Biomaterials (v.33, #2)

The use of a library of industrial materials to determine the nature of substrate-dependent performance of primary adherent human cells by Ming Ni; Philipp K. Zimmermann; Karthikeyan Kandasamy; Weizheng Lai; Yao Li; Meng Fatt Leong; Andrew C.A. Wan; Daniele Zink (353-364).
We developed a library of industrial materials, which can be applied to any adherent cell type for determining cell–material interactions. Bulk and surface chemistry as well as other material properties were characterized. The library covered broad ranges of various material properties. We applied the library to primary human endothelial and epithelial cells, which play important roles in tissue engineering and biomedical applications. The results revealed that substrate stiffness was the major determinant of cell performance. The ability to grow and differentiate on stiff or more compliant materials was cell type-dependent, but cell performance was consistently best on stiff and smooth materials. These results give new insights into the nature of substrate-dependent performance of primary human cells and are potentially useful for the development of improved biomaterials. The materials of the library can be easily accessed by the scientific community to determine cell–material interactions of any adherent cell type of interest.
Keywords: Compliance; Hydrophilicity; Membrane; Cell proliferation; Epithelial cell; Endothelial cell;

Biomaterial-associated infections remain a serious complication in orthopaedic surgery. Treatments, including the local use of antibiotic-loaded polymethylmethacrylate (PMMA) bone cement, are not always successful because of multiantibiotic-resistant organisms. In this study, we synthesised a new quaternised chitosan derivative (hydroxypropyltrimethyl ammonium chloride chitosan, HACC) that contains a series of substitutions of quaternary ammonium and demonstrated that HACC with a 26% degree of substitution (DS; referred to as 26%HACC) had a strong antibacterial activity and simultaneously good biocompatibility with osteogenic cells. We loaded 26%HACC at 20% by weight into PMMA bone cement to investigate whether HACC in PMMA prevents bacterial biofilm formation on the surface of bone cements. Chitosan-loaded PMMA (at the same weight ratio), gentamicin-loaded PMMA and PMMA with no antibiotic were also investigated and compared. Two clinical isolates, Staphylococcus epidermidis 389 and methicillin-resistant S. epidermidis (MRSE287), and two standard strains, S. epidermidis (ATCC35984) and methicillin-resistant Staphylococcus aureus (ATCC43300), were selected to evaluate the bacterial biofilm formation at 6, 12 and 24 h using the spread plate method, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The results showed that 26%HACC-loaded PMMA inhibited biofilm formation on its surface, while the PMMA control and chitosan-loaded PMMA were unable to inhibit biofilm formation. The gentamicin-loaded PMMA decreased the number of viable methicillin-resistant Staphylococcus strains, but its ability to inhibit biofilm formation was lower than 26%HACC-loaded PMMA. Real-time PCR demonstrated that 26%HACC-loaded PMMA markedly downregulated the expression of icaAD, which encodes essential enzymes for polysaccharide intercellular adhesion (PIA) biosynthesis, upregulated the expression level of icaR, which negatively mediates icaAD expression, and also downregulated the expression of MecA, which encodes membrane-bound enzymes known to be penicillin-binding proteins. Our study indicates that 26%HACC-loaded PMMA prevents biofilm formation of Staphylococcus, including antibiotic-resistant strains, on the surface of bone cement, and downregulates the virulence-associated gene expression of antibiotic-resistant staphylococcus, thus providing a promising new strategy for combating implant infections and osteomyelitis.
Keywords: PMMA bone cement; Quaternised chitosan; Antibiotic-resistant staphylococcus; Biofilm formation; Ica genes; MecA genes;

The field of optogenetics has been successfully used to understand the mechanisms of neuropsychiatric diseases through the precise spatial and temporal control of specific groups of neurons in a neural circuitry. However, it remains a great challenge to integrate optogenetic modulation with electrophysiological and behavioral read out methods as a means to explore the causal, temporally precise, and behaviorally relevant interactions of neurons in the specific circuits of freely behaving animals. In this study, an eight-channel chronically implantable optrode array was fabricated and modified with poly(3,4-ethylenedioxythiophene)/poly(styrenesulfonate)-poly(vinyl alcohol)/poly(acrylic acid) interpenetrating polymer networks (PEDOT/PSS-PVA/PAA IPNs) for improving the optrode-neural tissue interface. The conducting polymer-hydrogel IPN films exhibited a significantly higher capacitance and lower electrochemical impedance at 1 kHz as compared to unmodified optrode sites and showed significantly improved mechanical and electrochemical stability as compared to pure conducting polymer films. The cell attachment and neurite outgrowth of rat pheochromocytoma (PC12) cells on the IPN films were clearly observed through calcein-AM staining. Furthermore, the optrode arrays were chronically implanted into the hippocampus of SD rats after the lentiviral expression of synapsin-ChR2-EYFP, and light-evoked, frequency-dependant action potentials were obtained in freely moving animals. The electrical recording results suggested that the modified optrode arrays showed significantly reduced impedance and RMS noise and an improved SNR as compared to unmodified sites, which may have benefited from the improved electrochemical performance and biocompatibility of the deposited IPN films. All these characteristics are greatly desired in optogenetic applications, and the fabrication method of conducting polymer-hydrogel IPNs can be easily integrated with other modification methods to build a more advanced optrode-neural tissue interface.
Keywords: Optogenetics; Optrode; Channelrhodopsin-2 (ChR2); Conducting polymers (CPs); Hydrogel; Neural interface;

Characteristics of motility-based filtering of adherent cells on microgrooved surfaces by Hiromi Miyoshi; Taiji Adachi; Jungmyoung Ju; Sang Min Lee; Dong Jin Cho; Jong Soo Ko; Go Uchida; Yutaka Yamagata (395-401).
Topographical features are known to physically affect cell behavior and are expected to have great potential for non-invasive control of such behavior. To provide a design concept of a microstructured surface for elaborate non-invasive control of cell migration, we systematically analyzed the effect of microgrooves on cell migration. We fabricated silicon microstructured surfaces covered with SiO2 with microgrooves of various sizes, and characterized the behavior of cells moving from the flat surface to the grooved surface. The intersecting microgrooves with well-defined groove width absorbed or repelled cells precisely according to the angle of approach of the cell to the boundary with the grooved surface. This filtering process was explained by the difference in the magnitude of the lamellar dragging effect resulting from the number of the grooves interacting with the lamella of the cell. This study provides a framework to tailor the microgrooved surface for non-invasive control of cell migration with label-free detection of a specific property of the target cells. This should offer significant benefits to biomedical research and applications.
Keywords: Cell-substrate interaction; Non-invasive technique; Intersecting microgrooves; Cell migration; Motility-based filtering;

The triggering of apoptosis in macrophages by pristine graphene through the MAPK and TGF-beta signaling pathways by Yang Li; Ying Liu; Yujian Fu; Taotao Wei; Laurent Le Guyader; Ge Gao; Ru-Shi Liu; Yan-Zhong Chang; Chunying Chen (402-411).
With the development of nanotechnology and the wide use of graphene, it has become necessary to assess the potential biological adverse effects of graphene. However, most of the recent publications are focused on various modified graphenes. We demonstrated biological effects of commercial pristine graphene in murine RAW 264.7 macrophages, which is an important effector cells of the innate immune system. We found that the pristine graphene can induce cytotoxicity through the depletion of the mitochondrial membrane potential (MMP) and the increase of intracellular reactive oxygen species (ROS), then trigger apoptosis by activation of the mitochondrial pathway. The MAPKs (JNK, ERK and p38) as well as the TGF-beta-related signaling pathways were found to be activated in the pristine grapheme-treated cells, which activated Bim and Bax, two pro-apoptotic member of Bcl-2 protein family. Consequently, the caspase 3 and its downstream effector proteins such as PARP were activated and the execution of apoptosis was initiated. This study provides an insight for the suppression of the apoptosis induced by the graphene through the mitochondrial pathways, the MAPKs- and TGF-beta-related signaling pathways.
Keywords: Graphene; Apoptosis; Macrophage; Mitogen activated protein kinase; TGF-beta;

Activation of pluripotency-associated genes in mouse embryonic fibroblasts by non-viral transfection with in vitro-derived mRNAs encoding Oct4, Sox2, Klf4 and cMyc by Geertrui Tavernier; Katharina Wolfrum; Joseph Demeester; Stefaan C. De Smedt; James Adjaye; Joanna Rejman (412-417).
The first successful reprogramming of differentiated cells to a pluripotent state was done by retroviral introduction of four transcription factors (Oct4, Sox2, Klf4, cMyc) by the group of Yamanaka in 2006. Since then, scientists all over the world have attempted various methods to avoid insertional mutagenesis, a major limitation of the retrovirus-based method, however no technique was found to completely avoid DNA integration. Recently, a non-viral mRNA-based approach, inherent to avoid genomic integration, was implemented to generate stem cell-like cells, yet, seventeen daily transfections were required, inducing substantial stress on the cells. In this work, we demonstrate successful activation of pluripotency-associated genes in mouse embryonic fibroblasts by means of cationic lipid-mediated introduction of mRNAs encoding the four factors. Moreover, our transfection protocol required maximally three transfections. Up-regulation of the transfected factors as well as Nanog and SSEA-1, typical mouse pluripotency markers, was detected already after the first transfection. Nuclear localization of the introduced factors was confirmed. Positive alkaline phosphatase staining of cell clusters further confirmed the onset of the reprogramming process. In conclusion, the transfection method presented here holds great promise for safe generation of induced pluripotent stem cells of mouse origin.
Keywords: mRNA; Reprogramming; Non-viral; Liposome; Oct4;

A graphene-based platform for induced pluripotent stem cells culture and differentiation by G.-Y. Chen; D.W.-P. Pang; S.-M. Hwang; H.-Y. Tuan; Y.-C. Hu (418-427).
Induced pluripotent stem cells (iPSCs) hold great promise as a cell source for regenerative medicine yet its culture, maintenance of pluripotency and induction of differentiation remain challenging. Conversely, graphene (G) and graphene oxide (GO) have captured tremendous interests in the fields of materials science, physics, chemistry and nanotechnology. Here we report on that G and GO can support the mouse iPSCs culture and allow for spontaneous differentiation. Intriguingly, G and GO surfaces led to distinct cell proliferation and differentiation characteristics. In comparison with the glass surface, iPSCs cultured on the G surface exhibited similar degrees of cell adhesion and proliferation while iPSCs on the GO surface adhered and proliferated at a faster rate. Moreover, G favorably maintained the iPSCs in the undifferentiated state while GO expedited the differentiation. The iPSCs cultured on both G and GO surfaces spontaneously differentiated into ectodermal and mesodermal lineages without significant disparity, but G suppressed the iPSCs differentiation towards the endodermal lineage whereas GO augmented the endodermal differentiation. These data collectively demonstrated that the different surface properties of G and GO governed the iPSCs behavior and implicate the potentials of graphene-based materials as a platform for iPSCs culture and diverse applications.
Keywords: Graphene; Graphene oxide; Induced pluripotent stem cells; Differentiation; Proliferation; Stem cells;

Exploiting extracellular matrix-stem cell interactions: A review of natural materials for therapeutic muscle regeneration by Drew Kuraitis; Céline Giordano; Marc Ruel; Antonio Musarò; Erik J. Suuronen (428-443).
Myopathies of skeletal muscle are prevalent diseases worldwide. To address this, regenerative therapies are being developed to restore perfusion to ischemic muscle and to reverse muscle wasting. There are adult stem cell populations that inherently possess these therapeutic properties; however, cell transplantation trials in the clinic have shown modest results at best, being limited by poor cell persistence and viability post-transplantation, and by cell relocation to non-target sites. Many materials exist that can elicit and enhance beneficial cell responses – these materials can be applied directly, or used as stem cell delivery vehicles, for regenerative therapies. In particular, components of the body’s extracellular matrices may be advantageous for therapeutic application because cells already have a pre-disposition for recognizing them, and also because their usage carries a low probability of inducing negative immune responses. This review will survey the major components of the extracellular matrix and their interactions with relevant stem cell populations for the regeneration of muscle. Future material-based therapies will benefit from a more precise control over therapeutic cell populations implicated in the regenerative response.
Keywords: Angiogenesis; Extracellular matrix; Muscle; Stem cell;

The development of a serum-free medium utilizing the interaction between growth factors and biomaterials by Kumiko Iwata; Yukiyo Asawa; Satoru Nishizawa; Yoshiyuki Mori; Satoru Nagata; Tsuyoshi Takato; Kazuto Hoshi (444-454).
To promote clinical application of cartilage tissue engineering, we should establish a serum-free chondrocyte growth medium. The serum-free medium would increase the cell numbers by more than 20-fold within one week, which proliferation ability almost matches that of serum-based one. For that, we examined the combinations of growth factors and the methods to enhance their effects by making use of the interaction with biomaterials. From various growth factors that are contained within the serum, we made the cocktail of FGF-2 (100 ng/mL), insulin (5 μg/mL), EGF (10 pg/mL), PDGF (625 pg/mL) and TGF-β (5 pg/mL), which increased the chondrocyte numbers by approximately 3-fold for 7 days. Moreover, we used the biomaterials including albumin and hyaluronan as the carrier of those factors. By direct mixing of those factors with biomaterials before the administration to the medium, the medium containing those mixture showed the chondrocyte growth of approximately a 25-fold increase by day 10. In this medium, the FGF-2 or insulin concentration hardly decreased, and rather enhanced the activation of ERK. Due to the optimal usage of biomaterials, this serum-free medium will realize a constant harvest of chondrocytes and could contribute to the safety and quality in regenerative medicine.
Keywords: Cartilage tissue engineering; Growth factors; Cell proliferation; Hyaluronic acid; Albumin;

The effects of acellular amniotic membrane matrix on osteogenic differentiation and ERK1/2 signaling in human dental apical papilla cells by Yi-Jane Chen; Min-Chun Chung; Chung-Chen Jane Yao; Chien-Hsun Huang; Hao-Hueng Chang; Jiiang-Huei Jeng; Tai-Horng Young (455-463).
The amniotic membrane (AM) has been widely used in the field of tissue engineering because of the favorable biological properties for scaffolding material. However, little is known about the effects of an acellular AM matrix on the osteogenic differentiation of mesenchymal stem cells. In this study, it was found that both basement membrane side and collagenous stroma side of the acellular AM matrix were capable of providing a preferential environment for driving the osteogenic differentiation of human dental apical papilla cells (APCs) with proven stem cell characteristics. Acellular AM matrix potentiated the induction effect of osteogenic supplements (OS) such as ascorbic acid, β-glycerophosphate, and dexamethasone and enhanced the osteogenic differentiation of APCs, as seen by increased core-binding factor alpha 1 (Cbfa-1) phosphorylation, alkaline phosphatase activity, mRNA expression of osteogenic marker genes, and mineralized matrix deposition. Even in the absence of soluble OS, acellular AM matrix also could exert the substrate-induced effect on initiating APCs’ differentiation. Especially, the collagenous stroma side was more effective than the basement membrane side. Moreover, the AM-induced effect was significantly inhibited by U0126, an inhibitor of extracellular signaling-regulated kinase 1/2 (ERK1/2) signaling. Taken together, the osteogenic differentiation promoting effect on APCs is AM-specific, which provides potential applications of acellular AM matrix in bone/tooth tissue engineering.
Keywords: Amniotic membrane (AM); Apical papilla cells (APCs); Osteogenic differentiation; Extracellular signaling-regulated kinase 1/2 (ERK1/2);

Formation of post-confluence structure in human parotid gland acinar cells on PLGA through regulation of E-cadherin by Yen-Hui Chan; Tsung-Wei Huang; Ya-Shuan Chou; Sheng-Hao Hsu; Wei-Fang Su; Pei-Jen Lou; Tai-Horng Young (464-472).
As a potential solution for patients to retrieve their lost salivary gland functions, tissue engineering of an auto-secretory device is profoundly needed. Under serum-free environment, primary human parotid gland acinar (PGAC) cells can be obtained. After reaching confluence, PGAC cells spontaneously form three-dimension (3D) cell aggregations, termed post-confluence structure (PCS), and change their behaviors. Poly (lactic-co-glycolic acid) (PLGA) has been widely used in the field of biomedical applications because of its biodegradable properties for desired functions. Nonetheless, the role of PLGA in facilitating PGAC cells to form PCS has seldom been explored to recover epithelial characteristics. In this study, PGAC cells were found to have a greater tendency to form PCS on PLGA than on tissue culture polystyrene (TCPS). By tracing cell migration paths and modulating E-cadherin activity with specific inhibitor or antibody, we demonstrated that the static force of homophilic interaction on surfaces of individual cells, but not the dynamics of cell migration, played a more important role in PCS formation. Thus, PLGA was successfully confirmed to support PGAC cells to form more PCS through the effects on enhancing E-cadherin expression, which is associated with FAK/ILK/Snail expression in PGAC cells. This result indicates that selective appropriate biomaterials may be potentially useful in generating 3D PCS on two-dimension (2D) substrate without fabricating a complex 3D scaffold.
Keywords: Poly (lactic-co-glycolic acid) (PLGA); Human parotid gland acinar (PGAC) cells; Post-confluence structure (PCS); E-cadherin;

The use of BDNF to enhance the patency rate of small-diameter tissue-engineered blood vessels through stem cell homing mechanisms by Wen Zeng; Can Wen; Yangxiao Wu; Li Li; Zhenhua Zhou; Jianhong Mi; Wen Chen; Mingcan Yang; Chunli Hou; Jiansen Sun; Chuhong Zhu (473-484).
The patency rate of small-diameter tissue-engineered blood vessels is the determinant for their application in coronary artery bypass grafting. The coronary artery is innervated by vagus nerves. The origin of vagus nerves is rich in brain-derived neurotrophic factors (BDNF). We have investigated whether BDNF could improve the patency rate of small-diameter tissue-engineered blood vessels through promoting stem cell homing and paracrine activity. In vitro, we isolated early and late endothelial progenitor cells (EPCs) and found BDNF could promote single clone formation and paracrine function of EPCs, and could also induce the proliferation, migration and differentiation of late EPCs. BDNF could enhance the capturing of EPCs in parallel-plate flow chamber. Flow cytometric analysis and laser-scanning confocal microscope showed BDNF could enhance the mobilization and homing of C57BL/6 mouse EPCs after wire injury. Based on it, BDNF was coupled to the lumen surface of the blood vessel matrix material incubated with collagen through SPDP to construct BDNF-modified small-diameter tissue-engineered blood vessel. The blood vessel patency rate was significantly higher than that of control group 8 weeks after implantation in rats and the endothelialization level was superior to control. These results demonstrate that BDNF can effectively improve patency of small-diameter tissue-engineered blood vessels through stem cell homing and paracrine, and it is expected to play an important role in the construction of substitutes for coronary artery bypass grafting.
Keywords: Tissue-engineered blood vessels; Endothelial progenitor cells; Endothelialization; Vascular matrix material; Brain-derived neurotrophic factors; Patency rate;

The differential effects of aligned electrospun PHBHHx fibers on adipogenic and osteogenic potential of MSCs through the regulation of PPARγ signaling by Yang Wang; Rui Gao; Pei-Pei Wang; Jia Jian; Xian-Li Jiang; Chao Yan; Xiao Lin; Li Wu; Guo-Qiang Chen; Qiong Wu (485-493).
Cell-substrate interaction was functionally essential for phenotypic maintenance and multipotency remodeling of stem cells. For bone tissue engineering, electrospinning techniques are useful to create fibrous scaffolds mimicking natural mineralized collagen fibrous structure in bone. In this study, influence of electrospun fiber alignment on MSCs differentiation potential was investigated on PHBHHx electrospun meshes. Compared with randomly-oriented ones, the aligned fiber orientation increased elastic modulus and tension stress of the PHBHHx meshes. Most of the attached MSCs elongated along the aligned fibers. From the transcriptome microarray results, there were a total of 67 differentially expressed genes between aligned and random groups, and most of them were involved in cell adhesion and actin cytoskeleton regulation. In addition, PPAR signaling pathway was reduced on the aligned fibers, which might contribute to the impaired adipogenesis and enhanced osteogenesis. It was further confirmed by RT-PCR and western blotting. The PPARγ downregulation on the aligned fibers was related to phosphorylated activation of ERK, with no effect on total ERK expression. However, the induction of osteogenic by PHBHHx fiber alignment was relatively less significant that it could only support initial adipo-osteogenic switch and would be partially covered up by osteogenic or adipogenic inductive chemicals.
Keywords: Mesenchymal stem cells; Electrospun fibers; Polyhydroxyalkanoates; Osteogenic differentiation; PPARγ;

Engineered insulin-like growth factor-1 for improved smooth muscle regeneration by Kristen M. Lorentz; Lirong Yang; Peter Frey; Jeffrey A. Hubbell (494-503).
Insulin-like growth factor-1 (IGF-1) has been shown to induce potent mitogenic responses in various cell types, yet its sustained local delivery is still an underdeveloped domain in the clinic. We report here an engineered IGF-1 that facilitates extended local delivery to a site through its immobilization capacity within fibrin. Through recombinant fusion with a substrate sequence tag derived from α2-plasmin inhibitor (α2PI1-8), the resulting variant, α2PI1-8–IGF-1, was covalently incorporated into fibrin matrices during normal thrombin/factor XIIIa-mediated polymerization. Bioactivity of the variant was confirmed to be equivalent to wild type (WT) IGF-1 via IGF-1 receptor phosphorylation and cell proliferation studies in urinary tract-derived cells in 2-D. Assessment of functional retention within 3-D fibrin matrices demonstrated that incorporation of α2PI1–8–IGF-1 induced a 1.3- and 1.5-fold more robust proliferative response in smooth muscle cells (SMCs) than WT IGF-1 and negative control matrices, respectively, when release was not contained. Sustained α2PI1–8–IGF-1 availability at bladder lesion sites in vivo evoked a considerable increase in SMC proliferation and a favorable host tissue response after 28 days in rats. We conclude that the sustained local IGF-1 availability from fibrin provided by our variant protein enhances smooth muscle regeneration better than the WT form of the protein.
Keywords: Smooth muscle cell; Bladder tissue engineering; Fibrin; Growth factors; Recombinant protein; In vivo test;

Engineering of adult human neural stem cells differentiation through surface micropatterning by Amélie Béduer; Christophe Vieu; Florent Arnauduc; Jean-Christophe Sol; Isabelle Loubinoux; Laurence Vaysse (504-514).
Interaction between differentiating neural stem cells and the extracellular environment guides the establishment of cell polarity during nervous system development. Developing neurons read the physical properties of the local substrate in a contact-dependent manner and retrieve essential guidance cues. To restore damage brain area by tissue engineering, the biomaterial scaffold has to mimic this microenvironment to allow organized tissue regeneration. To establish the validity of using microgrooved surfaces in order to simultaneously provide to primary adult human neural stem cells a permissive growth environment and a guide for neurite outgrowth in a pre-established direction, we have studied the long-term culture of adult human neural stem cells from patient biopsies on microgrooved polymers. By exploiting polymer moulding techniques, we engineered non-cytotoxic deep microstructured surfaces of polydimethylsiloxane (PDMS) exhibiting microchannels of various widths. Our results demonstrate that precoated micropatterned PDMS surfaces can serve as effective neurite guidance surfaces for human neural stem cells. Immunocytochemistry analysis show that channel width can impact strongly development and differentiation. In particular we found an optimal microchannel width, that conciliates a high differentiation rate with a pronounced alignment of neurites along the edges of the microchannels. The impact of the microstructures on neurite orientation turned out to be strongly influenced by cell density, attesting that cell/surface interactions at the origin of the alignment effect, are in competition with cell/cell interactions tending to promote interconnected networks of cells. Considering all these effects, we have been able to design appropriate structures allowing to obtain neuron development and differentiation rate comparable to a plane unpatterned surface, with an efficient neurite guidance and a long-term cell viability.
Keywords: Adult human neural stem cells; Neurite outgrowth; Microtopography; Cell interactions; Polymer; Bioimplant;

The establishment of cardiomyocyte differentiation of embryonic stem cells (ESCs) is a useful strategy for cardiovascular regenerative medicine. Here, we report a strategy for cardiomyocyte differentiation of ESCs using substrate immobilization of insulin-like growth factor binding protein 4 (IGFBP4) with elastin-like polypeptides. Recently, IGFBP4 was reported to promote cardiomyocyte differentiation of ESCs through inhibition of the Wnt/β-catenin signaling. However, high amounts of IGFBP4 (approximately 1 μg/mL) were required to inhibit the Wnt/β-catenin signaling and induce differentiation to cardiomyocytes. We report herein induction of cardiomyocyte differentiation using IGFBP4-immobilized substrates. IGFBP4-immobilized substrates were created by fusion with elastin-like polypeptides. IGFBP4 was stably immobilized to polystyrene dishes through fusion of elastin-like polypeptides. Cardiomyocyte differentiation of ESCs was effectively promoted by strong and continuous inhibition of Wnt/β-catenin signaling with IGFBP4-immobilized substrates. These results demonstrated that IGFBP4 could be immobilized using fusion of elastin-like polypeptides. Our results also demonstrate that substrate immobilization of IGFBP4 is a powerful tool for differentiation of ESCs into cardiomyocytes. These findings suggest that substrate immobilization of soluble factors is a useful technique for differentiation of ESCs in regenerative medicine and tissue engineering.
Keywords: Cardiomyocyte; ES cells; Growth factors; Recombinant protein; Regenerative medicine;

Increasing the pore sizes of bone-mimetic electrospun scaffolds comprised of polycaprolactone, collagen I and hydroxyapatite to enhance cell infiltration by Matthew C. Phipps; William C. Clem; Jessica M. Grunda; Gregory A. Clines; Susan L. Bellis (524-534).
Bone-mimetic electrospun scaffolds consisting of polycaprolactone (PCL), collagen I and nanoparticulate hydroxyapatite (HA) have previously been shown to support the adhesion, integrin-related signaling and proliferation of mesenchymal stem cells (MSCs), suggesting these matrices serve as promising degradable substrates for osteoregeneration. However, the small pore sizes in electrospun scaffolds hinder cell infiltration in vitro and tissue-ingrowth into the scaffold in vivo, limiting their clinical potential. In this study, three separate techniques were evaluated for their capability to increase the pore size of the PCL/col I/nanoHA scaffolds: limited protease digestion, decreasing the fiber packing density during electrospinning, and inclusion of sacrificial fibers of the water-soluble polymer PEO. The PEO sacrificial fiber approach was found to be the most effective in increasing scaffold pore size. Furthermore, the use of sacrificial fibers promoted increased MSC infiltration into the scaffolds, as well as greater infiltration of endogenous cells within bone upon placement of scaffolds within calvarial organ cultures. These collective findings support the use of sacrificial PEO fibers as a means to increase the porosity of complex, bone-mimicking electrospun scaffolds, thereby enhancing tissue regenerative processes that depend upon cell infiltration, such as vascularization and replacement of the scaffold with native bone tissue.
Keywords: Bone tissue engineering; Cellular infiltration; Porosity; Scaffold; Biomimetic material; Organ culture;

Engineering fibrin polymers through engagement of alternative polymerization mechanisms by Sarah E. Stabenfeldt; Merek Gourley; Laxminarayanan Krishnan; James B. Hoying; Thomas H. Barker (535-544).
Fibrin is an attractive material for regenerative medicine applications. It not only forms a polymer but also contains cryptic matrikines that are released upon its activation/degradation and enhance the regenerative process. Despite this advantageous biology associated with fibrin, commercially available systems (e.g. TISSEEL) display limited regenerative capacity. This limitation is in part due to formulations that are optimized for tissue sealant applications and result in dense fibrous networks that limit cell infiltration. Recent evidence suggests that polymerization knob ‘B’ engagement of polymerization hole ‘b’ activates an alternative polymerization mechanism in fibrin, which may result in altered single fiber mechanical properties. We hypothesized that augmenting fibrin polymerization through the addition of PEGylated knob peptides with specificity to hole ‘b’ (AHRPYAAC-PEG) would result in distinct fibrin polymer architectures with grossly different physical properties. Polymerization dynamics, polymer architecture, diffusivity, viscoelasticity, and degradation dynamics were analyzed. Results indicate that specific engagement of hole ‘b’ with PEGylated knob ‘B’ conjugates during polymerization significantly enhances the porosity of and subsequent diffusivity through fibrin polymers. Paradoxically, these polymers also display increased viscoelastic properties and decreased susceptibility to degradation. As a result, fibrin polymer strength was significantly augmented without any adverse effects on angiogenesis within the modified polymers.
Keywords: Fibrin; Angiogenesis; Mechanical properties; Biodegradation;

A hybrid nanofiber matrix to control the survival and maturation of brain neurons by Shantanu Sur; Eugene T. Pashuck; Mustafa O. Guler; Masao Ito; Samuel I. Stupp; Thomas Launey (545-555).
Scaffold design plays a crucial role in developing graft-based regenerative strategies, especially when intended to be used in a highly ordered nerve tissue. Here we describe a hybrid matrix approach, which combines the structural properties of collagen (type I) with the epitope-presenting ability of peptide amphiphile (PA) nanofibers. Self-assembly of PA and collagen molecules results in a nanofibrous scaffold with homogeneous fiber diameter of 20–30 nm, where the number of laminin epitopes IKVAV and YIGSR can be varied by changing the PA concentrations over a broad range of 0.125–2 mg/ml. Granule cells (GC) and Purkinje cells (PC), two major neuronal subtypes of cerebellar cortex, demonstrate distinct response to this change of epitope concentration. On IKVAV hybrid constructs, GC density increases three-fold compared with the control collagen substrate at a PA concentration of ≥0.25 mg/ml, while PC density reaches a maximum (five-fold vs. control) at 0.25 mg/ml of PA and rapidly decreases at higher PA concentrations. In addition, adjustment of the epitope number allowed us to achieve fine control over PC dendrite and axon growth. Due to the ability to modulate neuron survival and maturation by easy manipulation of epitope density, our design offers a versatile test bed to study the extracellular matrix (ECM) contribution in neuron development and the design of optimal neuronal scaffold biomaterials.
Keywords: Self assembly; Laminin; Collagen; Peptide amphiphile; Nerve tissue engineering; Brain;

Spontaneous osteogenesis of MSCs cultured on 3D microcarriers through alteration of cytoskeletal tension by Pei-Chi Tseng; Tai-Horng Young; Ting-Ming Wang; Hsiao-Wen Peng; Sheng-Mou Hou; Men-Luh Yen (556-564).
3-dimensional microcarrier (3D-MC) cell culture systems are often used for expansion of stem cells including mesenchymal stem cells (MSCs) for high cell volumes required in clinical applications. However, compared to 2-dimensional (2D) cell culture, effects of 3D-MC systems on MSC differentiation have not been well studied. In this study, the behavior of various sources of MSCs from two species was observed and compared on 3D collagen I-coated-MCs (COL-MC) versus 2D culture. Proliferation of all MSCs cultured on 3D COL-MC was much decreased compared to 2D culture. Unexpectedly, COL-MC-cultured MSCs underwent spontaneous osteogenesis without exogenous addition of biochemical factors, as evidenced by increased osteogenic genes expression, ALP activity, calcium deposition, and collagen I secretion. Furthermore, MSCs cultured on 3D-MC alone without collagen I coating is sufficient to induce osteogenesis. The spontaneous lineage commitment induced by 3D-MC culture was mediated by increased cytoskeletal tension and actomyosin contraction of MSCs, which could be prevented by latrunculin B and blebbistatin, inhibitors of cytoskeletal tension and actomyosin contraction respectively. Our findings show that the combination of bioengineered MC and MSCs alone can induce specific lineage commitment very efficiently. These data have strong implications in simplifying tissue engineering strategies for therapeutic applications.
Keywords: Mesenchymal stem cells (MSCs); 3-Dimensional microcarrier (3D-MC); Differentiation; Osteogenesis; Cytoskeletal tension; Tissue engineering;

Mitochondrial targeting liposomes incorporating daunorubicin and quinacrine for treatment of relapsed breast cancer arising from cancer stem cells by Liang Zhang; Hong-Juan Yao; Yang Yu; Yan Zhang; Ruo-Jing Li; Rui-Jun Ju; Xiao-Xing Wang; Meng-Ge Sun; Ji-Feng Shi; Wan-Liang Lu (565-582).
Breast cancer stem cells play a crucial role in the relapse of breast cancers because they are resistant to a standard chemotherapy and the residual cancer stem cells are able to proliferate indefinitely. The objectives of present study were to construct a kind of mitochondrial targeting daunorubicin plus quinacrine liposomes for treating and for preventing the recurrence of breast cancer arising from the cancer stem cells. MCF-7 cancer stem cells were identified as CD44+/CD24 cells and cultured in free-serum medium. Evaluations were performed on MCF-7 cancer stem cells, MCF-7 cancer stem cell mammospheres, and the relapsed tumor by xenografting MCF-7 cancer stem cells into female NOD/SCID mice. The particle size of mitochondrial targeting daunorubicin plus quinacrine liposomes was approximately 98 nm. The mitochondrial targeting liposomes evidently increased the mitochondrial uptake of drugs, were selectively accumulated into mitochondria, activated the pro-apoptotic Bax protein, dissipated the mitochondrial membrane potential, opened the mitochondrial permeability transition pores, released cytochrome C by translocation, and initiated a cascade of caspase 9 and 3 reactions, thereby inducing apoptosis of MCF-7 cancer stem cells. The mitochondrial targeting liposomes showed the strongest efficacy in treating MCF-7 cancer cells in vitro, in treating MCF-7 cancer stem cells in vitro, and in treating the relapsed tumor in mice. Mitochondrial targeting daunorubicin plus quinacrine liposomes would provide a new strategy for treating and preventing the relapse of breast cancers arising from cancer stem cells.
Keywords: Relapse of breast cancer; Mitochondrial targeting daunorubicin plus quinacrine liposomes; Apoptosis; Mitochondrial signaling pathway; Breast cancer stem cells;

Octa-functional PLGA nanoparticles for targeted and efficient siRNA delivery to tumors by Jiangbing Zhou; Toral R. Patel; Michael Fu; James P. Bertram; W. Mark Saltzman (583-591).
Therapies based on RNA interference, using agents such as siRNA, are limited by the absence of safe, efficient vehicles for targeted delivery in vivo. The barriers to siRNA delivery are well known and can be individually overcome by addition of functional modules, such as conjugation of moieties for cell penetration or targeting. But, so far, it has been impossible to engineer multiple modules into a single unit. Here, we describe the synthesis of degradable nanoparticles that carry eight synergistic functions: 1) polymer matrix for stabilization/controlled release; 2) siRNA for gene knockdown; 3) agent to enhance endosomal escape; 4) agent to enhance siRNA potency; 5) surface-bound PEG for enhancing circulatory time; and surface-bound peptides for 6) cell penetration; 7) endosomal escape; and 8) tumor targeting. Further, we demonstrate that this approach can provide prolonged knockdown of PLK1 and control of tumor growth in vivo. Importantly, all elements in these octa-functional nanoparticles are known to be safe for human use and each function can be individually controlled, giving this approach to synthetic RNA-loaded nanoparticles potential in a variety of clinical applications.
Keywords: PLGA; Nanoparticle; Gene delivery; RNA interference;

Molecular mechanisms of antibacterial and antitumor actions of designed surfactant-like peptides by Cuixia Chen; Jing Hu; Shengzhong Zhang; Peng Zhou; Xichen Zhao; Hai Xu; Xiubo Zhao; Mohammed Yaseen; Jian R. Lu (592-603).
Biomimicry of antimicrobial peptides secreted by innate immune systems represents a major strategy in developing novel antibacterial treatments. There are however emerging concerns over the possible compromise of host natural defenses by these biomimetic peptides due to their structural similarity. In our recent work we have extended the search by exploring the potential from unnatural synthetic antimicrobial peptides. Here we show that a series of surfactant-like peptides (A m K n , m ≥ 3, n = 1, 2) can kill not only bacteria but also cancerous HeLa cells in similar manner. Under the same experimental conditions, however, these peptides showed little affinity to NIH 3T3 cells and human red blood cells (hRBCs), thus demonstrating high biocompatibility in selective responses to host mammalian cells and low hemolysis. A9K1 was most effective in killing HeLa cells, a trend consistent with their bactericidal effects against Escherichia coli and Bacillus subtilis. Mechanistic investigations through combined studies of SEM and fluorescence assays revealed that the killing of bacteria and cancerous cells was caused by disrupting cell membranes, initiated by electrostatic interactions between cationic peptides and negatively charged cell membranes. In contrast, the absence of such interactions in the case of NIH 3T3 and hRBCs over the same peptide concentration range rendered low cytotoxicity. The most effective killing power of A9K1 within this series benefited from the combined effects of several factors including modest micellar concentration and balanced amphiphilicity, consistent with its propensity of self-assembly and effective membrane lytic power.
Keywords: Antimicrobial peptides; Surfactant-like peptides; Peptide self assembly; Cell selectivity; Membrane permeability;

The biocompatibility of fatty acid modified dextran-agmatine bioconjugate gene delivery vector by Jianhai Yang; Yuan Liu; Hongbo Wang; Lu Liu; Wei Wang; Chuandong Wang; Qin Wang; Wenguang Liu (604-613).
A lauric acid modified dextran-agmatine bioconjugate (Dex-l-Agm) was prepared by 1,1′-carbonyldiimidazole (CDI) activation and the nucleophilic reaction between tosyl of tosylated dextran and primary amine of agmatine. Dextran-agmatine bioconjugates (Dex-Agm) were capable of condensing DNA into nanocomplexes, and combining lauric acid promoted the complexation with DNA supposedly due to the cooperative binding effect attributed to hydrophobic interaction. Higher degree substitution of agmatine and hydrophobic grafting resulted in increased luciferase activities expressed in COS-7 and HEK293 cells; Semiquantitative assay of GFP expression by flow cytometry in COS-7, HEK293 and CHOK1 cells further demonstrated that conjugation of fatty acid could remarkably increase gene transfection of Dex-Agm in spite of 1.1–2.3-fold lower efficiency compared to Exgen 500. The biocompatibilities of Dex-Agm and Dex-l-Agm were assessed in detail by hemolytic activity determination, red blood cell aggregation assay as well as MTT evaluation of degraded products. Dex-Agm and Dex-l-Agm were shown to be highly cytocompatible without causing hemolysis and red blood cell aggregation presumably owing to the bidentate hydrogen bonding of guanidine with the constituents present in cell membrane rather than electrostatic interactions alone which could cause cell damage. Importantly, cells cultured with the degraded products of Dex-Agm and Dex-l-Agm retained more than 80% viability, suggest their potential application as a gene delivery vector.
Keywords: Dextran; Agmatine; Lauric acid; Hydrophobic modification; Gene transfection;

Bioreducible poly(amidoamine)s as carriers for intracellular protein delivery to intestinal cells by Shmuel Cohen; Grégory Coué; Delila Beno; Rafi Korenstein; Johan F.J. Engbersen (614-623).
An effective intracellular protein delivery system was developed based on linear poly(amidoamine)s (PAAs) that form self-assembled cationic nanocomplexes with oppositely charged proteins. Two differently functionalized PAAs were synthesized by Michael-type polyaddition of 4-amino-1-butanol (ABOL) to cystamine bisacrylamide (CBA) and to bisacryloylpiperazine (BAP), yielding p(CBA-ABOL) and p(BAP-ABOL), respectively. These water-soluble PAAs efficiently condense human serum albumin (HSA) by self-assembly into stable nanoscaled and positively-charged complexes. The disulfide-containing p(CBA-ABOL)/HSA nanocomplexes exhibited high mucoadhesive properties and, while stable under neutral (extracellular) conditions, rapidly destabilized in a reductive (intracellular) environment due to the cleavage of the repetitive disulfide linkages in the CBA units of the polymer. Human-derived intestinal Caco-2/TC7 cells and HT29-MTX mucus secreting cells were exposed to these PAAs/HSA nanoparticles and the extent of their uptake and the localization within endosomal compartments were examined. The higher uptake of p(CBA-ABOL)/HSA than that of p(BAP-ABOL)/HSA suggests that the mucoadhesive properties of the p(CBA-ABOL) are beneficial to the uptake process. The transported HSA was located within early endosomes, lysosomes and the cytosol. The enhanced uptake of the p(CBA-ABOL)/HSA nanoparticles, observed in the presence of Cyclosporin A, a non-specific Multi Drug Resistance (MDR) blocker, indicates the possible efflux of these nanoparticles through MDR transporters. The results show that bioreducible PAAs have excellent properties for intracellular protein delivery, and should be applicative in oral protein delivery.
Keywords: Poly(amidoamine); Intracellular protein delivery; Mucoadhesive nanoparticles; Bioresponsive nanoparticles; Bioreducible polymer; Endosomal uptake;

We describe a hydrogel patterning and transferring (HPT) method that facilitates the quantitative analysis of synthetically engineered genetic circuits within bacterial cells. The HPT method encapsulates cells in the alginate hydrogel patterns by using polydimethylsiloxane (PDMS) template. Then, the hydrogel-encapsulated cell patterns are transferred onto an agarose hydrogel substrate that encapsulates inducer chemicals or bacterial cells. Using the HPT method, we demonstrate that inducers in the agarose hydrogel substrate regulate gene expression of the patterned cells for qualitative analysis by activating the promoters of fluorescence protein genes. In addition, we demonstrate that the HPT method can be used for the analysis of the cross-talk between genetic circuits and the concentration-dependent gene expression and regulation because the agarose hydrogel substrate can produce concentration gradients of inducers. Lastly, we demonstrate that the HPT method can be applied to investigating intercellular communication between neighboring cells with a wide range of cell densities. Since the HPT method is simple to deal with but versatile and powerful to quantitatively analyze genetic circuits in living cells in many controllable manners, we believe that the method can be widely used for the rapid advancement of synthetic, molecular, and systems biology.
Keywords: Hydrogel patterning; Hydrogel transferring; Extracellular induction; Intercellular communication; Gene expression and regulation;

Gene therapy of endometriosis introduced by polymeric micelles with glycolipid-like structure by Meng-Dan Zhao; Yan-Mei Sun; Guo-Fang Fu; Yong-Zhong Du; Feng-Ying Chen; Hong Yuan; Cai-Hong Zheng; Xin-Mei Zhang; Fu-Qiang Hu (634-643).
To reduce the side effects and improve the lack of clinical treatment countermeasures in endometriosis chemotherapy, a polymeric micelle gene delivery system composed of lipid grafted chitosan micelles (CSO-SA) and the pigment epithelium derived factor (PEDF) was designed. Due to the cationic property, the glycolipid-like micelles could compact the PEDF to form complexes nanoparticles. The complexes nanoparticles with an N/P at 9.6 had 135.6 nm volume average hydrodynamic diameters with a narrow size distribution, and 6.4 ± 0.1 mV surface potential. PEDF can be distributed to endometriotic lesions in a rat model of peritoneal endometriosis mediated by CSO-SA via the intravenous injection. It showed that the CSO-SA/PEDF nanoparticles gene therapy caused decrease in the sizes of the endometriotic lesions and atrophy and degeneration of ectopic endometrium significantly. And it showed no toxicity to the reproductive organs under electron microscope observation. In addition, a reduction in microvessel density labeled by Von Willebrand factor was observed and no decrease in α-Smooth Muscle Actine-positive mature vessels. And the index of apoptotic was increased significantly in endometriotic lesions of CSO-SA/PEDF group. So, glycolipid-like structure micelles mediated PEDF gene delivery system could be used as an effective treatment approach for endometriosis disease.
Keywords: Glycolipid-like; Polymeric micelles; PEDF; Endometriosis; Gene therapy;

Self-assembled carboxymethyl poly (l-histidine) coated poly (β-amino ester)/DNA complexes for gene transfection by Jijin Gu; Xiao Wang; Xinyi Jiang; Yanzuo Chen; Liangcen Chen; Xiaoling Fang; Xianyi Sha (644-658).
Biomaterials coated polymer/DNA complexes are developed as an efficient non-viral gene delivery system. It is able to circumvent the changes of various biophysical properties of the biomaterials and the corresponding polymer/DNA nanoparticles with covalent linkage. In the present study, we introduced pH-sensitive carboxymethyl poly (l-histidine) (CM-PLH) and poly (β-amino ester) (PbAE) as functional biomaterials to form CM-PLH/PbAE/DNA core-shell ternary complexes system based on electrostatically adsorbed coatings for gene efficient delivery and transfection. The preparation of the complexes was performed self-assembly in 25 mm sodium acetate buffer solution at pH 5.2. The complexes kept stable nano-size, behaving good condensation capacity and low toxicity, even provided a higher transfection efficiency than the binary complexes (PbAE/DNA without CM-PLH) and transfected up to (89.6 ± 4.45) % in HEK293 and (57.1 ± 2.10) % in B16-F10 in vitro. The ternary complexes significantly enhanced their cellular uptake and endosomal escape which were proved by the results that the complexes could evade the endosomal lumen and localize in the nucleus of treated cells visualized under Fluorescence Confocal Microscopy (FCM). The aforementioned results indicated that CM-PLH with pH-sensitive imidazole groups played an important role in enhancing the endosomal escape and transfection efficiency. The in vivo gene transfection confirmed that the ternary complexes with pGL3-promoter as led to effectively deposit at the tumor site by the EPR effect and shown 4 fold higher luciferase expression in B16-F10 tumor than the binary complexes. Consequently, CM-PLH/PbAE/DNA ternary complexes system exhibited significant improvements in transfection efficiency in comparison with non-coated PbAE/DNA both in vitro and in vivo, highlighting their functional prospect. Our approach and the gene delivery system fabrication could potentially be useful for effective gene delivery and therapies to targeted cells.
Keywords: Self-assembly; Non-viral vector; Carboxymethyl poly (l-histidine); Poly (β-amino ester); Ternary complexes;

Antisense inhibition of gene expression and growth in gram-negative bacteria by cell-penetrating peptide conjugates of peptide nucleic acids targeted to rpoD gene by Hui Bai; Yu You; Hua Yan; Jingru Meng; Xiaoyan Xue; Zheng Hou; Ying Zhou; Xue Ma; Guojun Sang; Xiaoxing Luo (659-667).
Gram-negative bacteria (GNB) cause common and severe hospital- and community-acquired infections with a high incidence of multidrug resistance (MDR) and mortality. The emergence and spread of MDR-GNB strains limit therapeutic options and highlight the need to develop new therapeutic strategies. In this study, the peptide (RXR)4XB- and (KFF)3K-conjugated peptide nucleic acids (PPNAs) were developed to target rpoD, which encodes an RNA polymerase primary σ70 that is thought to be essential for bacterial growth. Their antimicrobial activities were tested against different clinical isolates of MDR-GNB in vitro and in infection models. The (RXR)4XB- and (KFF)3K- conjugated PNAs were bactericidal against different strains of MDR-GNB in concentration-dependent and sequence-selective manner, whereas a PPNA with a scrambled base sequence had no effect on growth. Among tested PPNAs, (RXR)4XB conjugate PPNA06 showed more potent and broad spectrum inhibition in multidrug-resistant Escherichia coli, Salmonella enterica, Klebsiella pneumoniae, and Shigella flexneri in vitro and in vivo. The results were associated with suppression of rpoD mRNA and σ70 expression, as well as σ70 downstream regulated genes including ftsZ, mazF, prfB, rpoS, seqA, turfB and ygjD. The treatment of PPNA06 on mono- or multiple MDR-GBN infected human gastric mucosal epithelial cells demonstrated the complete inhibition on bacterial growth and no influence on morphology and growth of human cells. Also, PPNA06 did not show the induction of antibiotic resistance as compared with classical antibiotics in GNB. These findings firstly demonstrate that rpoD is potential target for developing antisense antibiotics, and indicate that peptide conjugates of anti-rpoD PNA are active against GNBs in vitro and in vivo. Our results offer a feasible strategy for treating MDR-GNB infections.
Keywords: Cell-penentrating peptide conjugated peptide nucleic acid; Antisense therapy; Gram negative bacteria; Antibiotic resistance; RNA polymerase primary σ70;

Hydrogel swelling as a trigger to release biodegradable polymer microneedles in skin by MinYoung Kim; Bokyung Jung; Jung-Hwan Park (668-678).
Biodegradable polymeric microneedles were developed as a method for achieving sustained transdermal drug release. These microneedles have potential as a patient-friendly substitute for conventional sustained release methods. However, they have limitations related to the difficulty of achieving separation of the needles into the skin. We demonstrated that microneedle separation into the skin was mediated by hydrogel swelling in response to contact with body fluid after the needles were inserted into the skin. The hydrogel microparticles were synthesized by an emulsification method using poly-N-isopropylacrylamide (PNIPAAm). The microneedles were fabricated by micromolding poly-lactic-co-glycolic acid (PLGA) after filling the cavities of the mold with the hydrogel microparticles. The failure of microneedle tips caused by hydrogel swelling was studied in regard to contact with water, insertion of microneedles into porcine cadaver skin in vitro, stress–strain behavior, and insertion into the back skin of a hairless mouse in vivo. The drug delivery property of the hydrogel particles was investigated qualitatively by inserting polymer microneedles into porcine cadaver skin in vitro, and the sustained release property of PLGA microneedles containing hydrogel microparticles was studied quantitatively using the Franz cell model. The hydrogel particles absorbed water quickly, resulting in the cracking of the microneedles due to the difference in volume expansion between the needle matrix polymer and the hydrogel particles. The swollen particles caused the microneedles to totally breakdown, leaving the microneedle tips in the porcine cadaver skin in vitro and in the hairless mouse skin in vivo. Model drugs encapsulated in biodegradable polymer microneedles and hydrogel microparticles were successfully delivered by releasing microneedles into the skin.
Keywords: Biodegradable polymer; Microneedle; Swelling of hydrogel; Transdermal drug delivery;

Tumor stem cells have emerged as the new targets for anti-cancer therapy, besides tumor cells themselves. To eradicate both breast cancer cells and breast cancer stem cells which can not be eliminated by the conventional chemotherapy, octreotide (Oct)-modified paclitaxel (PTX)-loaded PEG-b-PCL polymeric micelles (Oct-M-PTX) and salinomycin (SAL)-loaded PEG-b-PCL polymeric micelles (M-SAL) were developed and investigated in combination. In this study, Oct that targets somatostatin receptors (SSTR) overexpressed in tumors including breast cancer, was coupled to the PEG end of PEG-b-PCL, and all the micelles were prepared using thin film hydration method. Results showed that the particle size of all the micelles was approximately 25–30 nm, and the encapsulation efficiency was >90%. Quantitative and qualitative analysis demonstrated that Oct facilitates the uptake of micelles in SSTR overexpressed breast cancer MCF-7 cells while free Oct inhibited cellular uptake of Oct-modified micelles, revealing the mechanism of receptor-mediated endocytosis. Breast cancer stem cells (side population cells, SP cells) were sorted from MCF-7 cells and identified with the CD44+/CD24− phenotype. M-SAL was capable of decreasing the proportion of SP cells, and its suppression was more potent in SP cells than that in cancer cells. As compared to PTX-loaded micelles (M-PTX), the inhibition of Oct-M-PTX against MCF-7 cells was stronger while such effect significantly increased when applying Oct-M-PTX in combination with M-SAL. In the MCF-7 xenografts, the combination therapy with Oct-M-PTX plus M-SAL produced the strongest antitumor efficacy, in accord with the combination treatment in vitro. Compared with free SAL, M-SAL was found to be more effective in suppressing breast cancer stem cells in vivo. Thus, this combination therapy may provide a strategy to improve treatment of breast cancers for eradication of breast cancer cells together with breast cancer stem cells.
Keywords: Breast cancer; Paclitaxel; Polymeric micelles; Somatostatin receptors; Cancer stem cells; Salinomycin;

Tissue uptake of docetaxel loaded hydrophobically derivatized hyperbranched polyglycerols and their effects on the morphology of the bladder urothelium by Clement Mugabe; Peter A. Raven; Ladan Fazli; Jennifer H.E. Baker; John K. Jackson; Richard T. Liggins; Alan I. So; Martin E. Gleave; Andrew I. Minchinton; Donald E. Brooks; Helen M. Burt (692-703).
Recently, we have reported that docetaxel (DTX) loaded, amine terminated hyperbranched polyglycerol (HPG-C8/10-MePEG-NH2) nanoparticles significantly increased drug uptake in mouse bladder tissues and was the most effective formulation to significantly inhibit tumor growth in an orthotopic model of bladder cancer. The objective of this study was to investigate the effects of HPG-C8/10-MePEG-NH2 nanoparticles on bladder urothelial morphology and integrity, DTX uptake and permeability in bladder tissue and the extent of bladder urothelial recovery following exposure to, and then washout of, HPG-C8/10-MePEG-NH2 nanoparticles. HPG-C8/10-MePEG-NH2 nanoparticles significantly increased the uptake of DTX in both isolated pig bladder as well as in live mouse bladder tissues. Furthermore, HPG-C8/10-MePEG-NH2 nanoparticles were demonstrated to increase the permeability of the urinary bladder wall by causing changes to the urothelial barrier function and morphology through opening of tight junctions and exfoliation of the superficial umbrella cells. These data suggest that exfoliation may be triggered by an apoptosis mechanism, which was followed by a rapid recovery of the urothelium within 24 h post-instillation of HPG-C8/10-MePEG-NH2 nanoparticles. HPG-C8/10-MePEG-NH2 nanoparticles cause significant but rapidly recoverable changes in the bladder urothelial morphology, which we believe may make them suitable for increasing drug permeability of bladder tissue and intravesical drug delivery.
Keywords: Bladder cancer; Intravesical therapy; Mucoadhesive; Hydrophobically derivatized hyperbranched polyglycerols; Urothelium; Exfoliation;

Concurrent blood–brain barrier opening and local drug delivery using drug-carrying microbubbles and focused ultrasound for brain glioma treatment by Chien-Yu Ting; Ching-Hsiang Fan; Hao-Li Liu; Chiung-Yin Huang; Han-Yi Hsieh; Tzu-Chen Yen; Kuo-Chen Wei; Chih-Kuang Yeh (704-712).
Glioblastoma multiforme (GBM) is a highly malignant brain tumor. The blood–brain barrier (BBB) provides a major obstacle to chemotherapy since therapeutic doses cannot be achieved by traditional drug delivery without severe systemic cytotoxic effects. Recently, microbubble (MB)-enhanced focused ultrasound (FUS) was shown to temporally and locally disrupt the BBB thereby enhancing drug delivery into brain tumors. Here we propose the concept of smart, multifunctional MBs capable of facilitating FUS-induced BBB disruption while serving as drug-carrying vehicles and protecting drugs from rapid degradation. The designed MBs had a high loading capacity (efficiency of 68.01 ± 4.35%) for 1,3-bis(2-chloroethyl)-1- nitrosourea (BCNU). When combined with FUS (1-MHz), these BCNU-MBs facilitated local BBB disruption and simultaneously released BCNU at the target site, thus increasing local BCNU deposition. Encapsulation of BCNU in MBs prolonged its circulatory half-life by 5-fold, and accumulation of BCNU in the liver was reduced 5-fold due to the slow reticuloendothelial system uptake of BCNU-MBs. In tumor-bearing animals, BCNU-MBs with FUS controlled tumor progression (915.3%–39.6%) and improved median survival (29 days–32.5 days). This study provides a new approach for designing multifunctional MBs to facilitate FUS-mediated chemotherapy for brain tumor treatment.
Keywords: Glioblastoma multiforme (GBM); Focused ultrasound (FUS); Microbubble (MB); Chemotherapy; Blood–brain barrier (BBB);

Controllable mineral coatings on PCL scaffolds as carriers for growth factor release by Darilis Suárez-González; Kara Barnhart; Francesco Migneco; Colleen Flanagan; Scott J. Hollister; William L. Murphy (713-721).
In this study, we have developed mineral coatings on polycaprolactone scaffolds to serve as templates for growth factor binding and release. Mineral coatings were formed using a biomimetic approach that consisted in the incubation of scaffolds in modified simulated body fluids (mSBF). To modulate the properties of the mineral coating, which we hypothesized would dictate growth factor release, we used carbonate (HCO3) concentration in mSBF of 4.2 mm, 25 mm, and 100 mm. Analysis of the mineral coatings formed using scanning electron microscopy indicated growth of a continuous layer of mineral with different morphologies. X-ray diffraction analysis showed peaks associated with hydroxyapatite, the major inorganic constituent of human bone tissue in coatings formed in all HCO3 concentrations. Mineral coatings with increased HCO3 substitution showed more rapid dissolution kinetics in an environment deficient in calcium and phosphate but showed re-precipitation in an environment with the aforementioned ions. The mineral coating provided an effective mechanism for growth factor binding and release. Peptide versions of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP2) were bound with efficiencies up to 90% to mineral mineral-coated PCL scaffolds. We also demonstrated sustained release of all growth factors with release kinetics that were strongly dependent in the solubility of the mineral coating.
Keywords: Polycaprolactone; Growth factor; Bone morphogenetic protein 2; Vascular endothelial growth factor; Hydroxyapatite; Bone tissue engineering;

The effect of zoledronate-hydroxyapatite nanocomposites on osteoclasts and osteoblast-like cells in vitro by Elisa Boanini; Paola Torricelli; Massimo Gazzano; Milena Fini; Adriana Bigi (722-730).
This study demonstrates that zoledronate containing hydroxyapatite nanocrystals (HA-ZOL) can be synthesized as a single crystalline phase up to a zoledronate content of about 7 wt% by direct synthesis in aqueous solution, at variance with what previously found for alendronate-hydroxyapatite nanocrystals (HA-AL). On increasing zoledronate incorporation, the length of the coherent crystalline domains and the crystal dimensions of hydroxyapatite decrease, whereas the specific surface area increases. Full profile fitting of the powder X-ray diffraction patterns does not indicate major structural modifications, but an increase of the hydroxyapatite unit cell, on increasing zoledronate content. These data, together with a structural similarity between hydroxyapatite and calcium zoledronate, suggest a preferential interaction between zoledronate and the hydroxyapatite faces parallel to the c-axis direction. Osteoblast-like MG-63 cells and human osteoclasts were cultured on HA-ZOL nanocrystals and as a comparison on HA-AL nanocrystals containing almost the same (about 7 wt%) bisphosphonate amount. The beneficial influence of bisphosphonates on osteoblast proliferation and differentiation is enhanced when the tests are performed in co-cultures. Similarly, the reduction of osteoclast proliferation and the increase of Caspase 3 production are dramatically enhanced in co-cultures, which highlight an even greater influence of HA-ZOL than HA-AL on osteoclast apoptosis.
Keywords: Bisphosphonates; Hydroxyapatite; Osteoblast; Osteoclast; Co-cultures;