanism how nanofibrous scaffold modulated stem cells behavior. Spinal cord derived neural progenitor cells (NPCs) were cultured on electrospun aligned and randomly oriented collagen nanofibrous scaffolds. A 30% increase in proliferation and an elevation of BrdU incorporation were observed in NPCs on collagen nanofibers, compared to that on collagen-coated surface. In particular, NPCs expanded faster on aligned nanofibers in comparison with that on randomly oriented nanofibers. Moreover, an alteration in cell cycle progression with a reduced percentage of cells in G0/G1 phase and increased cell proliferation index (S phase plus G2/M phase) was also detected in NPCs cultured on collagen nanofibers. Incubating NPCs ! with anti-β1 integrin antibody or U1026 (an inhibitor of mitogen-activated protein kinase kinase, MEK) eliminated the altered cell cycle dynamics and BrdU incorporation induced by collagen nanofibers. In addition, cyclin D1 and cyclin dependent kinase 2 (CDK2), downstream genes of β1 integrin/mitogen-activated protein kinase (MAPK) pathway that control G1/S phase transition, were correspondingly regulated by nanofibers. Collectively, these data suggested that the property of substrate modulated NPCs proliferation by promoting cell cycle through β1 integrin/MAPK pathway. Our findings provide a better understanding of the interaction between NPCs and the substrate and therefore will pave way for regenerative medicine.
• A phosphate-dependent shift in redox state of cerium oxide nanoparticles and its effects on catalytic properties
  - Biomaterials 32(28):6745-6753 (2011)
  Cerium oxide nanoparticles (CeNPs) have shown promise as catalytic antioxidants in cell culture and animal models as both superoxide dismutase and catalase mimetics. The reactivity of the cerium (Ce) atoms at the surface of its oxide particle is critical to such therapeutic properties, yet little is known about the potential for a protein or small molecule corona to form on these materials in vivo. Moreover Ce atoms in these active sites have the potential to interact with small molecule anions, peptides, or sugars when administered in culture or animal models. Several nanomaterials have been shown to alter or aggregate under these conditions, rendering them less useful for biomedical applications. In this work we have studied the change in catalytic properties of CeNPs when exposed to various biologically relevant conditions in vitro. We have found that CeNPs are resistant to broad changes in pH and also not altered by incubation in cell culture medium. However to our! surprise phosphate anions significantly altered the characteristics of these nanomaterials and shifted the catalytic behavior due to the binding of phosphate anions to cerium. Given the abundance of phosphate in biological systems in an inorganic form, it is likely that the action of CeNPs as a catalyst may be strongly influenced by the local concentration of phosphate in the cells and/or tissues in which it has been introduced.
• Hydrogel-based biomimetic environment for in vitro modulation of branching morphogenesis
  - Biomaterials 32(28):6754-6763 (2011)
  The mechanical properties of the cellular microenvironment dramatically alter during tissue development and growth. Growing evidence suggests that physical microenvironments and mechanical stresses direct cell fate in developing tissue. However, how these physical cues affect the tissue morphogenesis remains a major unknown. We explain here that the physical properties of the cell and tissue microenvironment, biomimetically reproduced by using hydrogel, guide the tissue morphogenesis in the developmental submandibular gland (SMG). In particular, the softer gel enhances the bud expansion and cleft formation of SMG, whereas the stiffer gel attenuates them. These morphological changes in SMG tissue are led by soluble factors (FGF7/10) induction regulated by cell traction force derived from the tissue deformation. Our findings suggest that cells sense the mechanics of their surrounding environment and alter their properties for self-organization and the following tissue mo! rphogenesis. Also, physically designed hydrogel material is a valuable tool for producing the biomimetic microenvironment to explore how physical cues affect tissue morphogenesis and to modulate tissue morphogenesis for in vitro tissue synthesis.
• Irreversible optical clearing of rabbit dermis for autogenic corneal stroma transplantation
  - Biomaterials 32(28):6764-6772 (2011)
  Tissue engineering and transplantation of autogenic grafts have been widely investigated for solving problems on current allograft treatments (i.g., donor shortage and rejection). However, it is difficult to obtain an autogenic corneal stromal replacement that is composed of transparent, tough, and thick collagen constructs by current cell culture-based tissue engineering. Aim of this study is to develop transparent dermis for an autogenic corneal stroma transplantation. This study examined dehydration at 4–8 °C and carbodiimide cross-linking on cloudy rabbit dermis (approx. 1.8%–3.8% light transmittance at 550 nm) for dermis optical clearing. Transparency of dehydrated rabbit dermis was founded to be approx. 37.9%–41.4% at 550 nm. Additional cross-linking treatment on dehydrated dermis prevented from swelling and clouding in saline, and improved its transparency to be 56.9% at 550 nm. Rabbit corneal epithelium was found to regenerate on optically cleared dermis! in vitro. Furthermore, no abnormal biological response (i.e., inflammation, vascularization, and the barrier defect of epithelia) or no optical functional change on optically cleared dermis was observed during its 4-week autogenic transplantation into rabbit corneal stromal pocket.
• The balance between adipogenesis and osteogenesis in bone regeneration by platelet-rich plasma for age-related osteoporosis
  - Biomaterials 32(28):6773-6780 (2011)
  The aim of this study was to develop a new diagnostic and therapeutic approach for the treatment of osteoporosis. Previously, we demonstrated that intraosseous transplantation of platelet-rich plasma (PRP) treated-osteoblast-like cells into ovariectomized senescence-accelerated mice (OVX-SAMP8) prevented the development of osteoporosis. In continuation, we aimed to explore the complex etiology of osteoporosis using this platform. An inverse relationship between bone marrow adipogenesis and osteogenesis has been suggested in the development of osteoporosis but the underlying mechanisms remain poorly described. To address these issues, we used PRP to inhibit adipocyte differentiation by promoting osteoblastic differentiation in adipocytes. In addition, a positive correlation between an increase in bone marrow adipocytes and bone loss was established. We assessed this relationship using an osteoporotic animal disease model which consisted of young (for prevention) and old! (for treatment) OVX-SAMP8 mice. This animal model demonstrated that PRP treatment mainly exerted its action via promoting bone regeneration but also appeared to suppress adipogenesis within the marrow. The findings and methodology of this study could potentially be applied in the prevention and treatment of osteoporosis.
• Tumor eradication in rat glioma and bypass of immunosuppressive barriers using internal radiation with 188Re-lipid nanocapsules
  - Biomaterials 32(28):6781-6790 (2011)
  To date, glioblastoma treatments have only been palliative. In this context, locoregional drug delivery strategies, which allow for blood–brain barrier bypass and reduced systemic toxicity, are of major significance. Recent progress in nanotechnology has led to the development of colloidal carriers of radiopharmaceutics, such as lipid nanocapsules loaded with rhenium-188 (LNC188Re-SSS) that are implanted in the brain. In our study, we demonstrated that fractionated internal radiation using LNC188Re-SSS triggered remarkable survival responses in a rat orthotopic glioma model (cure rates of 83%). We also highlighted the importance of the radioactivity activity gradient obtained by combining a simple stereotactic injection (SI) with convection-enhanced delivery (CED).We assumed that the immune system played a role in the treatment's efficacy on account of the overproduction of peripheral cytokines, recruitment of immune cells to the tumor site, and memory response in ! long-term survivor animals. Hence, nanovectorized internal radiation therapy with activity gradients stimulating immune responses may represent a new and interesting alternative for the treatment of solid tumors such as glioblastomas.
• The influence of PEG chain length and targeting moiety on antibody-mediated delivery of nanoparticle vaccines to human dendritic cells
  - Biomaterials 32(28):6791-6803 (2011)
  Targeted delivery of nanoparticles (NPs) carrying vaccine components to dendritic cells (DCs) is a promising strategy to initiate antigen-specific immune responses. Improving the interactions between nanoparticle-carried ligands and receptors on DCs is a major challenge. These NPs are generally coated with poly(ethylene glycol) (PEG), to shield non-specific interactions, and antibodies, to facilitate specific delivery to DC surface receptors. We have devised a strategy to covalently link PEG molecules of various chain length (Mw 2000–20000 g/moL) to poly(lactic-co-)glycolic acid (PLGA) NP vaccines. We coated these NPs with various antibodies recognizing the DC-specific receptor DC-SIGN to study the effects of shielding and antibody type on antibody–receptor interactions. Chemical attachment of PEG to the particle surface was followed by detailed zeta potential, DLS and NMR studies, and analyzed by analytical chemistry. Increasing the PEG chain length increased part! icle size and polydispersity index and reduced the intracellular degradation rate of encapsulated antigens. Binding and uptake of NPs by human DCs was affected by both PEG chain length and antibody type. NPs coated with PEG-3000 had the optimal chain length for antibody–receptor interactions and induction of antigen-specific T-cell responses. Interestingly, clear differences were observed upon targeting distinct epitopes of the same receptor. Binding and uptake of NPs carrying antibodies recognizing the carbohydrate recognition domain of DC-SIGN was enhanced when compared to those carrying antibodies recognizing the receptor's neck region. In conclusion, our data show that PEG chains cannot be extended beyond a certain length for shielding purposes without compromising the efficacy of targeted delivery. Thereby, the implications of our findings are not limited to the future design of nanovaccines specifically targeted to DC-SIGN, but apply to the general design of targe! ted nanocarriers.
• Modulation of the immune-related gene responses to protect mice against Japanese encephalitis virus using the antimicrobial peptide, tilapia hepcidin 1-5
  - Biomaterials 32(28):6804-6814 (2011)
  Japanese encephalitis virus (JEV), a neurotropic flavivirus, is one of the major causes of acute encephalitis in humans. After infection, it is commonly associated with inflammatory reactions and neurological disease. There is still no effective antiviral drug available against Japanese encephalitis virus infection. Recently, a number of investigators found that antimicrobial peptide (AMPs) present a broad range of biological activities including antimicrobial and immunomodulatory activities. In this study, we found that an AMP, tilapia hepcidin (TH)1-5, caused no harm to either cells or test animals during the test course and could control JEV viral infection in BHK-21 cells. Mice co-injected with TH1-5/JEV and subsequently subjected to JEV re-challenge survived and behaved normally. The neuroprotective effects were associated with marked decreases in: (i) the viral load and viral replication within the brain, (ii) neuronal death, and (iii) secondary inflammation resu! lting from microglial activation. TH1-5 was also determined to enhance adaptive immunity by elevating levels of anti-JEV-neutralizing antibodies in the serum. The microarray data also showed that TH1-5 modulated Socs-6, interleukin (IL)-6, Toll-like receptor (TLR)-1, TLR-7, caspase-4, interferon (IFN)-β1, ATF-3, and several immune-responsive genes to protect mice against JEV infection. In addition, TH1-5 was confirmed to modulate the expressions of several proinflammatory and immune-responsive genes, such as IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, tumor necrosis factor (TNF)-α, IFN-γ and monocyte chemoattractant protein (MCP)-1 at both the transcriptional and translational levels in JEV-infected mice. In conclusion, our findings provide mechanistic insights into the actions of TH1-5 against JEV. Results from our in vivo and in vitro experiments clearly indicate that TH1-5 has antiviral, neuroprotective, anti-inflammatory, and immunomodulatory activities. Furthermore, TH1-5! successfully reduced the severity of disease induced by JEV. ! Our results point out that TH1-5 is a promising candidate for further development as an antiviral agent against JEV infection.
• Activation of innate immune responses in a pathogen-mimicking manner by amphiphilic polyanhydride nanoparticle adjuvants
  - Biomaterials 32(28):6815-6822 (2011)
  Techniques in materials design, immunophenotyping, and informatics can be valuable tools for using a molecular based approach to design vaccine adjuvants capable of inducing protective immunity that mimics a natural infection but without the toxic side effects. This work describes the molecular design of amphiphilic polyanhydride nanoparticles that activate antigen presenting cells in a pathogen-mimicking manner. Biodegradable polyanhydrides are well suited as vaccine delivery vehicles due to their adjuvant-like ability to: 1) enhance the immune response, 2) preserve protein structure, and 3) control protein release. The results of these studies indicate that amphiphilic nanoparticles possess pathogen-mimicking properties as evidenced by their ability to activate dendritic cells similarly to LPS. Specific molecular descriptors responsible for this behavior were identified using informatics analyses, including the number of backbone oxygen moieties, percent of hydroxyl ! end groups, polymer hydrophobicity, and number of alkyl ethers. Additional findings from this work suggest that the molecular characteristics mediating APC activation are not limited to hydrophobicity but vary in complexity (e.g., presentation of oxygen-rich molecular patterns to cells) and elicit unique patterns of cellular activation. The approach outlined herein demonstrates the ability to rationally design pathogen-mimicking nanoparticle adjuvants for use in next-generation vaccines against emerging and re-emerging diseases.
• Infusion pressure and pain during microneedle injection into skin of human subjects
  - Biomaterials 32(28):6823-6831 (2011)
  Infusion into skin using hollow microneedles offers an attractive alternative to hypodermic needle injections. However, the fluid mechanics and pain associated with injection into skin using a microneedle have not been studied in detail before. Here, we report on the effect of microneedle insertion depth into skin, partial needle retraction, fluid infusion flow rate and the co-administration of hyaluronidase on infusion pressure during microneedle-based saline infusion, as well as on associated pain in human subjects. Infusion of up to a few hundred microliters of fluid required pressures of a few hundred mmHg, caused little to no pain, and showed weak dependence on infusion parameters. Infusion of larger volumes up to 1 mL required pressures up to a few thousand mmHg, but still usually caused little pain. In general, injection of larger volumes of fluid required larger pressures and application of larger pressures caused more pain, although other experimental paramete! rs also played a significant role. Among the intradermal microneedle groups, microneedle length had little effect; microneedle retraction lowered infusion pressure but increased pain; lower flow rate reduced infusion pressure and kept pain low; and use of hyaluronidase also lowered infusion pressure and kept pain low. We conclude that microneedles offer a simple method to infuse fluid into the skin that can be carried out with little to no pain.
• Dual targeting effect of Angiopep-2-modified, DNA-loaded nanoparticles for glioma
  - Biomaterials 32(28):6832-6838 (2011)
  Gene therapy offers a promising cure of brain glioma and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to induce cell apoptosis of glioma selectively without affecting the normal cells. In this study, the nanoscopic high-branching dendrimer, polyamidoamine (PAMAM), was selected as the principal vector. Angiopep-2, which can target to the low-density lipoprotein receptor-related protein-1 (LRP1) expressed on BCECs and glial cells, was exploited as the targeting ligand to conjugate PAMAM via bifunctional polyethyleneglycol (PEG) and then complexed with the DNA, designated as PAMAM-PEG-Angiopep/DNA nanoparticles (NPs). The cellular uptake mechanism explored in glial cells showed that the DNA of PAMAM-PEG-Angiopep/DNA NPs entered into the nuclei through the endosome/lysosome pathway. The in vivo biodistribution of PAMAM-PEG-Angiopep/DNA NPs in the brain especially the tumor site was higher than that of PAMAM-PEG/DNA NPs and PAMAM/DNA NPs. Furtherm! ore, the TUNEL analysis showed a more wide-extended apoptosis in the PAMAM-PEG-Angiopep/pORF-TRAIL NPs treated group, compared to other groups including commercial Temozolomide-treated one. The median survival time of PAMAM-PEG-Angiopep/pORF-TRAIL NPs and Temozolomide treated on brain tumor-bearing mice was 61 and 49 days respectively, significantly longer than that of other groups. Besides, the NPs suggested low cytotoxicity after in vitro transfection. Thus, the results showed that Angiopep-2 could be exploited as a specific ligand to cross the BBB and targeted to glial cells, and PAMAM-PEG-Angiopep/DNA NPs can be a potential non-viral delivery system for gene therapy of glial tumor.
• Affinity hydrogels for controlled protein release using nucleic acid aptamers and complementary oligonucleotides
  - Biomaterials 32(28):6839-6849 (2011)
  Biomaterials for the precise control of protein release are important to the development of new strategies for treating human diseases. This study aimed to fundamentally understand aptamer–protein dissociation triggered by complementary oligonucleotides, and to apply this understanding to develop affinity hydrogels for controlled protein release. The results showed that the oligonucleotide tails of the aptamers played a critical role in inducing intermolecular hybridization and triggering aptamer–protein dissociation. In addition, the attachment of the oligonucleotide tails to the aptamers and the increase of hybridizing length could produce a synergistic effect on the dissociation of bound proteins from their aptamers. More importantly, pegylated complementary oligonucleotides could successfully trigger protein release from the aptamer-functionalized hydrogels at multiple time points. Based on these results, it is believed that aptamer-functionalized hydrogels and! complementary oligonucleotides hold great potential of controlling the release of protein drugs to treat human diseases.
• A strategy to establish a gene-activated matrix on titanium using gene vectors protected in a polylactide coating
  - Biomaterials 32(28):6850-6859 (2011)
  Bioactive implants are promising tools in regenerative medicine. Here we describe a versatile procedure for preparing a gene-activated matrix on titanium. Lyophilized copolymer-protected gene vectors (COPROGs) suspended in poly(d,l-lactide) (PDLLA) solutions in ethyl acetate were used to varnish solid surfaces. The gene-activated PDLLA surfaces were first established on polypropylene 96-well plates. Vector release from these surfaces in aqueous buffer, cell viability and gene transfer efficiency to NIH 3T3 fibroblasts was strongly dependent on the vector dose and its ratio to PDLLA film thickness. A detailed analysis of these relationships allowed establishing correlations which can be used to calculate suitable combinations of COPROGs and PDLLA yielding optimal gene transfer efficiency. This was verified with COPROG-activated PDLLA coatings on titanium foils. HEK 293 and mesenchymal stem cells expressed the BMP-2 gene comprised in the gene-activated surface in a manne! r that was consistent with the predicted dose–response and toxicity profiles found in NIH 3T3 cells. The systematic procedure presented here for identifying optimal coating compositions can be applied to any combination of vector type and coating material.
• Oral bioavailability, therapeutic efficacy and reactive oxygen species scavenging properties of coenzyme Q10-loaded polymeric nanoparticles
  - Biomaterials 32(28):6860-6874 (2011)
  The present investigation consists in the development and characterization of CoQ10 loaded PLGA nanoparticles (CoQ10-NPs, size < 100 nm) by a scalable emulsion-diffusion-evaporation method. Thermal and crystallinity analysis collectively corroborated that CoQ10 was entrapped into the NPs in amorphous form. The lyophilized CoQ10-NPs were found to be stable for a period of 6 months (at room temperature). In vitro cell culture studies indicated that CoQ10-NPs significantly quenched ROS with nearly 10 fold higher efficacy than free CoQ10. Further, positively charged CoQ10-NPs were localized in two major sources of ROS generation: mitochondria and lysosomes. CoQ10-NPs showed improved oral bioavailability (4.28 times) as compared to free CoQ10. Finally remarkably higher hepatoprotective and anti-inflammatory activity of CoQ10-NPs as compared to free CoQ10 was observed due to mitigation of deleterious effects associated with the generation of free radicals. As elucidated by l! ive noninvasive animal imaging, the higher anti-inflammatory activity of CoQ10-NPs can be attributed to significant accumulation of these NPs in the inflamed tissues.

Hot off the presses! Feb 01 Biomaterials

The Feb 01 issue of the Biomaterials is now up on Pubget (About Biomaterials): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• Editorial board
  - Biomaterials 33(4):IFC (2012)
  
• Microfluidic 3D bone tissue model for high-throughput evaluation of wound-healing and infection-preventing biomaterials
  - Biomaterials 33(4):999-1006 (2012)
  We report the use of a microfluidic 3D bone tissue model, as a high-throughput means of evaluating the efficacy of biomaterials aimed at accelerating orthopaedic implant-related wound-healing while preventing bacterial infection. As an example of such biomaterials, inkjet-printed micropatterns were prepared to contain antibiotic and biphasic calcium phosphate (BCP) nanoparticles dispersed in a poly(d,l-lactic-co-glycolic) acid matrix. The micropatterns were integrated with a microfluidic device consisting of eight culture chambers. The micropatterns immediately and completely killed Staphylococcus epidermidis upon inoculation, and enhanced the calcified extracellular matrix production of osteoblasts. Without antibiotic elution, bacteria rapidly proliferated to result in an acidic microenvironment which was detrimental to osteoblasts. These results were used to demonstrate the tissue model's potential in: (i) significantly reducing the number of biomaterial samples an! d culture experiments required to assess in vitro efficacy for wound-healing and infection prevention and (ii) in situ monitoring of dynamic interactions of biomaterials with bacteria as wells as with tissue cells simultaneously.
• Glycosaminoglycan (GAG) binding surfaces for characterizing GAG-protein interactions
  - Biomaterials 33(4):1007-1016 (2012)
  Glycosaminoglycans play an important role in tissue organisation through interactions with a diverse range of proteins, growth factors and other chemokines. In this report, we demonstrate the GAG-binding 'fingerprint' of two important GAG-binding proteins – osteoprotogerin and TIMP-3. The technique uses a straightforward method for attaching GAGs to assay surfaces in a non-covalent manner using plasma polymerization that leaves the adsorbed GAG able to participate in subsequent ligand binding. We show that OPG and TIMP-3 bind preferentially to different GAGs in a simple ELISA and that this binding does not correlate directly with simple GAG properties such as degree of sulfation. The methods outlined in this report can be easily applied to tissue engineering scaffolds in order to exploit the potential of surface-bound GAGs in influencing the structure of engineered tissues.
• Impact of processing parameters on the haemocompatibility of Bombyx mori silk films
  - Biomaterials 33(4):1017-1023 (2012)
  Silk has traditionally been used for surgical sutures due to its lasting strength and durability; however, the use of purified silk proteins as a scaffold material for vascular tissue engineering goes beyond traditional use and requires application-orientated biocompatibility testing. For this study, a library of Bombyx mori silk films was generated and exposed to various solvents and treatment conditions to reflect current silk processing techniques. The films, along with clinically relevant reference materials, were exposed to human whole blood to determine silk blood compatibility. All substrates showed an initial inflammatory response comparable to polylactide-co-glycolide (PLGA), and a low to moderate haemostasis response similar to polytetrafluoroethylene (PTFE) substrates. In particular, samples that were water annealed at 25 °C for 6 h demonstrated the best blood compatibility based on haemostasis parameters (e.g. platelet decay, thrombin-antithrombin complex,! platelet factor 4, granulocytes-platelet conjugates) and inflammatory parameters (e.g. C3b, C5a, CD11b, surface-associated leukocytes). Multiple factors such as treatment temperature and solvent influenced the biological response, though no single physical parameter such as β-sheet content, isoelectric point or contact angle accurately predicted blood compatibility. These findings, when combined with prior in vivo data on silk, support a viable future for silk-based vascular grafts.
• Three-dimensional paper-based electrochemiluminescence immunodevice for multiplexed measurement of biomarkers and point-of-care testing
  - Biomaterials 33(4):1024-1031 (2012)
  In this work, electrochemiluminescence (ECL) immunoassay was introduced into the recently proposed microfluidic paper-based analytical device (ﾎｼPADs) based on directly screen-printed electrodes on paper for the very first time. The screen-printed paper-electrodes will be more important for further development of this paper-based ECL device in simple, low-cost and disposable application than commercialized ones. To further perform high-performance, high-throughput, simple and inexpensive ECL immunoassay on ﾎｼPAD for point-of-care testing, a wax-patterned three-dimensional (3D) paper-based ECL device was demonstrated for the very first time. In this 3D paper-based ECL device, eight carbon working electrodes including their conductive pads were screen-printed on a piece of square paper and shared the same Ag/AgCl reference and carbon counter electrodes on another piece of square paper after stacking. Using typical tris-(bipyridine)-ruthenium (竇｡) - tri-n-prop! ylamine ECL system, the application test of this 3D paper-based ECL device was performed through the diagnosis of four tumor markers in real clinical serum samples. With the aid of a facile device-holder and a section-switch assembled on the analyzer, eight working electrodes were sequentially placed into the circuit to trigger the ECL reaction in the sweeping range from 0.5 to 1.1 V at room temperature. In addition, this 3D paper-based ECL device can be easily integrated and combined with the recently emerging paper electronics to further develop simple, sensitive, low-cost, disposable and portable ﾎｼPAD for point-of-care testing, public health and environmental monitoring in remote regions, developing or developed countries.
• Modulation of the migration and differentiation potential of adult bone marrow stromal stem cells by nitric oxide
  - Biomaterials 33(4):1032-1043 (2012)
  Nitric oxide (NO) is a diffusible free radical, which serves as a pluripotent intracellular messenger in numerous cell systems. NO has been demonstrated to regulate actin dependent cellular functions and functions as a putative inductive agent in directing stem cells differentiation. In this study, we investigated the effect of exogenous NO on the kinetics of movement and morphological changes in adult bone marrow stromal cells (BMSCs) in a wound healing model of cellular migration. Cellular migration and morphological changes were determined by measurement of changes in the area and fractal dimension of BMSCs monolayer as a function of time in the presence of an NO donor (S-Nitroso-N-Acetyl-D,l-Penicillamine, SNAP) compared to untreated BMSCs. Response of the BMSCs' actin cytoskeleton and desmin to NO was assessed by determining changes in their integrated optical density (IOD) and fractal dimension at 24 h and 7 days. NO suppressed BMSCs' migration accompanied by! a reduction in cell size, with maintenance of their stellate to polygonal morphology. In response to NO, the actin cytoskeleton expressed an increase in randomness but maintained a constant amount of F-actin relative to the cell size. The presence of NO also induced an increase in randomly organized cytoplasmic desmin. These data suggest that NO has an apparent inductive effect on adult BMSCs and is capable of initiating phenotypic change at the gross cellular, cytoskeletal and molecular levels. It is apparent, however, that additional factors or conditions are required to further drive the differentiation of adult BMSCs into specific phenotypes, such as cardiomyocytes.
• The mechanical stimulation of cells in 3D culture within a self-assembling peptide hydrogel
  - Biomaterials 33(4):1044-1051 (2012)
  The aim of this present study was to provide a scaffold as a tool for the investigation of the effect of mechanical stimulation on three-dimensionally cultured cells. For this purpose, we developed an artificial self-assembling peptide (SPG-178) hydrogel scaffold. The structural properties of the SPG-178 peptide were confirmed by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and transmission electron microscopy (TEM). The mechanical properties of the SPG-178 hydrogel were studied using rheology measurements. The SPG-178 peptide was able to form a stable, transparent hydrogel in a neutral pH environment. In the SPG-178 hydrogel, mouse skeletal muscle cells proliferated successfully (increased by 12.4 ± 1.5 times during 8 days of incubation; mean ± SEM). When the scaffold was statically stretched, a rapid phosphorylation of ERK was observed (increased by 2.8 ± 0.2 times; mean ± SEM). These results demonstrated that the developed self-! assembling peptide gel is non-cytotoxic and is a suitable tool for the investigation of the effect of mechanical stimulation on three-dimensional cell culture.
• Influence of perfusion and compression on the proliferation and differentiation of bone mesenchymal stromal cells seeded on polyurethane scaffolds
  - Biomaterials 33(4):1052-1064 (2012)
  In the present study, a porous meniscal-shaped scaffold consisting of polyurethane (PU)-based 1, 4-butanediisocyanate (BDI), which provided a 3-D culture condition for human bone mesenchymal stromal cells (hBMSC) was employed. A bioreactor was utilized to produce perfusion and mechanical stimulations. The viability, proliferation and fibro-cartilaginous differentiation of the hBMSC cultured on the PU-based meniscal scaffold were investigated during the perfusion and mechanical stimulation process. In addition, the mechanical properties of the cell-laden scaffolds were examined as well. Our finding indicated that the perfusion (10 ml/min) and on-off cyclic compressions mechanical stimulation (10% strain, 0.5 Hz, 4 times/day, 2 h/time with 4 h of rest thereafter) maintained the viability and promoted the proliferation of hBMSC over 2 weeks. The on-off cyclic compression caused a 1.85 fold increase in equilibrium modulus. Meanwhile, type I procollagen produced by hBMSC wa! s increased for 3.02-fold after 2 weeks culture. On the other hand, the irrigating medium enhanced the synthesis of type III procollagen for 2.24-fold after 2 weeks. Tensile modulus was elevated for 2.02-fold in perfusion group after 1 week, which was decreased after 2 weeks unexpectedly. Our study suggests that the perfusion and on-off compression are promising to enhance the functional properties of the hBMSC-laden PU-based meniscal scaffold.
• Three-dimensional cancer-bone metastasis model using ex-vivo co-cultures of live calvarial bones and cancer cells
  - Biomaterials 33(4):1065-1078 (2012)
  One of the major limitations of studying cancer-bone metastasis has been the lack of an appropriate ex-vivo model which can be used under defined conditions that simulates closely the in vivo live bone microenvironment in response to cancer-bone interactions. We have developed and utilized a three-dimensional (3D) cancer-bone metastasis model using free-floating live mouse calvarial bone organs in the presence of cancer cells in a roller tube system. In such co-cultures under hypoxia and a specifically defined bone remodeling stage, viz., resorption system, cancer cells showed a remarkable affinity and specificity for the "endosteal side" of the bone where they colonize and proliferate. This was concurrent with differentiation of resident stem/progenitor cells to osteoclasts and bone resorption. In contrast, under bone formation conditions this model revealed different pathophysiology where the breast cancer cells continued to induce osteoclastic bone resorption wh! ereas prostate cancer cells led to osteoblastic bone formation. The current 3D model was used to demonstrate its application to studies involving chemical and biochemical perturbations in the absence and presence of cancer cells and cellular responses. We describe proof-of-principle with examples of the broad versatility and multi-faceted application of this model that adds another dimension to the ongoing studies in the cancer-bone metastasis arena.
• Multifunctional nanoprobes for upconversion fluorescence, MR and CT trimodal imaging
  - Biomaterials 33(4):1079-1089 (2012)
  Early diagnosis probes that combine fluorescence, X-ray computed tomography (CT) and magnetic resonance (MR) imagings are anticipated to give three dimensional (3D) details of tissues and cells of high resolution and sensitivity. However, how to combine these three modalities together within a sub-50 nm sized structure is technically challenging. Here we report a trimodal imaging probe of PEGylated NaY/GdF4: Yb, Er, Tm @SiO2-Au@PEG5000 nanopaticles of uniform size of less than 50 nm. The as-designed nanoprobes showed (1) strong emissions ranging from the visible (Vis) to near infrared (NIR) for fluorescent imaging, (2) T1-weighted MRI by shorting T1 relaxation time and (3) enhanced HU value as a CT contrast agent. The structure was optimized based on a comprehensive investigation on the influence of the distance between the NaY/GdF4: Yb, Er, Tm core and Au nanoparticles (NPs) at the surface. The potential of trimodal imaging for cancerous cells and lesions was further ! demonstrated both in vitro and in vivo.
• Simultaneous electrochemical detection of multiple analytes based on dual signal amplification of single-walled carbon nanotubes and multi-labeled graphene sheets
  - Biomaterials 33(4):1090-1096 (2012)
  In this work, a sandwich-type electrochemical aptasensor for simultaneous sensitive detection of platelet-derived growth factor (PDGF) and thrombin is fabricated. Reduced graphene oxide sheets (rGS) are used as matrices to immobilize the redox probes, which are subsequently coated with platinum nanoparticles (PtNPs) to form the PtNPs-redox probes-rGS nanocomposites. With the employment of the as prepared nanocomposites, a signal amplification strategy was described based on bienzyme (glucose oxidase and horseradish peroxidase) modified PtNPs-redox probes-rGS nanocomposites as the tracer labels for secondary aptamers (Apt II) through sandwiched assay. Gold nanoparticles functionalized single-walled carbon nanotubes (AuNPs@SWCNTs) as the biosensor platform enhance the surface area to capture a large amount of primary aptamers (Apt I), thus amplifying the detection response. The experiment results show that the multi-labeled PtNPs-redox probes-rGS nanocomposites display s! atisfying electrochemical redox activity and highly electrocatalytic activity of PtNPs and bienzyme, which exhibit high sensitivity for detection of proteins. The linear range of PDGF is 0.01–35 nM with a detection limit of 8 pM, while the linear ranges from 0.02 to 45 nM and a detection limit of 11 pM for thrombin are obtained.
• Decorated graphene sheets for label-free DNA impedance biosensing
  - Biomaterials 33(4):1097-1106 (2012)
  An efficient DNA impedance biosensing platform is constructed, in which positively charged N,N-bis-(1-aminopropyl-3-propylimidazol salt)-3,4,9,10-perylene tetracarboxylic acid diimide (PDI) is anchored to graphene sheets. The π–π stacking and electronic interactions are elucidated by the distinct absorption features in UV–vis spectra and by quenching perylene fluorescence in contact with graphene. The rational design and tailoring of graphene surface invest it with desired properties (dispersive, structural, photoelectrical and conductive, etc.) and boost its application. Electrostatic interaction between PDI's positively charged imidazole rings and negatively charged phosphate backbones of single-stranded DNA (ssDNA) facilitates ssDNA immobilization. This manner is different from these mainly based on the attraction between the rings in DNA bases and the hexagonal cells of graphene, which is disturbed after hybridization and causes the leaving of formed double! -stranded DNA from graphene surface. The electrostatic ssDNA grafting occupies phosphate backbones and particularly leaves the bases available for efficient hybridization. DNA immobilization and hybridization lead to PDI/graphene interfacial property changes, which are monitored by electrochemical impedance spectroscopy and adopted as the analytical signal. The conserved sequence of the pol gene of human immunodeficiency virus 1 is satisfactorily detected via this PDI/graphene platform and shows high reproducibility, selectivity.
• PEGylated dendrimer-entrapped gold nanoparticles for in vivo blood pool and tumor imaging by computed tomography
  - Biomaterials 33(4):1107-1119 (2012)
  We report the synthesis and characterization of dendrimer-entrapped gold nanoparticles (Au DENPs) modified by polyethylene glycol (PEG) with enhanced biocompatibility for computed tomography (CT) imaging applications. In this study, amine-terminated poly(amidoamine) dendrimers of generation 5 (G5.NH2) modified by PEG monomethyl ether (G5.NH2-mPEG20) were used as templates to synthesize Au DENPs, followed by acetylation of the remaining dendrimer terminal amines to generate PEGylated Au DENPs. The partial PEGylation modification of dendrimer terminal amines allows high loading of Au within the dendrimer interior, and consequently by simply varying the Au salt/dendrimer molar ratio, the size of the PEGylated Au DENPs can be controlled at a range of 2–4 nm with a narrow size distribution. The formed PEGylated Au DENPs are water-dispersible, stable in a pH range of 5–8 and a temperature range of 0–50 °C, and non-cytotoxic at a concentration as high as 100 μm. X-ray! absorption coefficient measurements show that the attenuation intensity of the PEGylated Au DENPs is much higher than that of Omnipaque with iodine concentration similar to Au. With the sufficiently long half-decay time demonstrated by pharmacokinetics studies, the PEGylated Au DENPs enabled not only X-ray CT blood pool imaging of mice and rats after intravenous injection of the particles, but also effective CT imaging of a xenograft tumor model in nude mice. These findings suggest that the designed PEGylated Au DENPs can be used as a promising contrast agent with enhanced biocompatibility for CT imaging of various biological systems, especially in cancer diagnosis.
• The CD44/integrins interplay and the significance of receptor binding and re-presentation in the uptake of RGD-functionalized hyaluronic acid
  - Biomaterials 33(4):1120-1134 (2012)
  We have studied the interplay between two endocytic receptors for a carrier structure bearing two complementary ligands. Hyaluronic acid (HA; three different molecular weights) was functionalized with an RGD-containing peptide; this ancillary ligand allows the macromolecule to bind to 留v integrins in addition to the classical HA internalization receptor (CD44). The uptake of HA-RGD and of native HA was assessed in a phagocytic cell model (J774.2 murine macrophages), studying the kinetics of internalization and its mechanistic details. Indications of a synergic binding to integrins and CD44 emerged for HA-RGD; possibly, a first binding to integrins allows for a pre-concentration of the macromolecule on the cell surface, which is then followed by its binding to CD44. The endocytic mechanism and kinetics appeared then dominated by CD44, which has a much slower turnover than integrins. In this study we have demonstrated that the knowledge of the rate-determining steps of! the internalization of a carrier is necessary for assessing its performance. In this case, the presence of multiple ligands on a carrier was beneficial in some respect (e.g. in improved binding/targeting), but may not be sufficient to overcome penetration barriers that arise from slow receptor re-presentation.
• Intracellular pathways and nuclear localization signal peptide-mediated gene transfection by cationic polymeric nanovectors
  - Biomaterials 33(4):1135-1145 (2012)
  Polyethylenimine (PEI) - based polymers are promising cationic nanovectors. A good understanding of the mechanism by which cationic polymers/DNA complexes are internalized and delivered to nuclei helps to identify which transport steps may be manipulated in order to improve the transfection efficiency. In this work, cell internalization and trafficking of PEI-CyD (PC) composed of 硫-cyclodextrin (硫-CyD) and polyethylenimine (PEI, Mw 600) are studied. The results show that the PC transfected DNA is internalized by binding membrane-associated proteoglycans. The endocytic pathway of the PC particles is caveolae- and clathrin-dependent with both pathways converging to the lysosome. The intracellular fate of the PC provides visual evidence that it can escape from the lysosome. Lysosomal inhibition with chloroquine has no effect on PC mediated transfection implying that blocking the lysosomal traffic does not improve transfection. To improve the nuclear delivery of PC tra! nsfected DNA, nuclear localization signal (NLS) peptides are chosen to conjugate and combine with the PC. Compared to PC/pDNA, PC-NLS/pDNA, and PC/pDNA/NLS can effectively improve gene transfection in dividing and non-dividing cells.
• Highly dynamic biodegradable micelles capable of lysing Gram-positive and Gram-negative bacterial membrane
  - Biomaterials 33(4):1146-1153 (2012)
  The development of biodegradable antimicrobial polymers adds to the toolbox of attractive antimicrobial agents against antibiotic-resistant microbes. To this end, the potential of polycarbonate polymers as such materials were explored. A series of random polycarbonate polymers consisting of monomers MTC-OEt and MTC-CH2CH3Cl were designed and synthesized using metal-free organocatalytic ring-opening polymerization. Random polycarbonate polymers self-assembled in solution but appeared highly dynamic; such behaviors are desirable as ready disassembly of polymers at the microbial membrane facilitates membrane disruption. Their activities against clinically relevant Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (E.coli and Pseudomonas aeruginosa) revealed that the hydrophobic-hydrophilic composition balance in polymers are important to render antimicrobial potency. Scanning electron microscopy (SEM) studies indicated microbial cell surface damage after tr! eatment with polymers, and confocal microscopy studies also showed entry of FITC-dextran dye in Escherichia coli as a result of membrane disruption. On the other hand, the polymers exhibited minimal toxicity against red blood cells in hemolysis tests. Therefore, these random polycarbonate polymers are promising antimicrobial agents against both Gram-positive and Gram-negative bacteria for various biomedical applications.
• Sustained local delivery of siRNA from an injectable scaffold
  - Biomaterials 33(4):1154-1161 (2012)
  Controlled gene silencing technologies have significant, unrealized potential for use in tissue regeneration applications. The design described herein provides a means to package and protect siRNA within pH-responsive, endosomolytic micellar nanoparticles (si-NPs) that can be incorporated into nontoxic, biodegradable, and injectable polyurethane (PUR) tissue scaffolds. The si-NPs were homogeneously incorporated throughout the porous PUR scaffolds, and they were shown to be released via a diffusion-based mechanism for over three weeks. The siRNA-loaded micelles were larger but retained nanoparticulate morphology of approximately 100 nm diameter following incorporation into and release from the scaffolds. PUR scaffold releasate collected in vitro in PBS at 37 °C for 1–4 days was able to achieve dose-dependent siRNA-mediated silencing with approximately 50% silencing achieved of the model gene GAPDH in NIH3T3 mouse fibroblasts. This promising platform technology provid! es both a research tool capable of probing the effects of local gene silencing and a potentially high-impact therapeutic approach for sustained, local silencing of deleterious genes within tissue defects.
• The therapeutic efficacy of camptothecin-encapsulated supramolecular nanoparticles
  - Biomaterials 33(4):1162-1169 (2012)
  Nanomaterials have been increasingly employed as drug(s)-incorporated vectors for drug delivery due to their potential of maximizing therapeutic efficacy while minimizing systemic side effects. However, there have been two main challenges for these vectors: (i) the existing synthetic approaches are cumbersome and incapable of achieving precise control of their structural properties, which will affect their biodistribution and therapeutic efficacies, and (ii) lack of an early checkpoint to quickly predict which drug(s)-incorporated vectors exhibit optimal therapeutic outcomes. In this work, we utilized a new rational developmental approach to rapidly screen nanoparticle (NP)-based cancer therapeutic agents containing a built-in companion diagnostic utility for optimal therapeutic efficacy. The approach leverages the advantages of a self-assembly synthetic method for preparation of two different sizes of drug-incorporated supramolecular nanoparticles (SNPs), and a positr! on emission tomography (PET) imaging-based biodistribution study to quickly evaluate the accumulation of SNPs at a tumor site in vivo and select the favorable SNPs for in vivo therapeutic study. Finally, the enhanced in vivo anti-tumor efficacy of the selected SNPs was validated by tumor reduction/inhibition studies. We foresee our rational developmental approach providing a general strategy in the search of optimal therapeutic agents among the diversity of NP-based therapeutic agents.
• Multifunctional nanocarrier mediated co-delivery of doxorubicin and siRNA for synergistic enhancement of glioma apoptosis in rat
  - Biomaterials 33(4):1170-1179 (2012)
  As the most fatal malignancy in brain, glioma cannot be effectively treated with the conventional chemotherapy and thus techniques which may improve the chemotherapeutic effect are of great importance in clinical glioma treatment. Based on the folate-targeted multifunctional nanocarrier developed in our lab, effective co-delivery of DOX and siRNA into rat C6 glioma cells over-expressing folate receptors was achieved. Although cell apoptosis was initiated even at low DOX doses such as 0.5 μg/mL in the DOX-alone treatment mediated by the folate-targeted nanocarrier, anti-apoptotic response in C6 cells was activated as well, as revealed by molecular biological investigations. Delivery of BCL-2 siRNA using the folate-targeted nanocarrier can effectively suppress the anti-apoptotic response and sensitized C6 cells to DOX treatment both in vitro and in vivo. In particular, animal studies using the in situ rat C6 glioma model showed that the folate-targeted co-delivery of ! BCL-2 siRNA and DOX caused not only an obvious down-regulation of the anti-apoptotic BCL-2 gene but also a remarkable up-regulation of the pro-apoptotic Bax gene, resulting in the significantly elevated level of caspase-3 activation and remarkable cell apoptosis in tumor tissues. Our results strongly demonstrated the synergistic effect of siRNA and DOX in inducing glioma C6 cell apoptosis, upon which an excellent therapeutic effect was achieved using the folate-targeted co-delivery strategy as indicated by the effective tumor growth inhibition and prolonged rat survival time in the animal test.
• Gold nanoparticles functionalized with therapeutic and targeted peptides for cancer treatment
  - Biomaterials 33(4):1180-1189 (2012)
  Functionalization of nanostructures such as gold nanoparticles (AuNPs) with different biological molecules has many applications in biomedical imaging, clinical diagnosis and therapy. Researchers mostly employed AuNPs larger than 10 nm for different biological and medicinal applications in previous studies. Herein, we synthesized a novel small (2 nm) AuNPs, which were functionalized with the therapeutic peptide, PMI (p12), and a targeted peptide, CRGDK for selective binding to neuropilin-1(Nrp-1) receptors which overexpressed on the cancer cells and regulated the process of membrane receptor-mediated internalization. It was found that CRGDK peptides increased intracellular uptake of AuNPs compared to other surface conjugations quantified by ICP-MS. Interestingly, CRGDK functionalized AuNPs resulted in maximal binding interaction between the CRGDK peptide and targeted Nrp-1 receptor overexpressed on MDA-MB-321 cell surface, which improved the delivery of therapeutic P12! peptide inside targeted cells. Au@p12 + CRGDK nanoparticles indicated with highly effective cancer treatment by increasing p53 expression upregulated with intracellular enhanced p12 therapeutic peptide. These results have implications to design and functionalize different molecules onto AuNPs surfaces to make hybrid model system for selective target binding as well as therapeutic effects for cancer treatment.
• Polyethylene glycol-conjugated hyaluronic acid-ceramide self-assembled nanoparticles for targeted delivery of doxorubicin
  - Biomaterials 33(4):1190-1200 (2012)
  Polyethylene glycol (PEG)-conjugated hyaluronic acid-ceramide (HACE) was synthesized for the preparation of doxorubicin (DOX)-loaded HACE-PEG-based nanoparticles, 160 nm in mean diameter with a negative surface charge. Greater uptake of DOX from these HACE-PEG-based nanoparticles was observed in the CD44 receptor highly expressed SCC7 cell line, compared to results from the CD44-negative cell line, NIH3T3. A strong fluorescent signal was detected in the tumor region upon intravenous injection of cyanine 5.5-labeled nanoparticles into the SCC7 tumor xenograft mice; the extended circulation time of the HACE-PEG-based nanoparticle was also observed. Pharmacokinetic study in rats showed a 73.0% reduction of the in vivo clearance of DOX compared to the control group. The antitumor efficacy of the DOX-loaded HACE-PEG-based nanoparticles was also verified in a tumor xenograft mouse model. DOX was efficiently delivered to the tumor site by active targeting via HA and CD44 rece! ptor interaction and by passive targeting due to its small mean diameter (<200 nm). Moreover, PEGylation resulted in prolonged nanoparticle circulation and reduced DOX clearance rate in an in vivo model. These results therefore indicate that PEGylated HACE nanoparticles represent a promising anticancer drug delivery system for cancer diagnosis and therapy.

Hot off the presses! Oct 01 Biomaterials

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Latest Articles Include:

• Editorial board
  - Biomaterials 30(30):IFC (2009)
  
• On the nature of biomaterials
  Williams DF - Biomaterials 30(30):5897-5909 (2009)
  The situations in which biomaterials are currently used are vastly different to those of just a decade ago. Although implantable medical devices are still immensely important, medical technologies now encompass a range of drug and gene delivery systems, tissue engineering and cell therapies, organ printing and cell patterning, nanotechnology based imaging and diagnostic systems and microelectronic devices. These technologies still encompass metals, ceramics and synthetic polymers, but also biopolymers, self assembled systems, nanoparticles, carbon nanotubes and quantum dots. These changes imply that our original concepts of biomaterials and our expectations of their performance also have to change. This Leading Opinion Paper addresses these issues. It concludes that many substances which hitherto we may not have thought of as biomaterials should now be considered as such so that, alongside the traditional structural biomaterials, we have substances that have been engin! eered to perform functions within health care where their performance is directly controlled by interactions with tissues and tissue components. These include engineered tissues, cells, organs and even viruses. This essay develops the arguments for a radically different definition of a biomaterial.
• Scaffold-free vascular tissue engineering using bioprinting
  Norotte C Marga FS Niklason LE Forgacs G - Biomaterials 30(30):5910-5917 (2009)
  Current limitations of exogenous scaffolds or extracellular matrix based materials have underlined the need for alternative tissue-engineering solutions. Scaffolds may elicit adverse host responses and interfere with direct cell–cell interaction, as well as assembly and alignment of cell-produced ECM. Thus, fabrication techniques for production of scaffold-free engineered tissue constructs have recently emerged. Here we report on a fully biological self-assembly approach, which we implement through a rapid prototyping bioprinting method for scaffold-free small diameter vascular reconstruction. Various vascular cell types, including smooth muscle cells and fibroblasts, were aggregated into discrete units, either multicellular spheroids or cylinders of controllable diameter (300–500 μm). These were printed layer-by-layer concomitantly with agarose rods, used here as a molding template. The post-printing fusion of the discrete units resulted in single- and double-lay! ered small diameter vascular tubes (OD ranging from 0.9 to 2.5 mm). A unique aspect of the method is the ability to engineer vessels of distinct shapes and hierarchical trees that combine tubes of distinct diameters. The technique is quick and easily scalable.
• The use of glandular-derived stem cells to improve vascularization in scaffold-mediated dermal regeneration
  Egaña JT Danner S Kremer M Rapoport DH Lohmeyer JR Dye JF Hopfner U Lavandero S Kruse C Machens HG - Biomaterials 30(30):5918-5926 (2009)
  Clinical success in tissue regeneration requires improvements in vascularization capacity of scaffolds. Several efforts have been made in this field including cellular and acellular technologies. In this work we combined the use of stem cells derived from pancreas or submandibular glands expressing green fluorescent protein (GFP+) with a commercially available scaffold for dermal regeneration. Cells were isolated, characterized and seeded in a scaffold for dermal regeneration. Scaffolds containing cells were used to induce dermal regeneration in a full skin defect model. After 3 weeks of in vivo regeneration, tissues were harvested and vascularization was analyzed. Results showed that gland-derived stem cells displayed stem cell features and presented multipotential differentiation capacity because they were able to differentiate in cell types representing the 3 different germ layers. After seeding, cells were homogeneously distributed and formed focal adhesions with t! he scaffold. Metabolic assays showed that cells can be cultured for at least 3 weeks in the scaffold. In vivo, the presence of pancreatic or submandibular stem cells significantly enhanced the vascularization compared to empty scaffolds. Presence of gland-derived stem cells in the regenerating tissue was confirmed by the detection of GFP expression in the wound area. In order to explore the possible mechanisms behind the improvement in vascular regeneration, in vitro experiments were performed, showing that gland-derived stem cells could contribute by angiogenic and vasculogenic mechanisms to this process. Our results suggest that the combined use of stem cells derived from glands and scaffold for dermal regeneration could be a rational alternative to improve vascularization in scaffold-mediated dermal regeneration.
• Laminar-flow immediate-overlay hepatocyte sandwich perfusion system for drug hepatotoxicity testing
  Xia L Ng S Han R Tuo X Xiao G Leo HL Cheng T Yu H - Biomaterials 30(30):5927-5936 (2009)
  Drug hepatotoxicity testing requires in vitro hepatocyte culture to maintain the long-term and stable liver specific functions. We developed a drug testing platform based on laminar-flow immediate-overlay hepatocyte sandwich perfusion culture. The immediate-overlay sandwich (collagen-coated porous polymeric membrane as top overlay) protects the cells and integrity of the top collagen matrix from the impact of flow. A bioreactor was designed that allowed proper control of shear stress and mass transfer. The culture parameters such as the optimal perfusion initiation time and flow rate were systematically and mechanistically determined. The optimized system could re-establish hepatocyte polarity to support biliary excretion and to maintain other liver specific functions, such as the biotransformation enzyme activities, for two weeks that extended the usable in vitro hepatocyte-based drug testing window. When the perfusion cultured hepatocytes from days 7 or 14 were used ! for drug testing, the APAP-induced hepatotoxicity measurements were more sensitive and consistent over time than the static culture control, enabling further exploitations in large-scale drug testing applications.
• Enzymatically fabricated and degradable microcapsules for production of multicellular spheroids with well-defined diameters of less than 150 μm
  Sakai S Ito S Ogushi Y Hashimoto I Hosoda N Sawae Y Kawakami K - Biomaterials 30(30):5937-5942 (2009)
  Microcapsules with a single, spherical hollow core less than 150 μm in diameter were developed to obtain multicellular spheroids with well-defined sizes of less than 150 μm in diameter. An aqueous solution of phenolic hydroxyl derivative of carboxymethylcellulose (CMC-Ph) containing human hepatoma cell line (HepG2) cells and horse radish peroxidase (HRP) was injected into a coflowing stream of liquid paraffin, containing H2O2, resulting in cell-enclosing CMC-Ph microparticles, 135 μm in diameter, via a peroxidase-catalyzed crosslinking reaction. The CMC-Ph microparticles were then coated with a phenolic hydroxyl derivative of alginate (Alg-Ph) gel membrane several dozen micrometers in thickness, crosslinked via the same enzymatic reaction process, followed by further crosslinking between the carboxyl groups of alginate by Sr2+. A hollow core structure was achieved by immersing the resultant microcapsules in a medium containing cellulase, which degrades the enclosed ! CMC-Ph microparticles. The HepG2 cells in the microcapsules then grew and completely filled the hollow core. Multicellular spheroids the same size as the CMC-Ph microparticles, with living cells at their outer surface, were collected within 1 min by soaking them in a medium containing alginate lyase to degrade the Alg-Ph gel microcapsule membrane.
• Bioengineering of a functional sheet of islet cells for the treatment of diabetes mellitus
  Shimizu H Ohashi K Utoh R Ise K Gotoh M Yamato M Okano T - Biomaterials 30(30):5943-5949 (2009)
  The present study was designed to establish a novel tissue engineering approach for diabetes mellitus (DM) by fabricating a tissue sheet composed of pancreatic islet cells for in vivo transplantation. Pancreatic islet cell suspensions were obtained from Lewis rats, and plated onto temperature-responsive culture dishes coated with extracellular matrix (ECM) proteins. After the cells reached confluency, islet cells cultured on laminin-5 coated dishes were successfully harvested as a uniformly spread tissue sheet by lowering the culture temperature to 20 °C for 20 min. The functional activity of the islet cell sheets was confirmed by histological examination and Insulin secretion assay prior to in vivo transplantation. Histological examination revealed that the harvested islet cell sheet was comprised of insulin- (76%) and glucagon- (19%) positive cells, respectively. In vivo functionality of the islet cell sheet was maintained even 7 days after transplantation into the ! subcutaneous space of Lewis rats. The present study describes an approach to generate a functional sheet of pancreatic islet cells on laminin-5 coated temperature-responsive dishes, which can be subsequently transplanted in vivo. This study serves as the foundation for the creation of a novel cell-based therapy for DM to provide patients an alternative method.
• The growth and differentiation of mesenchymal stem and progenitor cells cultured on aligned collagen matrices
  Lanfer B Seib FP Freudenberg U Stamov D Bley T Bornhäuser M Werner C - Biomaterials 30(30):5950-5958 (2009)
  Cell-matrix interactions are paramount for the successful repair and regeneration of damaged and diseased tissue. Since many tissues have an anisotropic architecture, it has been proposed that aligned extracellular matrix (ECM) structures in particular could guide and support the differentiation of resident mesenchymal stem and progenitor cells (MSCs). We therefore created aligned collagen type I structures using a microfluidic set-up with the aim to assess their impact on MSC growth and differentiation. In addition, we refined our aligned collagen matrices by incorporating the glycosaminoglycan (GAG) heparin to demonstrate the versatility of the applied methodology to study multiple ECM components in a single system. Our reconstituted, aligned ECM structures maintained and allowed multilineage (osteogenic/adipogenic/chondrogenic) differentiation of MSCs. Most noticeable was the observation that during osteogenesis, aligned collagen substrates choreographed ordered mat! rix mineralization. Likewise, myotube assembly of C2C12 cells was profoundly influenced by aligned topographic features resulting in enhanced myotube organization and length. Our results shed light on the regulation of MSCs through directional ECM structures and demonstrate the versatility of these cell culture platforms for guiding the morphogenesis of tissue types with anisotropic structures.
• Rapid three-dimensional quantification of VEGF-induced scaffold neovascularisation by microcomputed tomography
  Schmidt C Bezuidenhout D Beck M Van der Merwe E Zilla P Davies N - Biomaterials 30(30):5959-5968 (2009)
  Microcomputed tomography (micro-CT) is increasingly being used to analyze the three-dimensional structure and architecture of microvascular networks. Therefore we have evaluated a micro-CT analysis of VEGF-induced vessel ingrowth into a porous polyurethane scaffold through comparison with analyses by CD31 immunohistochemistry, vascular perfusion by intravital Lycopersicon esculentum lectin perfusion and vascular corrosion casting. Micro-CT scanning found a similar level of vascularisation within the VEGF treated scaffolds to that determined by the other analytical methods. However, although the relative increase in vascularisation (17 fold above PBS controls p < 0.05) induced by VEGF determined by micro-CT was similar to the perfusion based analyses (20.1 and 10.4 fold for lectin perfusion and vascular corrosion respectively p < 0.05), it differed substantially from that determined by CD31 immunohistochemistry (3.2 fold p < 0.05). This difference was due to a large pro! portion of unperfused vessels in the PBS control that were not present in the VEGF group. The increase in perfusion probably resulted in part from an increase in average vessel diameter. Though this increase was detected by micro-CT, the actual diameters were overestimated by 60–90% most likely as a consequence of a merging effect for juxtaposed vessels. Thus whilst micro-CT gives an accurate three-dimensional quantification of the VEGF-induced increase in perfused vessels, resolution needs to be maximized for accurate sizing of a microvascular network's components.
• Antibiofilm activity of nanosized magnesium fluoride
  Lellouche J Kahana E Elias S Gedanken A Banin E - Biomaterials 30(30):5969-5978 (2009)
  The ability of bacteria to develop antibiotic resistance and colonize abiotic surfaces by forming biofilms is a major cause of medical implant-associated infections and results in prolonged hospitalization periods and patient mortality. This raises the urgent need to develop compounds that can inhibit bacterial colonization of surfaces. In this study, we present an unreported microwave-based synthesis of MgF2 nanoparticles (Nps) using ionic liquid. We demonstrate the antimicrobial activity of these fluoride nanomaterials and their ability to restrict biofilm formation of common bacterial pathogens. Scanning and transmission electron microscopic techniques indicated that the MgF2·Nps attach and penetrate into the cells. Flow cytometry analysis revealed that the Nps caused a disruption in the membrane potential. The MgF2·Nps also induced membrane lipid peroxidation and once internalized can interact with chromosomal DNA. Based on these findings we further explored the ! possibility of using the MgF2·Nps to coat surfaces and inhibit biofilm formation. A microwave synthesis and coating procedure was utilized to coat glass coupons. The MgF2 coated surfaces effectively restricted biofilm formation of the tested bacteria. Taken together these results highlight the potential for developing MgF2 nanoparticles in order to inhibit bacterial infections.
• The disruption of bacterial membrane integrity through ROS generation induced by nanohybrids of silver and clay
  Su HL Chou CC Hung DJ Lin SH Pao IC Lin JH Huang FL Dong RX Lin JJ - Biomaterials 30(30):5979-5987 (2009)
  Nanohybrids, synthesized via silver nitrate reduction in the presence of silicate clay, exhibit a high potency against bacterial growth. The plate-like clay, due to its anionic surface charges and a large surface area, serves as the support for the formation of silver nanoparticles (AgNPs) 30 nm in diameter. The nanohybrid consisting of Ag/silicate at a 7/93 weight ratio inhibited the growth of dermal pathogens including Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa and Streptococcus pyrogens, as well as the methicillin- and oxacillin-resistant S. aureus (MRSA and ORSA). Scanning electron microscope revealed that these nanohybrids were adherent on the surface of individual bacteria. The thin silicate plates provide a surface for immobilizing AgNPs in one highly concentrated area but prevent them from entering the cell membrane. Subsequent cytotoxicity studies indicated that surface contact with the reduced AgNPs on clay is sufficient to initiate cell death.! This toxicity is related to a loss in membrane integrity due to reactive oxygen species (ROS) generation. The hybridization of AgNPs on clay surface is viable for generating a new class of nanohybrids exhibiting mild cytotoxicity but high efficacy for battling drug-resistant bacteria.
• Cholesterol-mediated anchoring of enzyme-loaded liposomes within disulfide-stabilized polymer carrier capsules
  Chandrawati R Städler B Postma A Connal LA Chong SF Zelikin AN Caruso F - Biomaterials 30(30):5988-5998 (2009)
  Polymer capsules containing multiple liposomes, termed capsosomes, are a promising new concept toward the design of artificial cells. Herein, we report on the fundamental aspects underpinning the assembly of capsosomes. A stable and high loading of intact liposomal cargo into a polymer film was achieved by non-covalently sandwiching the liposomes between a tailor-made cholesterol-modified poly(l-lysine) (PLLc) precursor layer and a poly(methacrylic acid)-co-(cholesteryl methacrylate) (PMAc) capping layer. The film assembly, optimized on planar surfaces, was successfully transferred onto colloidal substrates, and a polymer membrane was subsequently assembled by the alternating adsorption of poly(N-vinyl pyrrolidone) (PVP) and thiol-modified poly(methacrylic acid) (PMASH) onto the pre-adsorbed layer of liposomes. Upon removal of the silica template, stable capsosomes encapsulating the enzyme luciferase or β-lactamase within their liposomal sub-compartments were obtained! at both assembly (pH 4) and physiological conditions (pH 7.4). Excellent retention of the liposomes and the enzymatic cargo within the polymer carrier capsules was observed for up to 14 days. These engineered capsosomes are particularly attractive as autonomous microreactors, which can be utilized to repetitively add smaller reactants to cause successive distinct reactions within the capsosomes and simultaneously release the products to the surrounding environment, bringing these systems one step closer toward constructing artificial cells.
• An electrochemical glucose biosensor exploiting a polyaniline grafted multiwalled carbon nanotube/perfluorosulfonate ionomer–silica nanocomposite
  Gopalan AI Lee KP Ragupathy D Lee SH Lee JW - Biomaterials 30(30):5999-6005 (2009)
  A glucose biosensor was fabricated with loading of glucose oxidase (GOx) into a new organic–inorganic hybrid nanocomposite. The preparation involves formation of silica network into a Nafion (perfluorosulfonate ionomer) and subsequent loading of polyaniline grafted multiwalled carbon nanotubes (MWNT-g-PANI) onto Nafion–silica nanocomposite. Field emission scanning electron microscopy (FE-SEM) of Nafion–silica/MWNT-g-PANI composite reveals the presence of spherical silica particles (sizes in the range 250 nm–1 μm) and tubular MWNT-g-PANI particles. Chronoamperometry and cyclic voltammetry were used to evaluate the performance of biosensor towards glucose. The Nafion–silica/MWCNT-g-PANI/GOx biosensor exhibited a linear response to glucose in the concentration range of 1–10 mm with a correlation coefficient of 0.9972, good sensitivity (5.01 μA/mm), a low response time (6 s), repeatability (R.S.D value of 2.2%) and along-term stability. The presence of silica! network within Nafion and MWNT-g-PANI synergistically contributes to the performance of the biosensor towards the electrochemical detection of glucose.
• A self-assembling nanoparticle for paclitaxel delivery in ovarian cancer
  Xiao K Luo J Fowler WL Li Y Lee JS Xing L Cheng RH Wang L Lam KS - Biomaterials 30(30):6006-6016 (2009)
  Paclitaxel (PTX) is one of the most effective chemotherapeutic drugs for the treatment of a variety of cancers. However, it is associated with serious side effects caused by PTX itself and the Cremophor EL emulsifier. In the present study, we report the development of a well-defined amphiphilic linear-dendritic copolymer (named as telodendrimer) composed of polyethylene glycol (PEG), cholic acid (CA, a facial amphiphilic molecule) and lysine, which can form drug-loaded core/shell micelles when mixed with hydrophobic drug, such as PTX, under aqueous condition. We have used PEG5k-CA8, a representive telodendrimer, to prepare paclitaxel-loaded nanoparticles (PTX-PEG5k-CA8 NPs) with high loading capacity (7.3 mg PTX/mL) and a size of 20–60 nm. This novel nanoformulation of PTX was found to exhibit similar in vitro cytotoxic activity against ovarian cancer cells as the free drug (Taxol®) or paclitaxel/human serum albumin nanoaggregate (Abraxane®). The maximum tolerated ! doses (MTDs) of PTX-PEG5k-CA8 NPs after single dose and five consecutive daily doses in mice were approximately 75 and 45 mg PTX/kg, respectively, which were 2.5-fold higher than those of Taxol®. In both subcutaneous and orthotopic intraperitoneal murine models of ovarian cancer, PTX-PEG5k-CA8 NPs achieved superior toxicity profiles and anti-tumor effects compared to Taxol® and Abraxane® at equivalent PTX doses, which were attributed to their preferential tumor accumulation, and deep penetration into tumor tissue, as confirmed by near infrared fluorescence (NIRF) imaging.
• Transdermal immunization with low-pressure-gene-gun mediated chitosan-based DNA vaccines against Japanese encephalitis virus
  Huang HN Li TL Chan YL Chen CL Wu CJ - Biomaterials 30(30):6017-6025 (2009)
  DNA vaccine is a milestone in contemporary vaccine development. It has considerably offset many shortcomings in conventional vaccines. Although DNA vaccines applied through 'traditional' high-pressure gene guns generally elicit high titers of protective immunity, such a practice however requires enormous investment in daunting instruments that often discourage vaccines due to an inevitable pain-eliciting effect. In this study, we exploited a less expensive yet low-pressure-gene-gun that can alleviate such phobia of pain. DNA vaccines were prepared by using the associative feature of cationic chitosan and anionic DNAs. The optimized N/P ratio is 3. The formulized complex sizes to nano-scale. The vaccine complexes were tested in C3H/HeN mice. The expression of GFP reporter gene was observable and traceable in epidermis and spleen over 3 days. The expressions of GFP and the activation of dendritic cells (DCs) were evident and co-localized in hair follicles and epiderm! is. C3H/HeN mice immunized with the developed chitosan-JEV DNA vaccines can elicit desired JEV specific antibodies, whereby the mice maintained high survival rates against 50 × LD50 JEV challenge. The low-pressure-gene-gun mediated chitosan-based JEV DNA vaccines have proven to be convenient and efficacious, thereby with high capacity in deployment for future prophylaxis against JEV outbreaks.
• Synthesis, characterization, and preliminary assessment of anti-Flt1 peptide–hyaluronate conjugate for the treatment of corneal neovascularization
  Oh EJ Park K Choi JS Joo CK Hahn SK - Biomaterials 30(30):6026-6034 (2009)
  Anti-Flt1 peptide of GNQWFI has been reported to inhibit vascular endothelial growth factor receptor 1 (VEGFR1) – mediated endothelial cell migration and tube formation. In this work, a protocol to synthesize anti-Flt1 peptide–hyaluronate (HA) conjugate was successfully developed for the treatment of corneal neovascularization. Using tetrabutyl ammonium salt of HA (HA-TBA), water-insoluble anti-Flt1 peptide could be conjugated with HA in dimethyl sulfoxide (DMSO) by the amide bond formation between carboxyl groups of HA and N-terminal amine groups of GGNQWFI. The formation of anti-Flt1 peptide–HA conjugate was confirmed by 1H NMR and fluorometric analyses. The average number of grafted peptide molecules in anti-Flt1 peptide–HA conjugates could be controlled from 3 to 30 per single HA chain by changing the feeding amount of peptide for the conjugation reaction. According to in vitro biological activity tests, anti-Flt1 peptide–HA conjugate exhibited a signific! ant inhibition effect on the binding of Flt1-Fc to VEGF165 coated on the well. Furthermore, in vivo biological activity of anti-Flt1 peptide–HA conjugate was confirmed from the inhibitory effect on corneal neovascularization in silver nitrate cauterized corneas of SD rats. The VEGF receptor 2 expression was also reduced after treatment with anti-Flt1 peptide–HA conjugate. The water-soluble anti-Flt1 peptide–HA conjugate was thought to have a potential to be developed as anti-angiogenic therapeutics for the treatment of corneal neovascularization.
• The antimicrobial activity of liposomal lauric acids against Propionibacterium acnes
  Yang D Pornpattananangkul D Nakatsuji T Chan M Carson D Huang CM Zhang L - Biomaterials 30(30):6035-6040 (2009)
  This study evaluated the antimicrobial activity of lauric acid (LA) and its liposomal derivatives against Propionibacterium acnes (P. acnes), the bacterium that promotes inflammatory acne. First, the antimicrobial study of three free fatty acids (lauric acid, palmitic acid and oleic acid) demonstrated that LA gives the strongest bactericidal activity against P. acnes. However, a setback of using LA as a potential treatment for inflammatory acne is its poor water solubility. Then the LA was incorporated into a liposome formulation to aid its delivery to P. acnes. It was demonstrated that the antimicrobial activity of LA was not only well maintained in its liposomal derivatives but also enhanced at low LA concentration. In addition, the antimicrobial activity of LA-loaded liposomes (LipoLA) mainly depended on the LA loading concentration per single liposomes. Further study found that the LipoLA could fuse with the membranes of P. acnes and release the carried LA directly! into the bacterial membranes, thereby killing the bacteria effectively. Since LA is a natural compound that is the main acid in coconut oil and also resides in human breast milk and liposomes have been successfully and widely applied as a drug delivery vehicle in the clinic, the LipoLA developed in this work holds great potential of becoming an innate, safe and effective therapeutic medication for acne vulgaris and other P. acnes associated diseases.
• Targeted delivery and controlled release of doxorubicin to cancer cells using modified single wall carbon nanotubes
  Zhang X Meng L Lu Q Fei Z Dyson PJ - Biomaterials 30(30):6041-6047 (2009)
  A targeted drug delivery system that is triggered by changes in pH based on single wall carbon nanotubes (SWCNTs), derivatized with carboxylate groups and coated with a polysaccharide material, can be loaded with the anticancer drug doxorubicin (DOX). The drug binds at physiological pH (pH 7.4) and is only released at a lower pH, for example, lysosomal pH and the pH characteristic of certain tumor environments. By manipulating the surface potentials of the modified nanotubes through modification of the polysaccharide coating, both the loading efficiency and release rate of the associated DOX can be controlled. Folic acid (FA), a targeting agent for many tumors, can be additionally tethered to the SWCNTs to selectively deliver DOX into the lysosomes of HeLa cells with much higher efficiency than free DOX. The DOX released from the modified nanotubes has been shown to damage nuclear DNA and inhibit the cell proliferation.
• Poly(ethylene glycol) hydrogels formed by thiol-ene photopolymerization for enzyme-responsive protein delivery
  Aimetti AA Machen AJ Anseth KS - Biomaterials 30(30):6048-6054 (2009)
  Degradable hydrogels have been extensively used in biomedical applications such as drug delivery, and recent interest has grown in hydrogels that degrade in recognition of a cellular response. This contribution describes a poly(ethylene glycol) (PEG) hydrogel platform with human neutrophil elastase (HNE) sensitive peptide cross-links formed using thiol-ene photopolymerization rendering the gel degradable at sites of inflammation. Further, protein therapeutics can be physically entrapped within the network and selectively released upon exposure to HNE. HNE-responsive hydrogels exhibited surface erosion where the degradation kinetics was influenced by changes in peptide kcat, concentration of HNE, and concentration of peptide within the gel. Using this platform, we were able to achieve controlled, zero-order release of bovine serum albumin (BSA) in the presence of HNE, and release was arrested in the absence of HNE. To further exploit the advantages of surface eroding de! livery systems, a smaller protein (carbonic anhydrase) was delivered at the same rate as BSA and only dependent on gel formulation and environmental conditions. Also, protein release was predicted from a 3-layered hydrogel device using mass loss data. Lastly, the bioactivity of lysozyme was maintained above 90% following the exposure to thiol-ene photopolymerization conditions.
• The use of pH-triggered leaky heterogeneities on rigid lipid bilayers to improve intracellular trafficking and therapeutic potential of targeted liposomal immunochemotherapy
  Karve S Alaouie A Zhou Y Rotolo J Sofou S - Biomaterials 30(30):6055-6064 (2009)
  During endocytosis, pH-triggered release of encapsulated therapeutics from delivery carriers may accelerate their intracellular trafficking increasing therapeutic efficacy. To improve the therapeutic potential of targeted immunochemotherapy using anti-HER2/neu liposomal doxorubicin, we exploit the formation of leaky heterogeneities on rigid lipid bilayers to extensively release doxorubicin during endocytosis. We have previously demonstrated that pH-dependent formation of phase-separated lipid heterogeneities on the plane of a bilayer membrane increases the permeability of bilayers when they are composed of lipid pairs with rigid non-matching acyl chain lengths. This was suggested to be due to defective packing among lipids residing at the interfaces of lipid domains. Here we design nanometer-size antiHER2/neu-labeled PEGylated vesicles composed of lipid pairs with longer non-matching acyl chain lengths (n = 18 and 21). These vesicles exhibit superior killing efficacy o! f cancer cells compared to established liposome formulations, and their killing efficacy is similar to the effect of combined free doxorubicin and free antiHER2/neu antibody. Other transport-related properties such as liposome blood circulation times, and specific binding and internalization by cancer cells are unaffected. These results demonstrate the potential of vesicles with pH-triggered leaky heterogeneities to increase the therapeutic potential of targeted immunochemotherapy.
• Gold nanoparticles with a monolayer of doxorubicin-conjugated amphiphilic block copolymer for tumor-targeted drug delivery
  Prabaharan M Grailer JJ Pilla S Steeber DA Gong S - Biomaterials 30(30):6065-6075 (2009)
  Gold (Au) nanoparticles (NPs) stabilized with a monolayer of folate-conjugated poly(l-aspartate-doxorubicin)-b-poly(ethylene glycol) copolymer (Au-P(LA-DOX)-b-PEG-OH/FA) was synthesized as a tumor-targeted drug delivery carrier. The Au-P(LA-DOX)-b-PEG-OH/FA NPs consist of an Au core, a hydrophobic poly(l-aspartate-doxorubicin) (P(LA-DOX)) inner shell, and a hydrophilic poly(ethylene glycol) and folate-conjugated poly(ethylene glycol) outer shell (PEG-OH/FA). The anticancer drug, doxorubicin (DOX), was covalently conjugated onto the hydrophobic inner shell by acid-cleavable hydrazone linkage. The DOX loading level was determined to be 17 wt%. The Au-P(LA-DOX)-b-PEG-OH/FA NPs formed stable unimolecular micelles in aqueous solution. The size of the Au-P(LA-DOX)-b-PEG-OH/FA micelles were determined as 24–52 and 10–25 nm by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. The conjugated DOX was released from the Au-P(LA-DOX)-b-PEG! -OH/FA micelles much more rapidly at pH 5.3 and 6.6 than at pH 7.4, which is a desirable characteristic for tumor-targeted drug delivery. Cellular uptake of the Au-P(LA-DOX)-b-PEG-OH/FA micelles facilitated by the folate-receptor-mediated endocytosis process was higher than that of the micelles without folate. This was consistent with the higher cytotoxicity observed with the Au-P(LA-DOX)-b-PEG-OH/FA micelles against the 4T1 mouse mammary carcinoma cell line. These results suggest that Au-P(LA-DOX)-b-PEG-OH/FA NPs could be used as a carrier with pH-triggered drug releasing properties for tumor-targeted drug delivery.
• Hyaluronic acid-based hydrogels as 3D matrices for in vitro evaluation of chemotherapeutic drugs using poorly adherent prostate cancer cells
  Gurski LA Jha AK Zhang C Jia X Farach-Carson MC - Biomaterials 30(30):6076-6085 (2009)
  The current investigation aimed to develop a biomimetic, three-dimensional (3D) culture system for poorly adherent bone metastatic prostate cancer cells (C4-2B) for use as an in vitro platform for anti-cancer drug screening. To this end, hyaluronic acid (HA) derivatives carrying complementary aldehyde (HAALD) and hydrazide (HAADH) groups were synthesized and characterized. In situ encapsulation of C4-2B cells was achieved by simple mixing of HAALD and HAADH in the presence of the cells. Unlike two-dimensional (2D) monolayer culture in which cells adopt an atypical spread morphology, cells residing in the HA matrix formed distinct clustered structures which grew and merged, reminiscent of real tumors. Anti-cancer drugs added to the media surrounding the cell/gel construct diffused into the gel and killed the embedded cells. The HA hydrogel system was used successfully to test the efficacy of anti-cancer drugs including camptothecin, docetaxel, and rapamycin, alone and i! n combination, including specificity, dose and time responses. Responses of cells to anti-neoplastics differed between the 3D HA hydrogel and 2D monolayer systems. We suggest that the data obtained from 3D HA systems is superior to that from conventional 2D monolayers as the 3D system better reflects the bone metastatic microenvironment of the cancer cells.
• Prolonged local antibiotics delivery from hydroxyapatite functionalised with cyclodextrin polymers
  Leprêtre S Chai F Hornez JC Vermet G Neut C Descamps M Hildebrand HF Martel B - Biomaterials 30(30):6086-6093 (2009)
  Per-operative infection is a common complication for bone-graft surgery. Combining antiseptic agents with graft materials may offer a solution by increasing local drug concentration at target sites. Aiming to achieve a sustained local antibiotic (ATB) delivery for a widely applied bone substitute material - hydroxyapatite (HA), we attempted incorporating hydroxypropyl-β-cyclodextrin polymer (polyHPβCD) into microporous HA via impregnating either in a CD monomers mixture solution or a pre-synthesized CD polymer solution, followed by thermal fixation processing. In such functionalised material (CD-HA), polyHPβCD could entrap ATBs and release them progressively. Infrared-spectroscopic analysis confirmed the presence of polyHPβCD in functionalised HA via both processing pathways; polyHPβCD functionalisation yields were quantitated by thermogravimetric analysis for optimising the processing regime. Ciprofloxacin (CFX) and vancomycin (VCM), commonly applied in orthopaed! ics, have been respectively loaded on CD-HA by dip-coating. For both ATBs, kinetic release test in phosphate buffered saline showed significantly increased initial-burst amount and prolonged release from CD-HA compared with those from non-functionalised HA. Encouragingly, ATBs loaded CD-HA also revealed a prolonged bacteriostatic activity against Staphylococcus aureus and progressively increased cytocompatibility to osteoblasts (MC3T3-E1). Overall, polyHPβCD functionalisation on HA could be an effective drug-delivery model for loading different drug molecules in prevention of infection.
• Biomimetic apatite-coated alginate/chitosan microparticles as osteogenic protein carriers
  Lee M Li W Siu RK Whang J Zhang X Soo C Ting K Wu BM - Biomaterials 30(30):6094-6101 (2009)
  Bone morphogenetic proteins (BMPs) are currently approved for spinal fusion, tibial fracture repair, and maxillofacial bone regeneration. However, BMP pleiotropism, paradoxical activities on precursor cells, and unexpected side effects at local and ectopic sites may limit their usage. Thus, the need remains for alternative osteoinductive factors that provide more bone-specific activities with fewer adverse effects. Nell-1 [Nel-like molecule-1; Nel (a protein highly expressed in neural tissue encoding epidermal growth factor like domain)] is a novel osteogenic protein believed to specifically target cells committed to the osteogenic lineage. The objective of this project is to incorporate Nell-1 into a moldable putty carrier that can adapt to bony defects and deliver Nell-1 to the local microenvironment. We show here that moldability can be achieved by mixing hyaluronan hydrogel with two types of particles: demineralized bone powder for osteoconductivity, and biomimetic! apatite-coated alginate/chitosan microparticles for controlled Nell-1 delivery. Besides enhancing overall osteoconductivity of the carrier, the biomimetic apatite coating also provides a more sustained release (15% cumulative release over 30 days) and greatly reduces the initial burst release that is observed with non-coated alginate/chitosan microparticles (40% release after 1 day). The efficacy of Nell-1 delivery from these carriers was evaluated in a rat spinal fusion model against Nell-free carriers as controls. At 4 weeks post-implantation, Nell-1 enhanced spinal fusion rates as assessed by manual palpation, radiographs, high-resolution micro-computerized tomography (μCT), and histology. This moldable putty carrier system appears to be a suitable carrier for promoting osteogenesis, and will be further evaluated in larger animal models over longer periods to follow the remodeling of the regenerated bone.
• The effect of osteoprotegerin gene modification on wear debris-induced osteolysis in a murine model of knee prosthesis failure
  Zhang T Yu H Gong W Zhang L Jia T Wooley PH Yang SY - Biomaterials 30(30):6102-6108 (2009)
  Using an in vivo adeno-associated virus (AAV)-mediated gene transfer technique, this study evaluated the therapeutic effects of an osteoprotegerin (OPG) transgene against orthopaedic wear debris-induced osteolysis in a long-term murine model. A titanium pin was surgically implanted into proximal tibia of Balb/c mice to mimic a weight-bearing knee arthroplasty, followed by an intra-articular challenge with Ti particles to provoke periprosthetic inflammation and osteolysis. rAAV-hOPG or AAV-LacZ vectors were injected into the prosthetic joint at 3 weeks post-op. The tissues were harvested at 2, 4, 12 and 24 weeks after transduction for histological and molecular analyses. Successful transgene expression at the local site was confirmed by real-time PCR and ELISA. Inflammatory pseudo-membranes were ubiquitously present at the interface between the Ti implant and the surrounding bone in both LacZ and virus-free control groups, while soft tissue was only observed sporadicall! y at the bone–implant interface in the OPG group. A significant reduction in TRAP+ osteoclast numbers was observed in the OPG treatment group. MicroCT assessment indicated a marked reversal in the loss of peri-implant bone mineral density (BMD) in the OPG-transduced group, when compared with the LacZ and virus-free controls. Further, OPG gene modification appeared to reduce local bone collagen loss by a mean of 40%. Real-time PCR examination confirmed that in vivo OPG gene transfer dramatically influenced the periprosthetic tissue gene expression profiles by diminishing the mRNA expression of TNF, IL-1, CPK and RANKL. There were no transgene-associated toxic effects apparent during the experiment, and the PCR detection of transgenes in remote organs such as lungs, kidneys, liver, and muscle of contralateral limb were consistently negative. Overall, rAAV-mediated OPG gene transfer effectively reversed Ti-particle-induced bone resorption in this experimental model. The ther! apeutic effects may be due to the blockage of local osteoclast! ogenesis and possibly the down-regulation of RANKL expression.
• PAMAM–Triamcinolone acetonide conjugate as a nucleus-targeting gene carrier for enhanced transfer activity
  Ma K Hu MX Qi Y Zou JH Qiu LY Jin Y Ying XY Sun HY - Biomaterials 30(30):6109-6118 (2009)
  The excellent transfection efficiency and viability are essential for successful gene therapy. It suggested that when bound to its glucocorticoid receptor, glucocorticoid steroid can dilate the nuclear pore complexes and facilitated the transport of pDNA into the nucleus. In this research, the two different degrees of substitution of PAMAM–triamcinolone acetonide (PAMAM–TA) conjugates were synthesised for efficient translocation of pDNA into the nucleus. The physicochemical properties of the polyplexes were investigated by agarose gel electrophoresis, Zeta-sizer and TEM. They both could form nano-size polyplexes with pDNA. The polyplexes were very stable and showed excellent buffering capacities, facilitating endosomal escape, and no obvious difference was found between them. The TA-conjugated PAMAM-mediated transfection of luciferase and EGFP genes showed better transfer activity than native PAMAM and was comparable to the PEI 25K (polyethylenimine), and lower cyt! otoxicity in HEK 293 and HepG 2 cells. Even with 10% serum, their transfer activity was still high relatively. In addition, confocal microscopy examination confirmed that the enhancing mechanism for enhanced gene transfer activity of PAMAM–TA conjugate may involve the nuclear translocation of the polyplex. The low substituted degree of TA to 0.22 did not interrupt its nuclear localization potency. These findings demonstrated that the TA-grafted PAMAM dendrimer is a potential candidate as a safe and efficient gene delivery carrier for gene therapy.
• Modeling the controllable pH-responsive swelling and pore size of networked alginate based biomaterials
  Chan AW Neufeld RJ - Biomaterials 30(30):6119-6129 (2009)
  Semisynthetic network alginate polymer (SNAP), synthesized by acetalization of linear alginate with di-aldehyde, is a pH-responsive tetrafunctionally linked 3D gel network, and has potential application in oral delivery of protein therapeutics and active biologicals, and as tissue bioscaffold for regenerative medicine. A constitutive polyelectrolyte gel model based on non-Gaussian polymer elasticity, Flory–Huggins liquid lattice theory, and non-ideal Donnan membrane equilibria was derived, to describe SNAP gel swelling in dilute and ionic solutions containing uni-univalent, uni-bivalent, bi-univalent or bi-bi-valent electrolyte solutions. Flory–Huggins interaction parameters as a function of ionic strength and characteristic ratio of alginates of various molecular weights were determined experimentally to numerically predict SNAP hydrogel swelling. SNAP hydrogel swells pronouncedly to 1000 times in dilute solution, compared to its compact polymer volume, while beha! ving as a neutral polymer with limited swelling in high ionic strength or low pH solutions. The derived model accurately describes the pH-responsive swelling of SNAP hydrogel in acid and alkaline solutions of wide range of ionic strength. The pore sizes of the synthesized SNAP hydrogels of various crosslink densities were estimated from the derived model to be in the range of 30–450 nm which were comparable to that measured by thermoporometry, and diffusion of bovine serum albumin. The derived equilibrium swelling model can characterize hydrogel structure such as molecular weight between crosslinks and crosslinking density, or can be used as predictive model for swelling, pore size and mechanical properties if gel structural information is known, and can potentially be applied to other point-link network polyelectrolytes such as hyaluronic acid gel.
• Molecular dynamics simulation study of P (VP-co-HEMA) hydrogels: Effect of water content on equilibrium structures and mechanical properties
  Lee SG Brunello GF Jang SS Bucknall DG - Biomaterials 30(30):6130-6141 (2009)
  Poly (N-vinyl-2-pyrrolidone-co-2-hydroxyethyl methacrylate) (P(VP-co-HEMA)) hydrogel system with a composition of VP:HEMA = 37:13 was studied using molecular dynamics simulations in order to investigate the effect of the water content on the equilibrium structures and the mechanical properties. The degree of randomness of the monomer sequence for the random and the blocky copolymers, were 1.170 and 0.104, respectively, and the degree of polymerization was fixed at 50. The equilibrated density of the hydrogel was found to be larger for the random sequence than for the blocky sequence at low water contents (<40 wt%), but this density difference decreased with increasing water content. The pair correlation function analysis shows that VP is more hydrophilic than HEMA and that the random sequence hydrogel is solvated more than the blocky sequence hydrogel at low water content, which disappears with increasing water content. Correspondingly, the water structure is more disr! upted by the random sequence hydrogel at low water content but eventually develops the expected bulk water-like structure with increasing water content. From mechanical deformation simulations, stress–strain analysis showed that the VP is found to relax more efficiently, especially in the blocky sequence, so that the blocky sequence hydrogel shows less stress levels compared to the random sequence hydrogel. As the water content increases, the stress level becomes identical for both sequences. The elastic moduli of the hydrogels calculated from the constant strain energy minimization show the same trend with the stress–strain analysis.
• Finite element study of scaffold architecture design and culture conditions for tissue engineering
  Olivares AL Marsal E Planell JA Lacroix D - Biomaterials 30(30):6142-6149 (2009)
  Tissue engineering scaffolds provide temporary mechanical support for tissue regeneration and transfer global mechanical load to mechanical stimuli to cells through its architecture. In this study the interactions between scaffold pore morphology, mechanical stimuli developed at the cell microscopic level, and culture conditions applied at the macroscopic scale are studied on two regular scaffold structures. Gyroid and hexagonal scaffolds of 55% and 70% porosity were modeled in a finite element analysis and were submitted to an inlet fluid flow or compressive strain. A mechanoregulation theory based on scaffold shear strain and fluid shear stress was applied for determining the influence of each structures on the mechanical stimuli on initial conditions. Results indicate that the distribution of shear stress induced by fluid perfusion is very dependent on pore distribution within the scaffold. Gyroid architectures provide a better accessibility of the fluid than hexago! nal structures. Based on the mechanoregulation theory, the differentiation process in these structures was more sensitive to inlet fluid flow than axial strain of the scaffold. This study provides a computational approach to determine the mechanical stimuli at the cellular level when cells are cultured in a bioreactor and to relate mechanical stimuli with cell differentiation.