Biomaterials (v.32, #15)

Amniotic liquid derived stem cells as reservoir of secreted angiogenic factors capable of stimulating neo-arteriogenesis in an ischemic model by Teodelinda Mirabella; Michele Cilli; Sebastiano Carlone; Ranieri Cancedda; Chiara Gentili (3689-3699).
Most urgent health problems are related to a blood vessel formation failure. The use of stem cells from different sources or species for both in vitro and in vivo engineering of endothelium does not necessarily imply their direct commitment towards a vascular phenotype. In the present study, we used human amniotic fluid stem cells (AFSC) to evoke a strong angiogenic response in murine recipients, in terms of host guided-regeneration of new vessels, and we demonstrated that the AFSC secretome is responsible for the vascularising properties of these cells. We indentified in AFSC conditioned media (ACM) pro-angiogenic soluble factors, such as MCP-1, IL-8, SDF-1, VEGF. Our in vitro results suggest that ACM are cytoprotective, pro-differentiative and chemoattractive for endothelial cells. We also tested ACM on a pre-clinical model of hind-limb ischemic mouse, concluding that ACM contain mediators that promote the neo-arteriogenesis, as remodelling of pre-existing collateral arteries to conductance vessels, thus preventing the capillary loss and the tissue necrosis of distal muscles. In line with the current regenerative medicine trend, in the present study we assert the concept that stem cell-secreted mediators can guide the tissue repair by stimulating or recruiting host reparative cells.
Keywords: Stem cells; Growth factors; Cytokines; In vivo test; Regenerative medicine;

Engineering the cell–material interface for controlling stem cell adhesion, migration, and differentiation by Ramses Ayala; Chao Zhang; Darren Yang; Yongsung Hwang; Aereas Aung; Sumeet S. Shroff; Fernando T. Arce; Ratnesh Lal; Gaurav Arya; Shyni Varghese (3700-3711).
The effective utilization of stem cells in regenerative medicine critically relies upon our understanding of the intricate interactions between cells and their extracellular environment. While bulk mechanical and chemical properties of the matrix have been shown to influence various cellular functions, the role of matrix interfacial properties on stem cell behavior is unclear. Here, we report the striking effect of matrix interfacial hydrophobicity on stem cell adhesion, motility, cytoskeletal organization, and differentiation. This is achieved through the development of tunable, synthetic matrices with control over their hydrophobicity without altering the chemical and mechanical properties of the matrix. The observed cellular responses are explained in terms of hydrophobicity-driven conformational changes of the pendant side chains at the interface leading to differential binding of proteins. These results demonstrate that the hydrophobicity of the extracellular matrix could play a considerably larger role in dictating cellular behaviors than previously anticipated. Additionally, these tunable matrices, which introduce a new control feature for regulating various cellular functions offer a platform for studying proliferation and differentiation of stem cells in a controlled manner and would have applications in regenerative medicine.
Keywords: Mesenchymal stem cells; Stem cell differentiation; Cell migration; Cytoskeletal organization; Hydrogel matrices; Hydrophobicity;

Bioorthogonal dual functionalization of self-assembling peptide fibers by Zahra N. Mahmoud; Smita B. Gunnoo; Andrew R. Thomson; Jordan M. Fletcher; Derek N. Woolfson (3712-3720).
The ability to modify peptide- and protein-based biomaterials selectively under mild conditions and in aqueous buffers is essential to the development of certain areas of bionanotechnology, tissue engineering and synthetic biology. Here we show that Self-Assembling peptide Fibers (SAFs) can incorporate multiple modified peptides non-covalently, stoichiometrically and without disrupting their structure or stability. The modified peptides contain groups suitable for post-assembly click reactions in water, namely azides and alkenes. Labeling of these groups is achieved using the orthogonal Cu(I)-catalyzed azide-alkyne and photoinitiated thiol-ene reactions, respectively. Functionalization is demonstrated through the conjugation of biotin followed by streptavidin-nanogold particles, or rhodamine, and visualized by electron and light microscopy, respectively. This has been shown for fibers harboring either or both of the modified peptides. Furthermore, the amounts of each modified peptide in the fibers can be varied with concomitant changes in decoration. This approach allows the design and assembly of fibers with multiple functional components, paving the way for the development of multi-component functionalized systems.
Keywords: Bioconjugation; Self-assembly; Coiled coil; Fibrous materials; Functionalization;

Viability and functionality of cells delivered from peptide conjugated scaffolds by Voranaddha Vacharathit; Eduardo A. Silva; David J. Mooney (3721-3728).
Many cell-based therapies aim to transplant functional cells to revascularize damaged tissues and ischemic areas. However, conventional cell therapy is not optimally efficient: massive cell death, damage, and non-localization of cells both spatially and temporally all likely contribute to poor tissue functionality. An alginate cell depot system has been proposed as an alternative means to deliver outgrowth endothelial cells (OECs) in a spatiotemporally controllable manner while protecting them in the early stages of tissue re-integration. Here OECs exiting the alginate scaffold were measured for viability, functionality, and migration speed and characterized for cytokine and surface marker profiles. OECs were highly viable in the alginate and were depleted from the scaffold via migration at a speed of 21 ± 6 μm/h following release. Prolonged interaction with the alginate scaffold microenvironment did not detrimentally change OECs; they retained high functionality, displayed a similar angiogenic cytokine profile as control OECs, and did not have significantly altered surface markers. These results suggest that alginate-OEC interactions do not adversely affect these cells, validating control of cellular migration as a means to control the cell delivery profile from the material system, and supporting usage of the alginate scaffold as an efficient cell delivery vehicle.
Keywords: Angiogenesis; RGD peptide; Cell adhesion; Cell activation; Biomimetic material; Alginate;

Embryonic body formation using the tapered soft stencil for cluster culture device by Hiroshi Yukawa; Hirofumi Noguchi; Shuji Hayashi (3729-3738).
Induced pluripotent stem (iPS) cells are expected to provide a source of tissue, a renewable cell source for tissue engineering, and a method for in vitro drug screening for patient-specific or disease-specific treatment. A simple technology by which iPS cells can be differentiated effectively and in large quantities is strongly desired. In this paper, a new device (Tapered Soft Stencil for Cluster Culture: TASCL) is proposed for the easy and efficient formation of EBs which can be used in regenerative medicine. This device was compared with the two major methods currently being evaluated, namely the HD method and the Terasaki® plate (MWC substitution), in terms of the efficiency, morphology and acquired number of EB formation. Using the TASCL device, the shape of the EBs formed was almost a perfect sphere, and the formation was also faster than for the two other methods. There was little variability in the number of cells. Moreover, EBs formed using the TASCL device had the ability to differentiate into all three germ layers, and differentiation of EBs from the TASCL culture into hepatic cells was confirmed. In conclusion, it appears that the TASCL device can be utilized for EB formation to generate cells for regenerative medicine applications.
Keywords: Embryonic body (EB); Induced pluripotent stem (iPS) cells; Hanging drop (HD); Multi-well chip (MWC); Tapered soft stencil for cluster culture (TASCL);

Maintenance of phenotype and function of cryopreserved bone-derived cells by Shaoyi Wang; Jun Zhao; Wenjie Zhang; Dongxia Ye; Wenwen Yu; Chao Zhu; Xiuli Zhang; Xiaojuan Sun; Chi Yang; Xinquan Jiang; Zhiyuan Zhang (3739-3749).
The emerging fields of tissue engineering and regenerative medicine require large numbers of cells for therapy. Although the properties of cells obtained from a variety of fresh tissues have been delineated, the knowledge regarding cryopreserved grafts-derived cells remains elusive. Previous studies have shown that living cells could be isolated from cryopreserved bone grafts. However, whether cryopreserved bone-derived cells can be applied in regenerative medicine is largely unknown. The present study was to evaluate the potential application of cryopreserved grafts-derived cells for tissue regeneration. We showed that cells derived from cryopreserved bone grafts could maintain good proliferation activity and osteogenic phenotype. The biological phenotype of these cells could be well preserved. The transplantation of cryopreserved bone-derived cells on scaffold could promote new bone formation in nude mice and enhance the osteointegration for dental implants in canine, which confirmed their osteogenic capacity, and showed that cells derived from cryopreserved bone were comparable to that of fresh bone in terms of the ability to promote osteogenesis in vivo. This work demonstrates that cryopreserved bone grafts may represent a novel, accessible source of cells for tissue regeneration therapy, and the results of our study may also stimulate the development of other cryopreservation techniques in basic and clinical studies.
Keywords: Cryopreservation; Bone; Cell; Tissue engineering; Regenerative medicine;

The influence of stereolithographic scaffold architecture and composition on osteogenic signal expression with rat bone marrow stromal cells by Kyobum Kim; David Dean; Jonathan Wallace; Rob Breithaupt; Antonios G. Mikos; John P. Fisher (3750-3763).
Scaffold design parameters, especially physical construction factors such as mechanical stiffness of substrate materials, pore size of 3D porous scaffolds, and channel geometry, are known to influence the osteogenic signal expression and subsequent differentiation of a transplanted cell population. In this study of photocrosslinked poly(propylene fumarate) (PPF) and diethyl fumarate (DEF) scaffolds, the effect of DEF incorporation ratio and pore size on the osteogenic signal expression of rat bone marrow stromal cells (BMSCs) was investigated. Results demonstrated that DEF concentrations and pore sizes that led to increased scaffold mechanical stiffness also upregulated osteogenic signal expression, including bone morphogenic protein-2 (BMP-2), fibroblast growth factors-2 (FGF-2), transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), and Runx2 transcriptional factor. Similar scaffold fabrication parameters supported rapid BMSC osteoblastic differentiation, as demonstrated by increased alkaline phosphatase (ALP) and osteocalcin expression. When scaffolds with random architecture, fabricated by porogen leaching, were compared to those with controlled architecture, fabricated by stereolithography (SLA), results showed that SLA scaffolds with the highly permeable and porous channels also have significantly higher expression of FGF-2, TGF-β1, and VEGF. Subsequent ALP expression and osteopontin secretion were also significantly increased in SLA scaffolds. Based upon these results, we conclude that scaffold properties provided by additive manufacturing techniques such as SLA fabrication, particularly increased mechanical stiffness and high permeability, may stimulate dramatic BMSC responses that promote rapid bone tissue regeneration.
Keywords: Osteogenic signal expression; Stereolithography; Stiffness; Pore geometry; Bone marrow stromal cells; Poly(propylene fumarate);

Peripheral nerve regeneration using a microporous polylactic acid asymmetric conduit in a rabbit long-gap sciatic nerve transection model by Shan-hui Hsu; Shan-Ho Chan; Chih-Ming Chiang; Clayton Chi-Chang Chen; Ching-Fen Jiang (3764-3775).
The performance of an asymmetric conduit made of microporous polylactic acid (PLA) in promoting the long-term peripheral nerve regeneration across a 20-mm-long sciatic nerve gap was evaluated by a rabbit sciatic nerve transection model. Magnetic resonance imaging (MRI) was employed to monitor the nerve regeneration process. The extents of nerve regeneration and conduit degradation were quantified by image analysis. Functional and histological analyses were followed to assess nerve reinnervation. MR images showed that the transected nerve was connected at about 4 months. The diameter of the regenerated nerve continued to increase while the conduit was gradually degraded. The conduit was completely degraded in 18 months. The degradation kinetics in vivo was estimated based on MR images. The functional recovery after 18 months was ∼82% based on electrophysiology. The extension range of the operated limb was slowly recuperated to ∼81% at 18 months. Histology showed that nerve bundles were self-assembled after 16–18 months, but the morphologies were still different from those of normal sciatic nerve. This was the first work on the long-term evaluation of peripheral nerve regeneration in a rabbit model, and the first to report the use of MRI to obtain the real-time images of regenerated nerve in a biomaterial conduit as well as to define the degradation rate of the conduit in vivo. The platform established in this study serves to evaluate the regeneration of larger-diameter (>3-mm) nerve across a long-gap bridged by a conduit.
Keywords: Nerve regeneration; Asymmetric nerve conduit; Magnetic resonance imaging (MRI); Functional recovery;

We developed a live imaging system enabling dynamic visualization of single cell alignment induced by external mechanical force in a 3-D collagen matrix. The alignment dynamics and migration of smooth muscle cells (SMCs) were studied by time lapse differential interference contrast and/or phase contrast microscopy. Fluorescent and reflection confocal microcopy were used to study the SMC morphology and the microscale collagen matrix remodeling induced by SMCs. A custom developed program was used to quantify the cell migration and matrix remodeling. Our system enables cell concentration-independent alignment eliminating cell-to-cell interference and enables dynamic cell tracking, high magnification observation and rapid cell alignment accomplished in a few hours compared to days in traditional models. We observed that cells sense and response to the mechanical signal before cell spreading. Under mechanical stretch the migration directionality index of SMCs is 46.3% more than those cells without external stretch; the dynamic direction of cell protrusion is aligned to that of the mechanical force; SMCs showed directional matrix remodeling and the alignment index calculated from the matrix in front of cell protrusions is about 3 fold of that adjacent to cell bodies. Our results indicate that the mechanism of cell alignment is directional cell protrusion. Mechano-sensing, directionality in cell protrusion dynamics, cell migration and matrix remodeling are highly integrated. Our system provides a platform for studying the role of mechanical force on the cell matrix interactions and thus finds strategies to optimize selected properties of engineered tissues.
Keywords: Molecular imaging; Cell signaling; ECM; Confocal microscopy; Collagen; Smooth muscle cell;

Electrospun sulfated silk fibroin nanofibrous scaffolds for vascular tissue engineering by Haifeng Liu; Xiaoming Li; Gang Zhou; Hongbin Fan; Yubo Fan (3784-3793).
One of the major downfalls of tissue-engineered small-diameter vascular grafts is the inability to obtain a confluent endothelium on the lumenal surface. Loosely attached endothelial cells (ECs) are easily separated from the vessel wall when exposed to the in vivo vascular system. Thus any denuded areas on the lumenal surface of vascular grafts may lead to thrombus formation via platelet deposition and activation. If the denuded areas could express anticoagulant activity until the endothelial cell lining is fully achieved, it may greatly improve the chances of successful vascular reconstruction. In this study, we fabricate sulfated silk fibroin nanofibrous scaffolds (S-silk scaffolds) and assess the anticoagulant activity and cytocompatibility of S-silk scaffolds in vitro in order to improve the antithrombogenicity and get some insights into its potential use for vascular tissue engineering. Sulfated silk fibroin was prepared by reaction with chlorosulphonic acid in pyridine, and then was developed to form an S-silk scaffold by electrospinning technique. FTIR analyses identified the successful incorporation of sulfate groups in silk fibroin molecules. It was found that the anticoagulant activity of S-silk scaffolds was significantly enhanced compared with silk fibroin nanofibrous scaffolds (Silk scaffolds). Vascular cells, including ECs and smooth muscle cells (SMCs), demonstrated strong attachment to S-silk scaffolds and proliferated well with higher expression of some phenotype-related marker genes and proteins. Overall, the data in this study suggest the suitability of S-silk scaffolds used along with vascular cells for the development of tissue-engineered vascular grafts.
Keywords: Sulfated silk fibroin; Electrospinning; Anticoagulant activity; Small-diameter; Vascular grafts;

The potential of celecoxib-loaded hydroxyapatite-chitosan nanocomposite for the treatment of colon cancer by P. Venkatesan; Nagaprasad Puvvada; Rupesh Dash; B.N. Prashanth Kumar; Devanand Sarkar; Belal Azab; Amita Pathak; Subhas C. Kundu; Paul B. Fisher; Mahitosh Mandal (3794-3806).
Celecoxib has shown potential anticancer activity against most carcinomas, especially in patients with familial adenomatous polyposis and precancerous disease of the colon. However, serious side effects of celecoxib restrict its generalized use for cancer therapy. In order to resolve these issues and develop an alternative strategy/preliminary approach, chitosan modified hydroxyapatite nanocarriers-mediated celecoxib delivery represents a viable strategy. We characterized the nanoparticle for morphology, particle size, zeta potential, crystalinity, functional group analysis, entrapment efficiency, drug release and hemocompatibility. The effects of celecoxib-loaded nanoparticles on colon cancer cell proliferation, morphology, cytoskeleton, cellular uptake and apoptosis were analysed in vitro. Further, we evaluated the antiproliferative, apoptotic and tumor inhibitory efficacy of celecoxib-loaded nanocarriers in a nude mouse human xenograft model. Nanoparticles exhibited small, narrow hydrodynamic size distributions, hemocompatibility, high entrapment efficiencies and sustained release profiles. In vitro studies showed significant antiproliferation, apoptosis and time-dependent cytoplasmic uptake of celecoxib-loaded Hap-Cht nanoparticles in HCT 15 and HT 29 colon cancer cells. Additional in vivo studies demonstrated significantly greater inhibition of tumor growth following treatment with this modified nanoparticle system. The present study indicates a promising, effective and safe means of using celecoxib, and potentially other therapeutic agents for colon cancer therapy.
Keywords: Nanocomposite; Celecoxib; Colon cancer; Inhibition; Xenograft;

MicroRNAs as participants in cytotoxicity of CdTe quantum dots in NIH/3T3 cells by Shuchun Li; Yong Wang; Haitao Wang; Yunfei Bai; Gaofeng Liang; Yuanyuan Wang; Ningping Huang; Zhongdang Xiao (3807-3814).
Epigenetic aspects of the cytotoxicity of CdTe quantum dots (QDs) recently have attracted more attention for their ability to reprogram gene expression after initial signals have been removed. And the involvement of epigenetic mechanisms in microRNA (miRNA) biogenesis suggests that miRNAs act as participants in the cytotoxicity of CdTe QDs. According to the results of SOLiD sequencing, the expression patterns of miRNAs are widely affected after CdTe QD exposure, resulting in the apoptosis-like cell death. Compared with 86 miRNAs with down-regulated expression, the expression levels of 121 miRNAs are up-regulated by CdTe QD treatment. The Z-test is used to find out miRNAs with significantly regulated expression, and the results indicate that the expression levels of 16 and 35 miRNAs are down- and up-regulated, respectively. And the expression levels of some significantly regulated miRNAs have time- and dose-dependent tendencies, which are similar to cell survival ratios affected by CdTe QDs. The fluctuations of miRNA expression start from the transcription of pri-miRNA, and are strengthened by the processing of pri-miRNA to pre-miRNA. As a regulator in miRNA biogenesis, p53 is involved in the transcription and processing of pri-miRNA. With no significant changes in the mRNA levels of p53, the increase in overall p53 protein levels and its post-translational modification by phosphorylation at Ser-15 are induced by CdTe QD treatment. Therefore, the differential expression of miRNAs are induced by CdTe QDs at the processing of miRNA biogenesis, which is an adaptive process of cells to external stimuli.
Keywords: MicroRNA; CdTe QDs; Cytotoxicity; p53; miRNA biogenesis;

Multifunctional 4-bit biomemory chip consisting of recombinant azurin variants by Taek Lee; Junhong Min; Sang-Uk Kim; Jeong-Woo Choi (3815-3821).
We developed a multi-functional 4-bit biomemory chip that consisted of recombinant azurin variants. The azurin was modified to introduce cysteine-residues. In addition, the Cu ion in this recombinant azurin protein was substituted with various other metal ions such as Co, Mn, Fe and Ni ion to allow the protein to perform various memory functions. Each metal-substituted recombinant protein was directly self-assembled attached onto Au surface via the thiol group of the cysteine. UV–VIS spectroscopy was performed to confirm the metal substitution. Atomic force microscopy was used to measure the film organization. Also, the 4 different azurin variants were investigated to assess the electrochemical behavior. Cyclic voltammetry and an open circuit potential indicated that the azurin variants had different redox peaks and specific open circuit potential values. Using these parameters, memory function was verified by chronoamperometry and open circuit potential amperometry. Therefore, a multi-bit biomemory chip was successfully developed. The results presented here provide a new approach, concept and material combination for the development of biomemory systems using recombinant protein. If a low electrochemical signal from a few single proteins could be achieved, it may be possible to substitute silicon-based memory devices with biological-based memory devices.
Keywords: Biomemory; Recombinant azurin; Atomic force microscopy; Cyclic voltammetry; Nanobiochip;

Effect of the dopant anion in polypyrrole on nerve growth and release of a neurotrophic protein by Brianna C. Thompson; Simon E. Moulton; Rachael T. Richardson; Gordon G. Wallace (3822-3831).
The dopant anion in polypyrrole plays a critical role in determining the physical and chemical properties of these conducting polymers. Here we demonstrate an additional effect on the ability to incorporate and release a neurotrophic protein – neurotrophin-3. The multi-faceted role of the dopant is critical in ensuring optimal performance of polypyrroles in their use as platforms for nerve growth. In this paper, the effect of changing the co-dopant used in electrochemical polypyrrole synthesis on the compatibility with primary auditory nerve tissue is considered and compared to some of the physical properties of the films. Significant differences in the controlled-release properties of the films were also observed. The ability of the polymers to enhance nerve growth and survival in vitro with neurotrophin-3 release was also studied, which is a function of both compatibility with the neural tissue and the ability of the polymer to release sufficient neurotrophic protein to affect cell growth. A small synthetic dopant, para-toluene sulphonate, was found to perform favourably in both aspects and ultimately proved to be the most suitable material for the application at hand, which is the delivery of neurotrophins for inner-ear therapies.
Keywords: Polypyrrole; Neurotrophin-3; Dopant; Electrical stimulation; Neural;

The fine-tuning of thermosensitive and degradable polymer micelles for enhancing intracellular uptake and drug release in tumors by Wei Li; Jinfeng Li; Jie Gao; Bohua Li; Yu Xia; Yanchun Meng; Yongsheng Yu; Huaiwen Chen; Jianxin Dai; Hao Wang; Yajun Guo (3832-3844).
Focusing on high temperature and low pH of tumor tissue, we prepared temperature and pH responsive poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide-b-lacitde) (PID 118 -b-PLA 59) and poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide-b-ε-caprolactone) (PID 118 -b-PCL 60) diblock copolymers with symmetric hydrophobic blocks by the reversible addition-fragmentation chain transfer (RAFT). The corresponding dual functional polymeric micelles were fabricated by dialysis methods. Their well-defined core-shell structure was characterized by 1H NMR in D2O and further confirmed by TEM. Their structural and physical chemistry properties such as diameters (D), core corona dimension (R core, R shell), distribution (PDI), M w, aggregation number (N agg), second virial coefficient (A 2), critical micellization concentration (CMC) and z-potential were firstly systemically investigated by dynamic and static laser light scattering. The volume phase transition temperature (VPTT) was around 40 °C above which the intracellular uptake of adriamycin (ADR) was significantly enhanced. Both flow cytometry and fluorescent microscopy showed that the ADR transported by these micelles was about 4 times higher than that by the commercial ADR formulation Taxotere®. In vitro cytotoxicity assay against N-87 cancer cell and confocal laser scanning microscopy (CLSM) also confirmed such promoting efficiency. In addition, it was interesting to find that cell surviving bounced back as T = 42 °C due to the inter-micellar aggregation. The well clarified mechanism strongly support that our finely tailored dual functional core-shell micelles are potent in enhancing cellular uptake and drug release.
Keywords: Fine synthesis and characterization; Block copolymer; Polymer micelle; Drug delivery;

Reconstitutable charged polymeric (PLGA)2-b-PEI micelles for gene therapeutics delivery by Deepa Mishra; Han Chang Kang; You Han Bae (3845-3854).
This study investigated the potential of creating a charged polymeric micelle-based nucleic acid delivery system that could easily be reconstituted by the addition of water. (PLGA36kDa)2-b-bPEI25kDa (PLGA MW 36 kDa, bPEI Mw 25 kDa, PLGA:bPEI block ratio = 2) was synthesized and used to prepare cationic micelles. The copolymer retained proton-buffering capability from the bPEI block within the endosomal pH range. Micelle/pDNA complexes retained their particle size (100–150 nm) and surface charge (30–40 mV) following reconstitution. It was found that adding a small amount of low molecular weight bPEI (1.8 kDa) completely shielded pDNA in the micelle/pDNA complexes and enhanced transfection efficiency 50–100 fold for both fresh and reconstituted complexes without affecting complex size. Transfection efficiency for “reconstituted” micelle/pDNA/bPEI1.8kDa (WR 1) complexes was 16-fold higher than its “fresh” counterpart. Although transfection levels achieved using “reconstituted” micelle/pDNA/bPEI1.8kDa complexes were 3.6-fold lower than control “fresh” bPEI25kDa/pDNA (N/P 5) complexes, transfection levels were 39-fold higher than “reconstituted” bPEI25kDa/pDNA (N/P 5) complexes. The micelle/pDNA/bPEI1.8kDa system showed very low cytotoxicity in MCF7 cells even with pDNA doses up to 20 μg, and transfection levels increased linearly with increasing pDNA dose. These results indicate that this PLGA-b-bPEI polymeric micelle-based system is well suited as a reconstitutable gene delivery system, and has high potential for use as a delivery system for gene therapy applications.
Keywords: Layer-by-layer; Polyethyleneimine; Poly(lactide-co-glycolide); Polymeric gene delivery; Polymeric micelles; Reconstitution;

The effect of BMP-2 on the osteoconductive properties of β-tricalcium phosphate in rat calvaria defects by Eloa R. Luvizuto; Stefan Tangl; Gerald Zanoni; Tetuo Okamoto; Celso K. Sonoda; Reinhard Gruber; Roberta Okamoto (3855-3861).
Bone formation in critical-sized calvaria defects is strongly dependent on the osteoconductive properties of grafts. It remains a matter of controversy whether biomaterials can replace autografts and whether the supplementation of biomaterials with Bone Morphogenetic Proteins (BMPs) is necessary to enhance bone formation. We examined rat calvaria critical-sized defects (5-mm-diameter) treated with β-tricalcium phosphate (TCP; Cerasorb® M), polylactic and polyglycolic acid gel (PLA/PGA; Fisiograft®) and calcium phosphate cement (CPC; Norian® CRS®), either alone or in the presence of 5 μg of BMP-2 after 45 days. Autografts and untreated defects served as controls. Bone formation was evaluated based on μCT analysis, histomorphometric analysis and fluorescence analysis. We report that TCP supported bone formation more efficiently than did autografts. Bone formation in the presence of TCP alone reached a maximal level, as BMP-2 supplementation failed to enhance bone formation. By contrast, no significant difference in bone formation was observed when PLA/PGA and CPC were compared to autografts. Moreover, the presence of BMP-2 did not substantially change the osteoconductive properties of PLA/PGA or CPC. We conclude that the osteoconductive properties of TCP are superior to those of autografts and that TCP does not require BMP-2 supplementation. Our findings also show that the decreased osteoconductive properties of PLA/PGA and CPC cannot be overcome by BMP-2 supplementation in rat calvaria defects.
Keywords: Animal model; Bone regeneration; BMP (bone morphogenetic protein); Calcium phosphate cement; Polylactic acid; Bone tissue engineering;

Integrin-assisted drug delivery of nano-scaled polymer therapeutics bearing paclitaxel by Anat Eldar-Boock; Keren Miller; Joaquin Sanchis; Ruth Lupu; María J. Vicent; Ronit Satchi-Fainaro (3862-3874).
Angiogenesis plays a prominent role in cancer progression. Anti-angiogenic therapy therefore, either alone or in combination with conventional cytotoxic therapy, offers a promising therapeutic approach. Paclitaxel (PTX) is a widely-used potent cytotoxic drug that also exhibits anti-angiogenic effects at low doses. However, its use, at its full potential, is limited by severe side effects. Here we designed and synthesized a targeted conjugate of PTX, a polymer and an integrin-targeted moiety resulting in a polyglutamic acid (PGA)-PTX-E-[c(RGDfK)2] nano-scaled conjugate. Polymer conjugation converted PTX to a macromolecule, which passively targets the tumor tissue exploiting the enhanced permeability and retention effect, while extravasating via the leaky tumor neovasculature. The cyclic RGD peptidomimetic enhanced the effects previously seen for PGA–PTX alone, utilizing the additional active targeting to the αvβ3 integrin overexpressed on tumor endothelial and epithelial cells. This strategy is particularly valuable when tumors are well-vascularized, but they present poor vascular permeability. We show that PGA is enzymatically-degradable leading to PTX release under lysosomal acidic pH. PGA-PTX-E-[c(RGDfK)2] inhibited the growth of proliferating αvβ3-expressing endothelial cells and several cancer cells. We also showed that PGA-PTX-E-[c(RGDfK)2] blocked endothelial cells migration towards vascular endothelial growth factor; blocked capillary-like tube formation; and inhibited endothelial cells attachment to fibrinogen. Orthotopic studies in mice demonstrated preferential tumor accumulation of the RGD-bearing conjugate, leading to enhanced anti-tumor efficacy and a marked decrease in toxicity as compared with free PTX-treated mice.
Keywords: Angiogenesis; Polymer therapeutics; Polyglutamic acid; Paclitaxel; RGD peptidomimetic; Integrin;

A simple nanocarrier of polyethylenimine-grafted graphene nanoribbon (PEI-g-GNR) was proposed as an effective gene vector. The GNR was formed by longitudinally unzipping multiwalled carbon nanotubes (MWCNTs), and treated with strong acids and sonication to obtain surface carboxylic acid groups for graft of PEI via electrostatic assembly. The PEI-g-GNR appeared to protect locked nucleic acid modified molecular beacon (LNA-m-MB) probes from nuclease digestion or single-strand binding protein interaction, thus could be used as a nanocarrier of the probes for more efficient transfection of cells than PEI or PEI-g-MWCNTs due to the large surface area of the GNR and high charge density of PEI. The cytotoxicity and apoptosis induced by the PEI-g-GNR were negligible under optimal transfection conditions. Combining with the remarkable affinity and specificity of LNA to microRNA (miRNA), a delivery system by the LNA-m-MB/PEI-g-GNR was proposed for effectively transferring LNA-m-MB into the cells to recognize the target miRNA. Using HeLa cells as model, a method for detection of miRNA in single cell was developed. These results suggested that PEI-g-GNR would be a promising nonviral vector for in situ detection of gene in cytoplasm and gene therapy in clinical application.
Keywords: Graphene nanoribbons; Nonviral gene vector; Locked nucleic acid; MicroRNA; Polyethylenimine; Cell transfection;