Biomaterials (v.32, #17)

Size dependent induction of proinflammatory cytokines and cytotoxicity of particulate beta-tricalciumphosphate in vitro by Tobias Lange; Arndt F. Schilling; Fabian Peters; Jan Mujas; Daniel Wicklein; Michael Amling (4067-4075).
Cellular responses to particulate calcium phosphate ceramics can lead to inflammatory reactions under certain conditions that depend on particle composition, size and morphology. In this context, the potential influence of varying sizes of particulate beta-tricalciumphosphate (beta-TCP) on the induction of inflammation and cytotoxicity remains to be determined. The present work investigates the effects of beta-TCP particles of five different sizes (1, 3, 13, 32 and 40 μm) on human peripheral blood mononuclear cells (PBMC) in vitro concerning the release of TNF-alpha, IL-1beta and IL-8 after six and 18 h of incubation (ELISA) as well as intracellular TNF-alpha, IFN-gamma, IL-1alpha, IL-1beta and IL-8 levels within distinct PBMC subpopulations after 12 h (FACS). Potential cytotoxic effects were determined by assaying lactate dehydrogenase (LDH) and morphological analyses (electron microscopy). Beta-TCP 1 μm did not induce any cytokine after 6 h but slightly increases TNF-alpha, IL-1beta and IL-8 release after 18 h. Larger particles (32 and 40 μm) consistently caused higher levels of cytokine release by increasing the fraction of cytokine producing monocytes. They also caused higher levels of LDH release as did smaller, phagocytosable particles. These data suggest a less inflammatory and cytotoxic profile of beta-TCP devices with a smaller primary particle size when compared to larger particles.
Keywords: Beta-tricalciumphosphate; Particle size; Human PBMC; Inflammation; Cytotoxicity; Phagocytosis;

Human corneal endothelial cell growth on a silk fibroin membrane by Peter W. Madden; Jonathan N.X. Lai; Karina A. George; Talia Giovenco; Damien G. Harkin; Traian V. Chirila (4076-4084).
Tissue engineering of the cornea could overcome shortages of donor corneas for transplantation and improve quality. Our aim was to grow an endothelial layer on a substratum suitable for transplant. Silkworm (Bombyx mori) fibroin was prepared as 5 μm thick transparent membranes. The B4G12 cell line was used to assess attachment and growth of human corneal endothelial cells on fibroin and compare this with a reference substratum of tissue-culture plastic. To see if cell attachment and proliferation could be improved, we assessed coatings of collagen IV, FNC Coating Mix® and a chondroitin sulphate–laminin mixture. All the coatings improved the final mean cell count, but consistently higher cell densities were achieved on a tissue-culture plastic rather than fibroin substratum. Collagen-coated substrata were the best of both groups and collagen-coated fibroin was comparable to uncoated tissue-culture plastic. Only fibroin with collagen coating achieved cell confluency. Primary human corneal endothelial cells were then grown using a sphere-forming technique and when seeded onto collagen-coated fibroin they grew to confluency with polygonal morphology. We report the first successful growth of primary human corneal endothelial cells on coated fibroin as a step in evaluating fibroin as a substratum for the transplantation of tissue-constructs for endothelial keratoplasty.
Keywords: Silk; Fibroin; Cornea; Endothelium; Cell culture; Collagen;

A proteomic analysis of engineered tendon formation under dynamic mechanical loading in vitro by Yongkang Jiang; Hongwei Liu; Hong Li; Fangjun Wang; Kai Cheng; Guangdong Zhou; Wenjie Zhang; Mingliang Ye; Yinlin Cao; Wei Liu; Hanfa Zou (4085-4095).
Previous studies have demonstrated the beneficial effect of mechanical loading on in vitro tendon engineering. To understand the mechanism, human tenocytes and polyglycolic acid long fibers were used for in vitro tendon engineering in a bioreactor system for 12 weeks with and without dynamic loading. The engineered neo-tendons were subjected to proteomic analysis using mass spectrometry along with shotgun strategy. As expected, mechanical loading resulted in a more mature tendon tissue characterized by a firmer tissue texture and densely deposited matrices which formed longitudinally aligned collagen fibers in a highly compact fashion. In contrast, non-loaded neo-tendon revealed loosely and less deposited matrices in a relatively less organized pattern. Proteins isolated from two groups of tissues exhibited similar distribution of isoeletric point and molecular weight indicating the similarity and comparability of the tissue specimens. Further, proteomic analysis showed that total 758 proteins were identified from both groups with 194 and 177 proteins uniquely presented in loaded and non-loaded tendons, respectively. Comparison of loaded and non-loaded tendons revealed 195 significantly up-regulated proteins and 189 significantly down-regulated proteins. The differentially expressed proteins could generally be classified into the categories of extracellular matrix, intra-cellular signaling, cytoskeleton and inflammatory response. Among them, significantly up-regulated collagens I and VI, MMP-14, WNT5A, microfilament molecules and some inflammatory factors suggest that the possible mechanism for this particular biological phenomenon may involve increased production of tendon specific matrices, enhanced cross-link of collagens and other matrix molecules, proper matrix remodeling for tissue maturation and mechanotransduction (including non-canonical Wnt signal pathway) mediated other biological processes.
Keywords: Mechanical loading; Proteomics; Human tenocytes; Tendon engineering; In vitro;

The pro-angiogenic properties of multi-functional bioactive glass composite scaffolds by Lutz-Christian Gerhardt; Kate L. Widdows; Melek M. Erol; Charles W. Burch; José A. Sanz-Herrera; Ignacio Ochoa; Rolf Stämpfli; Iman S. Roqan; Simon Gabe; Tahera Ansari; Aldo R. Boccaccini (4096-4108).
The angiogenic properties of micron-sized (m-BG) and nano-sized (n-BG) bioactive glass (BG) filled poly(D,L lactide) (PDLLA) composites were investigated. On the basis of cell culture work investigating the secretion of vascular endothelial growth factor (VEGF) by human fibroblasts in contact with composite films (0, 5, 10, 20 wt %), porous 3D composite scaffolds, optimised with respect to the BG filler content capable of inducing angiogenic response, were produced. The in vivo vascularisation of the scaffolds was studied in a rat animal model and quantified using stereological analyses. The prepared scaffolds had high porosities (81–93%), permeability (k = 5.4–8.6 × 10−9 m2) and compressive strength values (0.4–1.6 MPa) all in the range of trabecular bone. On composite films containing 20 wt % m-BG or n-BG, human fibroblasts produced 5 times higher VEGF than on pure PDLLA films. After 8 weeks of implantation, m-BG and n-BG containing scaffolds were well-infiltrated with newly formed tissue and demonstrated higher vascularisation and percentage blood vessel to tissue (11.6–15.1%) than PDLLA scaffolds (8.5%). This work thus shows potential for the regeneration of hard-soft tissue defects and increased bone formation arising from enhanced vascularisation of the construct.
Keywords: Angiogenesis; Bioactive glass; Composite scaffold; Vascular endothelial growth factor; Bone; Vascularisation;

The role of single cell derived vascular resident endothelial progenitor cells in the enhancement of vascularization in scaffold-based skin regeneration by Ziyang Zhang; Wulf D. Ito; Ursula Hopfner; Björn Böhmert; Mathias Kremer; Ann K. Reckhenrich; Yves Harder; Natalie Lund; Charli Kruse; Hans-Günther Machens; José T. Egaña (4109-4117).
Increasing evidence suggests that vascular resident endothelial progenitor cells (VR-EPCs) are present in several organs, playing an important role in postnatal neovascularization. Here, we isolated and characterized VR-EPCs from cardiac tissue in vitro, evaluating their regenerative potential in vivo. VR-EPCs showed to be highly clonogenic and expressed several stem and differentiation markers. Under endothelial differentiation conditions, cells form capillary-like structures, in contrast to osteogenic or adipogenic differentiation conditions where no functional changes were observed. After seeding in scaffolds, cells were distributed homogeneously and directly attached to the scaffold. Then, cell seeded scaffolds were used to induce dermal regeneration in a nude mice full skin defect model. The presence of VR-EPCs enhanced dermal vascularization. Histological assays showed increased vessel number (p < 0.05) and cellularization (p < 0.05) in VR-EPCs group. In order to explore possible mechanisms of vascular regeneration, in vitro experiments were performed. Results showed that pro-angiogenic environments increased the migration capacity (p < 0.001) and ability to form capillary-like structures (p < 0.05) of VR-EPC. In addition, VR-EPCs secreted several pro-angiogenic molecules including VEGF and PDGF. These results indicate that a highly clonogenic population of VR-EPCs might be established in vitro, representing a new source for therapeutic vascularization in tissue engineering and regeneration.
Keywords: Tissue engineering; Tissue regeneration; Endothelial progenitor cells; Dermis; Vascularization;

N-acetylgalactosamine-functionalized dendrimers as hepatic cancer cell-targeted carriers by Scott H. Medina; Venkatesh Tekumalla; Maxim V. Chevliakov; Donna S. Shewach; William D. Ensminger; Mohamed E.H. El-Sayed (4118-4129).
There is an urgent need for novel polymeric carriers that can selectively deliver a large dose of chemotherapeutic agents into hepatic cancer cells to achieve high therapeutic activity with minimal systemic side effects. PAMAM dendrimers are characterized by a unique branching architecture and a large number of chemical surface groups suitable for coupling of chemotherapeutic agents. In this article, we report the coupling of N-acetylgalactosamine (NAcGal) to generation 5 (G5) of poly(amidoamine) (PAMAM-NH2) dendrimers via peptide and thiourea linkages to prepare NAcGal-targeted carriers used for targeted delivery of chemotherapeutic agents into hepatic cancer cells. We describe the uptake of NAcGal-targeted and non-targeted G5 dendrimers into hepatic cancer cells (HepG2) as a function of G5 concentration and incubation time. We examine the contribution of the asialoglycoprotein receptor (ASGPR) to the internalization of NAcGal-targeted dendrimers into hepatic cancer cells through a competitive inhibition assay. Our results show that uptake of NAcGal-targeted G5 dendrimers into hepatic cancer cells occurs via ASGPR-mediated endocytosis. Internalization of these targeted carriers increased with the increase in G5 concentration and incubation time following Michaelis–Menten kinetics characteristic of receptor-mediated endocytosis. These results collectively indicate that G5-NAcGal conjugates function as targeted carriers for selective delivery of chemotherapeutic agents into hepatic cancer cells.
Keywords: Nanoparticle; Dendrimers; Drug delivery; Flow cytometry; Hepatocyte;

Bioengineering embryonic stem cell microenvironments for exploring inhibitory effects on metastatic breast cancer cells by Nurazhani Abdul Raof; Waseem K. Raja; James Castracane; Yubing Xie (4130-4139).
The recreation of an in vitro microenvironment to understand and manipulate the proliferation and migration of invasive breast cancer cells may allow one to put a halt to their metastasis capacity. Invasive cancer cells have been linked to embryonic stem (ES) cells as they possess certain similar characteristics and gene signatures. Embryonic microenvironments have the potential to reprogram cancer cells into a less invasive phenotype and help elucidate tumorigenesis and metastasis. In this study, we explored the feasibility of reconstructing embryonic microenvironments using mouse ES cells cultured in alginate hydrogel and investigated the interactions of ES cells and highly invasive breast cancer cells in 2D, 2&1/2D, and 3D cultures. Results showed that mouse ES cells inhibited the growth and tumor spheroid formation of breast cancer cells. The mouse ES cell microenvironment was further constructed and optimized in 3D alginate hydrogel microbeads, and co-cultured with breast cancer cells. Migration analysis displayed a significant reduction in the average velocity and trajectory of breast cancer cell locomotion compared to control, suggesting that bioengineered mouse ES cell microenvironments inhibited the proliferation and migration of breast cancer cells. This study may act as a platform to open up new options to understand and harness tumor cell plasticity and develop therapeutics for metastatic breast cancer.
Keywords: Stem cell; Breast cancer cell; Microenvironment; Co-culture; Alginate; Metastasis;

MRI assessment of blood outgrowth endothelial cell homing using cationic magnetoliposomes by Stefaan J. Soenen; Simon F. De Meyer; Tom Dresselaers; Greetje Vande Velde; Inge M. Pareyn; Kevin Braeckmans; Marcel De Cuyper; Uwe Himmelreich; Karen I. Vanhoorelbeke (4140-4150).
The use of contrast material to stimulate magnetic resonance imaging (MRI) of migrating cells has become an important area of research. In the present study, cationic magnetoliposomes (MLs) were used to magnetically label human blood outgrowth endothelial cells (BOECs) and follow their homing by magnetic resonance imaging (MRI). The biodistribution and functional integration capacity of BOECs, which have shown extensive promise as gene delivery vehicles, have thus far only rarely been investigated. MLs were avidly internalized by BOECs giving clear MRI contrast in phantom studies and the magnetic labeling did not affect cell proliferation, viability, morphology or homeostasis and elicited only minor reactive oxygen species levels. Intravenous injection of labeled BOECs was compared with injection of free MLs and unlabeled BOECs, resulting in homing of BOECs toward the liver and spleen, which was confirmed by histology. The MLs used offer great potential for cellular tracking studies by MRI when low levels of widely distributed cells are present. In particular, the use of these MLs will allow to evaluate the efficacy of new methods to enhance BOEC homing and integration to optimize their use as efficient vehicles for gene therapy.
Keywords: Blood outgrowth endothelial cells; Magnetic resonance imaging; Cytotoxicity; Nanoparticle; Magnetoliposome; Cell viability;

cRGD-functionalized, DOX-conjugated, and 64Cu-labeled superparamagnetic iron oxide nanoparticles for targeted anticancer drug delivery and PET/MR imaging by Xiaoqiang Yang; Hao Hong; Jamison J. Grailer; Ian J. Rowland; Alireza Javadi; Samuel A. Hurley; Yuling Xiao; Yunan Yang; Yin Zhang; Robert J. Nickles; Weibo Cai; Douglas A. Steeber; Shaoqin Gong (4151-4160).
Multifunctional and water-soluble superparamagnetic iron oxide (SPIO) nanocarriers were developed for targeted drug delivery and positron emission tomography/magnetic resonance imaging (PET/MRI) dual-modality imaging of tumors with integrin αvβ3 expression. An anticancer drug was conjugated onto the PEGylated SPIO nanocarriers via pH-sensitive bonds. Tumor-targeting ligands, cyclo(Arg-Gly-Asp-d-Phe-Cys) (c(RGDfC)) peptides, and PET 64Cu chelators, macrocyclic 1,4,7-triazacyclononane-N, N′, N″-triacetic acid (NOTA), were conjugated onto the distal ends of the PEG arms. The effectiveness of the SPIO nanocarriers as an MRI contrast agent was evaluated via an in vitro r2 MRI relaxivity measurement. cRGD-conjugated SPIO nanocarriers exhibited a higher level of cellular uptake than cRGD-free ones in vitro. Moreover, cRGD-conjugated SPIO nanocarriers showed a much higher level of tumor accumulation than cRGD-free ones according to non-invasive and quantitative PET imaging, and ex vivo biodistribution studies. Thus, these SPIO nanocarriers demonstrated promising properties for combined targeted anticancer drug delivery and PET/MRI dual-modality imaging of tumors.
Keywords: Superparamagnetic iron oxide; Drug delivery; Positron emission tomography (PET); Magnetic resonance imaging (MRI); Nanomedicine;

Monodisperse core–shell structured up-conversion Yb(OH)CO3@YbPO4:Er3+ hollow spheres as drug carriers by Zhenhe Xu; Ping’an Ma; Chunxia Li; Zhiyao Hou; Xuefeng Zhai; Shanshan Huang; Jun Lin (4161-4173).
In this work, we report a facile solution–phase synthesis of monodisperse core–shell structured Yb-(OH)CO3@YbPO4 hollow spheres (size around 380 nm) by utilizing the colloidal sphere of Yb(OH)CO3 as the sacrificial template via the Kirkendall effect. The Er3+ doped Yb(OH)CO3@YbPO4 core–shell hollow spheres can be prepared similarly, which exhibit strong green emission under 980 nm NIR laser excitation even after loading with drug molecules. Most importantly, the sample can be used as an effective drug delivery carrier. The biocompatibility test on L929 fibroblast cells using MTT assay reveals low cytotoxicity of the system. A typical anticancer drug, doxorubicin hydrochloride (DOX), is used for drug loading, and the release properties, cytotoxicity, uptake behavior and therapeutic effects were examined. It is found that DOX is shuttled into cell by core–shell hollow spheres carrier and released inside cells after endocytosis, and the DOX-loaded spheres exhibited greater cytotoxicity than free DOX. These results indicate that the core–shell Er3+ doped Yb(OH)CO3@YbPO4 hollow spheres have potential for drug loading and delivery into cancer cells to induce cell death.
Keywords: Core–shell; Doxorubicin; Drug delivery; Hydrothermal synthesis; Up-conversion luminescence;

A gene delivery system based on the N-terminal domain of human topoisomerase I by Yi-An Chen; Hsiao-Che Kuo; Young-Mao Chen; Shin-Yi Huang; Yu-Ru Liu; Su-Ching Lin; Huey-Lang Yang; Tzong-Yueh Chen (4174-4184).
The N-terminal 200 amino acid residues of topoisomerase I (TopoN) is highly positive in charge and has DNA binding activity, without DNA sequence and topological specificity. Here, a fusion protein (6 × His-PTD-TopoN) containing a hexahistidine (6 × His) tag, a membrane penetration domain and TopoN (amino acid 3–200) was designed and developed. The protein can bind to different sizes (3.0–8.0 kb) and forms (circular and linear) of DNA and translocates the bound DNA to the nucleus. The protein also showed low cytotoxicity to GF-1 grouper fish fin cells that were previously very sensitive and difficult to transfect in vitro. Maintaining the hexahistidine tag increased the protein’s transfection efficiency in COS7 African green monkey kidney cells and simplified the purification process. The plasmid pEGFP-N1 was delivered into COS7 cells by the protein in ATP- and temperature-dependent manners. The results indicate that the binding ability of TopoN is very useful for DNA delivery and the carrier protein can be expressed in Escherichia coli without removal of the hexahistidine tag.
Keywords: Gene delivery system; Protein transduction domain; Topoisomerase I; Membrane penetration domain; DNA binding;

Enhanced in vivo gene transfer into the placenta using RGD fiber-mutant adenovirus vector by Kazufumi Katayama; Rie Furuki; Hideaki Yokoyama; Makoto Kaneko; Masashi Tachibana; Ichiro Yoshida; Hisamitsu Nagase; Keiichi Tanaka; Fuminori Sakurai; Hiroyuki Mizuguchi; Shinsaku Nakagawa; Tsuyoshi Nakanishi (4185-4193).
Among viral vectors, the fiber-mutant adenovirus vector carrying the Arg-Gly-Asp (RGD) peptide sequence (Ad-RGD) seems to have potential for both clinical gene therapy and basic research. As a part of a thorough evaluation of Ad-RGD in preclinical studies, we designed an experiment to investigate in detail the distribution of Ad-RGD compared with conventional adenovirus vector (WT-Ad) in pregnant mice. Surprisingly, Ad-RGD had substantial placental tropism, at 10–100 times that of WT-Ad. Transgene expression was sustained for at least 7 days, and Ad-RGD expressing firefly luciferase or red fluorescent protein has so far caused no placental dysfunction leading to fetal death. Ad-RGD showed high levels of transduction efficiency in in vitro-differentiated trophoblast stem cells, in which higher expression of αvβ3 integrin than in undifferentiated cells was observed. Our results suggest that the use of Ad-RGD or another RGD-mediated targeting strategy holds promise for drug delivery to the placenta.
Keywords: Gene expression; Gene transfer; Adenovirus; RGD peptide; Drug delivery;