Biomaterials (v.33, #12)

Polycaprolactone electrospun mesh conjugated with an MSC affinity peptide for MSC homing in vivo by Zhenxing Shao; Xin Zhang; Yanbin Pi; Xiaokun Wang; Zhuqing Jia; Jingxian Zhu; Linghui Dai; Wenqing Chen; Ling Yin; Haifeng Chen; Chunyan Zhou; Yingfang Ao (3375-3387).
Mesenchymal stem cell (MSC) is a promising cell source candidate in tissue engineering (TE) and regenerative medicine. However, the inability to target MSCs in tissues of interest with high efficiency and engraftment has become a significant barrier for MSC-based therapies. The mobilization and transfer of MSCs to defective/damaged sites in tissues or organs in vivo with high efficacy and efficiency has been a major concern. In the present study, we identified a peptide sequence (E7) with seven amino acids through phage display technology, which has a high specific affinity to bone marrow-derived MSCs. Subsequent analysis suggested that the peptide could efficiently interact specifically with MSCs without any species specificity. Thereafter, E7 was covalently conjugated onto polycaprolactone (PCL) electrospun meshes to construct an “MSC-homing device” for the recruitment of MSCs both in vitro and in vivo. The E7-conjugated PCL electrospun meshes were implanted into a cartilage defect site of rat knee joints, combined with a microfracture procedure to mobilize the endogenous MSCs. After 7 d of implantation, immunofluorescence staining showed that the cells grown into the E7-conjugated PCL electrospun meshes yielded a high positive rate for specific MSC surface markers (CD44, CD90, and CD105) compared with those in arginine-glycine-aspartic acid (RGD)-conjugated PCL electrospun meshes (63.67% vs. 3.03%; 59.37% vs. 2.98%; and 61.45% vs. 3.82%, respectively). Furthermore, the percentage of CD68 positive cells in the E7-conjugated PCL electrospun meshes was much lower than that in the RGD-conjugated PCL electrospun meshes (5.57% vs. 53.43%). This result indicates that E7-conjugated PCL electrospun meshes absorb much less inflammatory cells in vivo than RGD-conjugated PCL electrospun meshes. The results of the present study suggest that the identified E7 peptide sequence has a high specific affinity to MSCs. Covalently conjugating this peptide on the synthetic PCL mesh significantly enhanced the MSC recruitment of PCL in vivo. This method provides a wide range of potential applications in TE.
Keywords: Phage display; Tissue engineering (TE); MSC-homing; Peptide-modified polycaprolactone electrospun mesh;

Adult stem cell-based tissue engineered blood vessels: A review by Jeffrey T. Krawiec; David A. Vorp (3388-3400).
With the high occurrence of cardiovascular disease and increasing numbers of patients requiring vascular access, there is a significant need for a small-diameter (<6 mm inner diameter) vascular graft that can provide long-term patency. Tissue engineering provides a very promising solution to this need. Approaches utilizing adult stem cells can address limitations previously realized in the use of terminally differentiated vascular cells, without introducing the ethical concerns that continue to limit the exploration and use of embryonic stem cells. This review summarizes the exciting work that has been reported on the application of adult stem cells to tissue engineered vascular grafts. Work in this area to date has employed bone marrow mononuclear progenitor cells, mesenchymal stem cells from various sources, and endothelial precursor cells.
Keywords: Arterial tissue engineering; Vascular grafts; Stem cell; Mesenchymal stem cell; Smooth muscle cell; Endothelial cell;

A rat decellularized small bowel scaffold that preserves villus-crypt architecture for intestinal regeneration by Giorgia Totonelli; Panagiotis Maghsoudlou; Massimo Garriboli; Johannes Riegler; Giuseppe Orlando; Alan J. Burns; Neil J. Sebire; Virpi V. Smith; Jonathan M. Fishman; Marco Ghionzoli; Mark Turmaine; Martin A. Birchall; Anthony Atala; Shay Soker; Mark F. Lythgoe; Alexander Seifalian; Agostino Pierro; Simon Eaton; Paolo De Coppi (3401-3410).
Management of intestinal failure remains a clinical challenge and total parenteral nutrition, intestinal elongation and/or transplantation are partial solutions. In this study, using a detergent-enzymatic treatment (DET), we optimize in rats a new protocol that creates a natural intestinal scaffold, as a base for developing functional intestinal tissue. After 1 cycle of DET, histological examination and SEM and TEM analyses showed removal of cellular elements with preservation of the native architecture and connective tissue components. Maintenance of biomechanical, adhesion and angiogenic properties were also demonstrated strengthen the idea that matrices obtained using DET may represent a valid support for intestinal regeneration.
Keywords: Intestinal failure; Decellularization; Natural acellular matrix; Tissue engineering; Regenerative medicine; Gut transplantation;

A hormone-responsive 3D human tissue-like culture system was developed in which human primary mammary epithelial cells (MECs) were co-cultured with two types of predominant mammary stromal cells on silk protein scaffolds. Silk porous scaffolds with incorporated extracellular matrix provided a compatible environment for epithelial structure morphogenesis and differentiation. The presence of stromal cells promoted MEC proliferation, induced both alveolar and ductal morphogenesis and enhanced casein expression. In contrast, only alveolar structures were observed in monocultures. The alveolar structures generated from the heterotypic cultures in vitro exhibited proper polarity similar to human breast tissue in vivo. Consistent with their phenotypic appearance, more functional differentiation of epithelial cells was also observed in the heterotypic cultures, where casein-α and mRNA expression were increased significantly. Additionally, this 3D multicellular culture model displayed an estrogen-responsive physiologically relevant response, evidenced by enhanced cell proliferation, aberrant morphology, changes in gene expression profile and few polarized lumen structures after estrogen treatment. This culture system offers an excellent opportunity to explore the role of cell–cell and cell–substrate interactions during mammary gland development, the consequences of hormone receptor activation on MEC behavior and morphogenesis, as well as their alteration during neoplastic transformation in human breast tissue.
Keywords: 3D tissue; Silk; Breast; Casein; Epithelial cells; Morphogenesis;

Ion-induced cell sheet detachment from standard cell culture surfaces coated with polyelectrolytes by Raphael Zahn; Elsa Thomasson; Orane Guillaume-Gentil; János Vörös; Tomaso Zambelli (3421-3427).
Polyelectrolyte multilayers (PEMs), formed by alternating layer-by-layer deposition of polyanions and polycations, are an ideal substrate for controlling cellular adhesion and behavior. In the present study we propose a simple mechanism for the controlled detachment of C2C12 myoblasts cell sheets from PEMs consisting of poly(l-lysine) and hyaluronic acid with a topmost layer of fibronectin. The multilayers were deposited on two standard cell culture surfaces: glass and polystyrene. Adding a low concentration of nontoxic ferrocyanide to the cell culture medium resulted in erosion of the polyelectrolyte multilayer and rapid detachment of viable cell sheets. Additional Quartz Crystal Microbalance and Atomic Force Microscopy measurements indicated that the detached cells retained their extracellular matrix and that no polyelectrolyte molecules remained bound to the cell sheets. The dissolution of polyelectrolyte multilayers by multivalent ions is a promising approach to cell sheet engineering that could potentially be used for regenerative medicine.
Keywords: Cell sheet; Tissue engineering; Polyelectrolyte multilayer films; Layer-by-layer films; Surface modification;

The use of hyaluronan to regulate protein adsorption and cell infiltration in nanofibrous scaffolds by Linhao Li; Yuna Qian; Chao Jiang; Yonggang Lv; Wanqian Liu; Li Zhong; Kaiyong Cai; Song Li; Li Yang (3428-3445).
Electrospun nanofibers are prepared with mixtures of natural and synthetic polymers that can behave cooperatively to demonstrate combinations of mechanical, structural and biochemical properties for tissue engineering applications. However, the large surface area and inherent small pores of these structures give nanofibrous scaffolds high non-specific protein adsorption and poor cell infiltration. In this study, we developed a protein resistant and porous nanofibrous scaffold composed of hyaluronan (HA), silk fibroin (SF), and polycaprolactone (PCL) blends via one-step emulsion electrospinning. The scaffolds were characterized and evaluated for nanostructures, chemical composition, mechanical properties, hydrophilicity, and protein adsorption. Swelling and degradation studies revealed the formation of oriented pore structures within the body of the scaffolds and increasing the pore size between fibers. Addition of HA component transformed current PCL/SF components into hydrophilic fibers, which caused the suppression of non-specific protein adsorption, resulting in the reduction of fibrosis tissue thickness and macrophages adhesion in vivo. Importantly, HA-based scaffolds significantly enhanced cell infiltration in vitro and tissue ingrowth in vivo. In vitro cultivation of human primary skin fibroblasts on the HA-based scaffolds showed a significant increase in cell proliferation and filopodia protrusions, but decreased in collagen I production. Furthermore, HA and HA-based scaffolds interacted with cell surface receptor CD44 to activate TGF-β1/MMPs signaling pathways that conducive to cell migration. These findings suggest that such an HA-based nanofibrous scaffold resists protein adsorption and enhances cell infiltration, may offer possibilities to overcome the limitations of electrospinning technology.
Keywords: Electrospun; Hyaluronan/hyaluronic acid; Porous; Protein adsorption; Cell infiltration;

Transplanted or endogenous neural stem cells often lack appropriate matrix in cavitary lesions in the central nervous system. In this study, gelatin-hydroxyphenylpropionic acid (Gtn-HPA), which could be enzymatically crosslinked with independent tuning of crosslinking degree and gelation rate, was explored as an injectable hydrogel for adult neural stem cells (aNSCs). The storage modulus of Gtn-HPA could be tuned (449–1717 Pa) to approximate adult brain tissue. Gtn-HPA was cytocompatible with aNSCs (yielding high viability >93%) and promoted aNSC adhesion. Gtn-HPA demonstrated a crosslinking-based approach for preconditioning aNSCs and increased the resistance of aNSCs to oxidative stress, improving their viability from 8-15% to 84% when challenged with 500 μM H2O2. In addition, Gtn-HPA was able to modulate proliferation and migration of aNSCs in relation to the crosslinking degree. Finally, Gtn-HPA exhibited bias for neuronal cells. In mixed differentiation conditions, Gtn-HPA increased the proportion of aNSCs expressing neuronal marker β-tubulin III to a greater extent than that for astrocytic marker glial fibrillary acidic protein, indicating an enhancement in differentiation towards neuronal lineage. Between neuronal and astrocytic differentiation conditions, Gtn-HPA also selected for higher survival in the former. Overall, Gtn-HPA hydrogels are promising injectable matrices for supporting and influencing aNSCs in ways that may be beneficial for brain tissue regeneration after injuries.
Keywords: Neural stem cell; Hydrogel; Oxidative stress resistance; Proliferation; Migration; Differentiation;

Target delivery of a gene into the brain using the RVG29-oligoarginine peptide by Cheng Gong; Xiangning Li; Lingling Xu; Yu-Hui Zhang (3456-3463).
The development of non-viral delivery systems that are capable of mediating an efficient, exclusive, and non-invasive transfer of DNA across the blood–brain barrier into the brain is challenging, but essential for the clinical application of gene therapy to brain diseases. Compared with other non-viral DNA carriers (e.g., lipids or polymers), peptide-based DNA delivery systems have many advantages including the ease of synthesis, low immunogenicity, biocompatibility, and biodegradability in vivo. However, all of the existing peptide-based vehicles for DNA delivery lack selectivity toward cells or tissues, which largely limited their applications in vivo. In this study, we demonstrated that an RVG29-9rR peptide-based DNA delivery system was able to transfect Neuro 2a cells in vitro more efficiently and specifically than Lipofectamine LTX & Plus, one of the most efficient commercially available transfection reagents. More significantly, the peptide mediated efficient and brain-targeting reporter gene expression after intravenous injection into mice. Thus, the results herein suggest a new strategy for brain-targeting DNA delivery in vivo.
Keywords: Brain targeting; DNA delivery; RVG29; Peptide vector; Gene therapy;

Creation and biochemical analysis of a broad-specific claudin binder by Azusa Takahashi; Yumiko Saito; Masuo Kondoh; Kyohei Matsushita; Susanne M. Krug; Hidehiko Suzuki; Hirofumi Tsujino; Xiangru Li; Hiroshi Aoyama; Koji Matsuhisa; Tadayuki Uno; Michael Fromm; Takao Hamakubo; Kiyohito Yagi (3464-3474).
Claudins (CL) are a family of tetra-transmembrane proteins that are the structural and functional components of tight junctions (TJ). CLs are promising targets for drug development because of their role in mucosal drug absorption and cancer. However, CL-targeted drug development has been delayed because CLs have low antigenicity and preparing CL proteins is difficult. We developed a CL binder by using the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) and a baculoviral display system. After screening CL binders from a C-CPE mutant-displaying library by using CL-displaying budded baculovirus (BV) we isolated a C-CPE mutant called m19, which bound to CL1, CL2, CL4 and CL5. A 3-dimensional analysis showed that m19 has a structural backbone similar to C-CPE. The charge density of the CL-binding domains of m19 and C-CPE differed, suggesting that electrostatic interactions may occur between m19 and CLs. Treatment of epithelial cells with m19 decreased the paracellular but not transcellular integrity, and m19 enhanced jejunal absorption. Thus, we successfully created a CL binder with broad specificity. These findings will contribute to future preparation of CL binders for CL-targeted drug development.
Keywords: Claudin; Drug delivery system; Baculovirus; Phage display; Tight junction;

Specific VEGF sequestering and release using peptide-functionalized hydrogel microspheres by Nicholas A. Impellitteri; Michael W. Toepke; Sheeny K. Lan Levengood; William L. Murphy (3475-3484).
Growth factor signaling plays an essential role in regulating processes such as tissue development, maintenance, and repair. Gene expression levels, diffusion, degradation, and sequestration by extracellular matrix components all play a role in regulating the concentration of growth factors within the cellular microenvironment. Herein, we describe the synthesis and characterization of hydrogel microspheres that mimic the ability of the native extracellular matrix to reversibly bind vascular endothelial growth factor (VEGF) out of solution. A peptide ligand derived from the VEGF receptor 2 (VEGFR2) was covalently incorporated into the hydrogel microspheres in order to achieve binding affinity and specificity. In addition to being able to both bind and release VEGF in a controllable manner, the microspheres were also shown to affect human umbilical vein endothelial cell (HUVEC) proliferation. The resulting microspheres may enable new strategies to specifically upregulate or downregulate growth factor signaling in the cellular microenvironment.
Keywords: Biomimetic; Bioinspired; Microparticle; ECM; Extracellular matrix; Growth factors;

The movement of self-assembled amphiphilic polymeric nanoparticles in the vitreous and retina after intravitreal injection by Heebeom Koo; Hyungwon Moon; Hyounkoo Han; Jin Hee Na; Myung Sook Huh; Jae Hyung Park; Se Joon Woo; Kyu Hyung Park; Ick Chan Kwon; Kwangmeyung Kim; Hyuncheol Kim (3485-3493).
The purpose of this study is to determine the correlation between the distribution of nanoparticles in the vitreous and retina and their surface properties after intravitreal injection. For this purpose, we synthesized seven kinds of nanoparticles through self-assembly of amphiphilic polymer conjugates in aqueous condition. They showed similar size but different surface properties. They were labeled with fluorescent dyes for efficient tracking. After intravitreal injection of these nanoparticles into a rodent eye, their time-dependent distribution in the vitreous and retina was determined in stacking tissue images by confocal microscopy. The results demonstrated that the surface property of nanoparticles is a key factor in determining their distribution in the vitreous and retina after intravitreal injection. In addition, immunohistochemistry and TEM images of retina tissues suggested the important mechanism related with Mülller cells for intravitreally administered nanoparticles to overcome the physical barrier of inner limiting membrane and to penetrate into the deeper retinal structures. Therefore, we expect that this study can provide valuable information for biomedical researchers to develop optimized nanoparticles as drug or gene carriers for retinal and optic nerve disorders such as glaucoma, age-related macular degeneration, and diabetic retinopathy.
Keywords: Ocular; Intravitreal; Nanoparticle; Drug delivery; Vitreous; Retina;

Theranostic liposomes of TPGS coating for targeted co-delivery of docetaxel and quantum dots by Madaswamy S. Muthu; Sneha A. Kulkarni; Anandhkumar Raju; Si-Shen Feng (3494-3501).
The aim of this work was to develop a new type of d-alpha-tocopheryl polyethylene glycol 1000 succinate mono-ester (TPGS) coated multi-functional (theranostic) liposomes, which contain both docetaxel and quantum dots (QDs) for cancer imaging and therapy. Non-targeting and folate receptor targeting TPGS coated theranostic liposomes were prepared by the solvent injection method and characterized for their particle size, polydispersity, zeta potential, surface chemistry and drug encapsulation efficiency. MCF-7 breast cancer cells of folate receptor overexpression were employed as an in vitro model to assess cellular uptake and cytotoxicity of the drug and QDs loaded liposomes. The mean particle size of the non-targeting and the targeting liposomes was found to be 202 and 210 nm, respectively. High resolution field emission transmission electron microscopy (FETEM) confirmed the presence of quantum dots in the peripheral hydrophobic membranes of the liposomes. The qualitative internalization of multi-functional liposomes by MCF-7 cells was visualized by confocal laser scanning microscopy (CLSM). The IC50 value, which is the drug concentration needed to kill 50% cells in a designated time period, was found to be 9.54 ± 0.76, 1.56 ± 0.19 and 0.23 ± 0.05 μg/ml for the commercial Taxotere®, non-targeting and targeting liposomes, respectively after 24 h culture with MCF-7 cells. The targeting multi-functional liposomes showed greater efficacy than the non-targeting liposomes and thus great potential to improve the cancer imaging and therapy.
Keywords: Cancer nanotechnology; Molecular biomaterials; Nanomedicine; Quantum dots; Stealth liposomes; Theranostic approach;