Biomaterials (v.30, #13)

Microscale mechanical properties of single elastic fibers: The role of fibrillin–microfibrils by Mieke M.J.F. Koenders; Lanti Yang; Ronnie G. Wismans; Kees O. van der Werf; Dieter P. Reinhardt; Willeke Daamen; Martin L. Bennink; Pieter J. Dijkstra; Toin H. van Kuppevelt; Jan Feijen (2425-2432).
Micromechanical properties of single elastic fibers and fibrillin–microfibrils, isolated from equine ligamentum nuchae using chemical and enzymatic methods, were determined with atomic force microscopy (AFM). Young's moduli of single elastic fibers immersed in water, devoid of or containing fibrillin–microfibrils, were determined using bending tests. Bending freely suspended elastic fibers on a micro-channeled substrate by a tip-less AFM cantilever generated a force versus displacement curve from which Young's moduli were calculated. For single elastic fibers, Young's moduli in the range of 0.3–1.5 MPa were determined, values not significantly affected by the presence of fibrillin–microfibrils. To further understand the role of fibrillin–microfibrils in vertebrate elastic fibers, layers of fibrillin–microfibrils were subjected to nano-indentation tests. From the slope of the force versus indentation curves, Young's moduli ranging between 0.56 and 0.74 MPa were calculated. The results suggest that fibrillin–microfibrils are not essential for the mechanical properties of single vertebrate elastic fibers.
Keywords: AFM (atomic force microscopy); Elastin; Mechanical properties; Mechanical test; Nano-indentation;

The influence of poly(ethylene oxide) grafting via siloxane tethers on protein adsorption by Ranjini Murthy; Courtney E. Shell; Melissa A. Grunlan (2433-2439).
Amphiphilic PEO–silanes (a–c) having siloxane tethers of varying lengths with the general formula α-(EtO)3Si–(CH2)2–oligodimethylsiloxane n -block-poly(ethylene oxide)8–OCH3 [n  = 0 (a), n  = 4 (b), and n  = 13 (c)] were grafted onto silicon wafers and resistance to adsorption of plasma proteins was measured. Distancing the PEO segment from the hydrolyzable triethoxysilane [(EtO)3Si] grafting group by a oligodimethylsiloxane tether represents a new method of grafting PEO chains to surfaces. Properties of surfaces grafted with a–c were compared to surfaces grafted with a traditional PEO–silane containing a propyl spacer [(EtO)3Si–(CH2)3–poly(ethylene oxide)8–OCH3, PEO control]. As the siloxane tether length increased, chain density of PEO–silanes grafted onto oxidized silicon wafers decreased and hydrophobicity of the PEO–silane increased which led to a decrease in surface hydrophilicity. Despite decreased surface hydrophilicity, resistance to the adsorption of bovine serum albumin (BSA) increased in the order: PEO control < a < b ≈ c and to human fibrinogen (HF) increased in the order: PEO control < a < b < c.
Keywords: Polyethylene oxide; Siloxane; Protein adsorption; Surface grafting;

The association of silicon microparticles with endothelial cells in drug delivery to the vasculature by Rita E. Serda; Jianhua Gu; Rohan C. Bhavane; XueWu Liu; Ciro Chiappini; Paolo Decuzzi; Mauro Ferrari (2440-2448).
Endothelial targeting is an approach evolving for drug delivery to the vasculature of pathological lesions. Nano-porous silicon-based multi-functional particles are of particular interest, since they can be manufactured in essentially any size and shape, employing methods of photolithography, to optimize their ability to localize on target endothelia. In this study we tested the impact of surface charge, serum opsonization, and inflammation on the ability of vascular endothelial cells to associate with nano-porous silicon microparticles. Vascular endothelial cells were capable of rapidly internalizing both positive and negative silicon microparticles by an actin-dependent mechanism involving both phagocytosis and macropinocytosis. However, following serum opsonization, internalization was selective for APTES (originally positive) modified microparticles, despite the finding that all opsonized microparticles had a net negative charge. Conversely, macrophages displayed a preference for internalization of serum opsonized oxidized (originally negative) microparticles, supporting the choice of positive microparticles for endothelial targeting. The internalization of opsonized microparticles by endothelial cells was further enhanced by the presence of inflammatory cytokines. These findings suggest that it may be possible to bioengineer silicon microparticles to favor opsonization with proteins that enhance uptake by endothelial cells, without a concurrent enhanced uptake by macrophages.
Keywords: Endothelia; Phagocytosis; Silicon; Microparticles;

In this paper we report a method for biomaterial surface modification that utilizes the self-assembly of block copolymers of poly(styrene-block-ethylene oxide) (PS–PEO) to generate micro-phase separated surfaces with varying density PEO domains. These PS–PEO self-assembled surfaces showed a significant reduction in protein adsorption compared to control polystyrene surfaces. The adhesion of NIH-3T3 fibroblast cells was shown to be significantly affected by the surface coverage of PEO nano-domains formed by copolymer self-assembly. These nano-domains, when presented at high number density (almost 1000 domains per square micron), were shown to completely prevent cellular attachment, even though small amounts of protein were able to bind to the surface.
Keywords: Protein adsorption; Biocompatibility; Self-assembly; Surface modification; Block copolymer;

A small diameter, fibrous vascular conduit generated from a poly(ester urethane)urea and phospholipid polymer blend by Yi Hong; Sang-Ho Ye; Alejandro Nieponice; Lorenzo Soletti; David A. Vorp; William R. Wagner (2457-2467).
The thrombotic and hyperplastic limitations associated with synthetic small diameter vascular grafts have generated sustained interest in finding a tissue engineering solution for autologous vascular segment generation in situ. One approach is to place a biodegradable scaffold at the site that would provide acute mechanical support while vascular tissue develops. To generate a scaffold that possessed both non-thrombogenic character and mechanical properties appropriate for vascular tissue, a biodegradable poly(ester urethane)urea (PEUU) and non-thrombogenic bioinspired phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine-co-methacryloyloxyethyl butylurethane) (PMBU) were blended at PMBU weight fractions of 0–15% and electrospun to create fibrous scaffolds. The composite scaffolds were flexible with breaking strains exceeding 300%, tensile strengths of 7–10 MPa and compliances of 2.9–4.4 × 10−4  mmHg−1. In vitro platelet deposition on the scaffold surfaces significantly decreased with increasing PMBU content. Rat smooth muscle cell proliferation was also inhibited on PEUU/PMBU blended scaffolds with greater inhibition at higher PMBU content. Fibrous vascular conduits (1.3 mm inner diameter) implanted in the rat abdominal aorta for 8 weeks showed greater patency for grafts with 15% PMBU blending versus PEUU without PMBU (67% versus 40%). A thin neo-intimal layer with endothelial coverage and good anastomotic tissue integration was seen for the PEUU/PMBU vascular grafts. These results are encouraging for further evaluation of this technique in larger diameter applications for longer implant periods.
Keywords: Polyurethane; Phospholipid copolymer; Electrospinning; Small diameter blood vessel; Scaffold;

The cytocompatibility and adhesion of cells to biomaterials are key to their success in the clinic. Here we report a study of the toxicity, cell-adhesive properties and biocompatibility of a range of alkyl-aminated hydrogels and amphiphilic conetworks comprising 1,2-propandiol-3-methacrylate (GMA) as the hydrophilic component. Previously we had shown that addition of amines containing alkyl spacers of at 3–6 carbons or addition of oligo(butyl methacrylate) sequences to crosslinked polyGMA hydrogels could be used to produce a step change in cell adhesion. In this work we produced two series of polymer networks, based on polyGMA, which contained both of these structural features and we examined the effects that these materials had on A549 epithelial cells and human dermal fibroblasts. No toxicity was observed from either direct contact or from supernatants extracted over 48 h. Each of the alkyl-aminated materials provided a good substrate for adhesion of both cell types whereas the non-alkyl-aminated materials were essentially non-cell-adhesive. Peritoneal murine macrophages were present on all of the materials and the activation of these adhered macrophages was investigated by determining the production of the pro-inflammatory cytokines, TNF-α, IL1β and IL-6. All of the materials behaved similarly to a clinically acceptable control, Permacol® (a decellularized collagen-based porcine derived material), and each material was much less activating than when the macrophages were in contact with lipopolysaccharide endotoxin. There were no differences in the capacity of the materials to activate TNF-α production by macrophages however, there was a trend towards stimulation of lower levels of IL-6 and IL-1β by the alkyl-aminated materials.
Keywords: Biocompatibility; Hydrogels; Cell adhesion; Macrophage;

The stimulation of healing within a rat calvarial defect by mPCL–TCP/collagen scaffolds loaded with rhBMP-2 by A.A. Sawyer; S.J. Song; E. Susanto; P. Chuan; C.X.F. Lam; M.A. Woodruff; D.W. Hutmacher; S.M. Cool (2479-2488).
Bone morphogenetic proteins (BMPs) have been widely investigated for their clinical use in bone repair and it is known that a suitable carrier matrix to deliver them is essential for optimal bone regeneration within a specific defect site. Fused deposited modeling (FDM) allows for the fabrication of medical grade poly ɛ-caprolactone/tricalcium phosphate (mPCL–TCP) scaffolds with high reproducibility and tailor designed dimensions. Here we loaded FDM fabricated mPCL–TCP/collagen scaffolds with 5 μg recombinant human (rh)BMP-2 and evaluated bone healing within a rat calvarial critical-sized defect. Using a comprehensive approach, this study assessed the newly regenerated bone employing micro-computed tomography (μCT), histology/histomorphometry, and mechanical assessments. By 15 weeks, mPCL–TCP/collagen/rhBMP-2 defects exhibited complete healing of the calvarium whereas the non-BMP-2-loaded scaffolds showed significant less bone ingrowth, as confirmed by μCT. Histomorphometry revealed significantly increased bone healing amongst the rhBMP-2 groups compared to non-treated scaffolds at 4 and 15 weeks, although the % BV/TV did not indicate complete mineralisation of the entire defect site. Hence, our study confirms that it is important to combine microCt and histomorphometry to be able to study bone regeneration comprehensively in 3D. A significant up-regulation of the osteogenic proteins, type I collagen and osteocalcin, was evident at both time points in rhBMP-2 groups. Although mineral apposition rates at 15 weeks were statistically equivalent amongst treatment groups, micro-compression and push-out strengths indicated superior bone quality at 15 weeks for defects treated with mPCL–TCP/collagen/rhBMP-2. Consistently over all modalities, the progression of healing was from empty defect < mPCL–TCP/collagen < mPCL–TCP/collagen/rhBMP-2, providing substantiating data to support the hypothesis that the release of rhBMP-2 from FDM-created mPCL–TCP/collagen scaffolds is a clinically relevant approach to repair and regenerate critically-sized craniofacial bone defects.
Keywords: Bone morphogenetic protein; Bone repair and regeneration; Composite scaffold; Polycaprolactone; Calvarial defect;

Vertical alveolar ridge augmentation with β-tricalcium phosphate and autologous osteoblasts in canine mandible by Shaoyi Wang; Zhiyuan Zhang; Jun Zhao; Xiuli Zhang; Xiaojuan Sun; Lunguo Xia; Qing Chang; Dongxia Ye; Xinquan Jiang (2489-2498).
A tissue-engineered bone has become a viable alternative to autologous bone for bone augmentation in atrophy alveolar ridge. The aim of the present study was to evaluate porous β-tricalcium phosphate (β-TCP) combined with autologous osteoblasts to augment edentulous alveolar ridge in a canine model. Autologous osteoblasts were expanded and combined with β-TCP scaffold to fabricate a tissue-engineered bone. 12 bilateral alveolar ridge augmentation surgeries were carried out in 6 beagle dogs with the following 3 groups: β-TCP/osteoblasts, β-TCP alone and autogenous iliac bone control (n  = 4 per group). Sequential fluorescent labeling and radiographs were used to compare new bone formation and mineralization in each group. 24 weeks later, animals were sacrificed and non-decalcified and decalcified sections were evaluated histologically and histomorphometrically. Results indicated that the tissue-engineered bone dramatically enhanced new bone formation and mineralization, increase the new bone area, and maintain the height and thickness of the augmented alveolar ridge when compared with β-TCP alone group. More importantly, the tissue-engineered bone achieved an elevated bone height and thickness comparable to that of autogenous iliac bone graft. This study demonstrated the potential of porous β-TCP as a substrate for autogenous osteoblasts in bone tissue engineering for alveolar ridge augmentation.
Keywords: Tissue engineering; Autologous osteoblasts; β-Tricalcium phosphate; Alveolar ridge augmentation;

Injectable in situ forming biodegradable chitosan–hyaluronic acid based hydrogels for cartilage tissue engineering by Huaping Tan; Constance R. Chu; Karin A. Payne; Kacey G. Marra (2499-2506).
Injectable, biodegradable scaffolds are important biomaterials for tissue engineering and drug delivery. Hydrogels derived from natural polysaccharides are ideal scaffolds as they resemble the extracellular matrices of tissues comprised of various glycosaminoglycans (GAGs). Here, we report a new class of biocompatible and biodegradable composite hydrogels derived from water-soluble chitosan and oxidized hyaluronic acid upon mixing, without the addition of a chemical crosslinking agent. The gelation is attributed to the Schiff base reaction between amino and aldehyde groups of polysaccharide derivatives. In the current work, N-succinyl-chitosan (S-CS) and aldehyde hyaluronic acid (A-HA) were synthesized for preparation of the composite hydrogels. The polysaccharide derivatives and composite hydrogels were characterized by FTIR spectroscopy. The effect of the ratio of S-CS and A-HA on the gelation time, microstructure, surface morphology, equilibrium swelling, compressive modulus, and in vitro degradation of composite hydrogels was examined. The potential of the composite hydrogel as an injectable scaffold was demonstrated by the encapsulation of bovine articular chondrocytes within the composite hydrogel matrix in vitro. The results demonstrated that the composite hydrogel supported cell survival and the cells retained chondrocytic morphology. These characteristics provide a potential opportunity to use the injectable, composite hydrogels in tissue engineering applications.
Keywords: Chitosan; Hyaluronic acid; Biodegradable hydrogel; Bovine articular chondrocytes; Tissue engineering;

Homogeneous and organized differentiation within embryoid bodies induced by microsphere-mediated delivery of small molecules by Richard L. Carpenedo; Andrés M. Bratt-Leal; Ross A. Marklein; Scott A. Seaman; Nathan J. Bowen; John F. McDonald; Todd C. McDevitt (2507-2515).
Cell specification and tissue formation during embryonic development are precisely controlled by the local concentration and temporal presentation of morphogenic factors. Similarly, pluripotent embryonic stem cells can be induced to differentiate in vitro into specific phenotypes in response to morphogen treatment. Embryonic stem cells (ESCs) are commonly differentiated as 3D spheroids referred to as embryoid bodies (EBs); however, differentiation of cells within EBs is typically heterogeneous and disordered. In this study, we demonstrate that in contrast to soluble morphogen treatment, delivery of morphogenic factors directly within EB microenvironments in a spatiotemporally controlled manner using polymer microspheres yields homogeneous, synchronous and organized ESC differentiation. Degradable PLGA microspheres releasing retinoic acid were incorporated directly within EBs and induced the formation of cystic spheroids uniquely resembling the phenotype and structure of early streak mouse embryos (E6.75), with an exterior of FOXA2+ visceral endoderm enveloping an epiblast-like layer of OCT4+ cells. These results demonstrate that controlled morphogen presentation to stem cells using degradable microspheres more efficiently directs cell differentiation and tissue formation than simple soluble delivery methods and presents a unique route to study the spatiotemporal effects of morphogenic factors on embryonic developmental processes in vitro.
Keywords: Embryonic stem cells; Embryoid bodies; Microspheres; Retinoic acid;

Embryonic stem cells represent a potentially unlimited cell source for tissue engineering applications. However, in order to be used for such applications, embryonic stem cells' differentiation must be controlled to only the desired lineages. In this study, we examine the effects of nanofibrous architecture and biochemical cues on the osteogenic differentiation of embryonic stem cells compared to the more traditional architecture without the nanofibrous features in two dimensions (thin matrix or flat films) and three dimensions (scaffolds) in vitro. After three weeks of culture the nanofibrous thin matrices were capable of supporting mRNA expression of osteogenic differentiation markers in embryonic stem cells without osteogenic supplements, while solid films required osteogenic supplements and growth factors to achieve mRNA expression of osteogenic differentiation markers. Nanofibrous scaffolds substantially enhanced mRNA expression of osteogenic differentiation markers compared to solid-walled scaffolds, nanofibrous thin matrices or solid films. After 4 weeks of culture, nanofibrous scaffolds were found to contain 3 times more calcium and stronger osteocalcin stain throughout the scaffolds than the solid-walled scaffolds. Overall, the nanofibrous architecture enhanced the osteogenic differentiation and mineralization of embryonic stem cells compared to the solid-walled architecture in both two and three-dimensional cultures.
Keywords: Nanofibers; Embryonic stem cells; Bone tissue engineering; Osteogenesis;

Self-assembled peptide-based hydrogels as scaffolds for anchorage-dependent cells by Mi Zhou; Andrew M. Smith; Apurba K. Das; Nigel W. Hodson; Richard F. Collins; Rein V. Ulijn; Julie E. Gough (2523-2530).
We report here the design of a biomimetic nanofibrous hydrogel as a 3D-scaffold for anchorage-dependent cells. The peptide-based bioactive hydrogel is formed through molecular self-assembly and the building blocks are a mixture of two aromatic short peptide derivatives: Fmoc-FF (Fluorenylmethoxycarbonyl-diphenylalanine) and Fmoc-RGD (arginine-glycine-aspartate) as the simplest self-assembling moieties reported so far for the construction of small-molecule-based bioactive hydrogels. This hydrogel provides a highly hydrated, stiff and nanofibrous hydrogel network that uniquely presents bioactive ligands at the fibre surface; therefore it mimics certain essential features of the extracellular matrix. The RGD sequence as part of the Fmoc-RGD building block plays a dual role of a structural component and a biological ligand. Spectroscopic and imaging analysis using CD, FTIR, fluorescence, TEM and AFM confirmed that FF and RGD peptide sequences self-assemble into β-sheets interlocked by π–π stacking of the Fmoc groups. This generates the cylindrical nanofibres interwoven within the hydrogel with the presence of RGDs in tunable densities on the fibre surfaces. This rapid gelling material was observed to promote adhesion of encapsulated dermal fibroblasts through specific RGD–integrin binding, with subsequent cell spreading and proliferation; therefore it may offer an economical model scaffold to 3D-culture other anchorage-dependent cells for in-vitro tissue regeneration.
Keywords: Minimalist design; Self-assembly; Biomimetic materials; RGD ligands; 3D culture; Anchorage-dependent cells;

Human hepatocyte functions in a crossed hollow fiber membrane bioreactor by Loredana De Bartolo; Simona Salerno; Efrem Curcio; Antonella Piscioneri; Maria Rende; Sabrina Morelli; Franco Tasselli; Augustinus Bader; Enrico Drioli (2531-2543).
An important challenge in liver tissue engineering is the development of bioartificial systems that are able to favour the liver reconstruction and to modulate liver cell behaviour.A crossed hollow fiber membrane bioreactor was developed to support the long-term maintenance and differentiation of human hepatocytes. The bioreactor consists of two types of hollow fiber (HF) membranes with different molecular weight cut-off (MWCO) and physico-chemical properties cross-assembled in alternating manner: modified polyetheretherketone (PEEK-WC) and polyethersulfone (PES), used for the medium inflow and outflow, respectively. The combination of these two fiber set produces an extracapillary network for the adhesion of cells and a high mass exchange through the cross-flow of culture medium. The transport of liver specific products such as albumin and urea together with the transport of drug such as diazepam was modelled and compared with the experimental metabolic data. The theoretical metabolite concentration differed 7.5% for albumin and 5% for urea with respect to experimental data. The optimised perfusion conditions of the bioreactor allowed the maintenance of liver functions in terms of urea synthesis, albumin secretion and diazepam biotransformation up to 18 days of culture. In particular the good performance of the bioreactor was confirmed by the high rate of urea synthesis (28.7 μg/h 106 cells) and diazepam biotransformation. In the bioreactor human hepatocytes expressed at high levels the individual cytochrome P450 isoenzymes involved in the diazepam metabolism. The results demonstrated that crossed HF membrane bioreactor is able to support the maintenance of primary human hepatocytes preserving their liver specific functions for all investigated period. This device may be a potential tool in the liver tissue engineering for drug metabolism/toxicity testing and study of disease pathogenesis alternatively to animal experimentation.
Keywords: Hollow fiber; Membrane bioreactor; Mass transport; Hepatocytes; Liver functions; Diazepam biotransformation;

Injectable chitosan-based hydrogels for cartilage tissue engineering by R. Jin; L.S. Moreira Teixeira; P.J. Dijkstra; M. Karperien; C.A. van Blitterswijk; Z.Y. Zhong; J. Feijen (2544-2551).
Water-soluble chitosan derivatives, chitosan-graft-glycolic acid (GA) and phloretic acid (PA) (CH-GA/PA), were designed to obtain biodegradable injectable chitosan hydrogels through enzymatic crosslinking with horseradish peroxidase (HRP) and H2O2. CH-GA/PA polymers were synthesized by first conjugating glycolic acid (GA) to native chitosan to render the polymer soluble at pH 7.4, and subsequent modification with phloretic acid (PA). The CH-GA43/PA10 with a degree of substitution (DS, defined as the number of substituted NH2 groups per 100 glucopyranose rings of chitosan) of GA of 43 and DS of PA of 10 showed a good solubility at pH values up to 10. Short gelation times (e.g. 10 s at a polymer concentration of 3 wt%), as recorded by the vial tilting method, were observed for the CH-GA43/PA10 hydrogels using HRP and H2O2. It was shown that these hydrogels can be readily degraded by lysozyme. In vitro culturing of chondrocytes in CH-GA43/PA10 hydrogels revealed that after 2 weeks the cells were viable and retained their round shape. These features indicate that CH-GA/PA hydrogels are promising as an artificial extracellular matrix for cartilage tissue engineering.
Keywords: Hydrogel; Biodegradable; Enzymatic crosslinking; Chitosan; Chondrocytes;

Biomedical device-associated infection is one of the most common and problematic complications faced by millions of patients worldwide. The current antibiotic therapy strategies face challenges, the most serious of which is antibiotic resistance. Studies have shown that the systemic level of interleukin 12 (IL-12) decreases following major injuries resulting in decreased cell-mediated immune response. Here we report the development of IL-12 nanoscale coatings using electrostatic layer-by-layer self-assembly nanotechnology. We found that IL-12 nanoscale coatings at the implant/tissue interface substantially decrease infections in vivo, and IL-12 nanoscale coatings are advantageous over traditional treatments. This approach could be a revolutionary step toward preventing device-associated infections using a non-antibiotic approach.
Keywords: Antimicrobial; Bone; Cytokine; Drug delivery; Infection; Surface modification;

Multiscale requirements for bioencapsulation in medicine and biotechnology by Paul de Vos; Marek Bučko; Peter Gemeiner; Marián Navrátil; Juraj Švitel; Marijke Faas; Berit Løkensgard Strand; Gudmund Skjak-Braek; Yrr A. Morch; Alica Vikartovská; Igor Lacík; Gabriela Kolláriková; Gorka Orive; Dennis Poncelet; Jose Luis Pedraz; Marion B. Ansorge-Schumacher (2559-2570).
Bioencapsulation involves the envelopment of tissues or biological active substances in semipermeable membranes. Bioencapsulation has been shown to be efficacious in mimicking the cell's natural environment and thereby improves the efficiency of production of different metabolites and therapeutic agents. The field of application is broad. It is being applied in bioindustry and biomedicine. It is clinically applied for the treatment of a wide variety of endocrine diseases. During the past decades many procedures to fabricate capsules have been described. Unfortunately, most of these procedures lack an adequate documentation of the characterization of the biocapsules. As a result many procedures show an extreme lab-to-lab variation and many results cannot be adequately reproduced. The characterization of capsules can no longer be neglected, especially since new clinical trials with bioencapsulated therapeutic cells have been initiated and the industrial application of bioencapsulation is growing. In the present review we discuss novel Approached to produce and characterize biocapsules in view of clinical and industrial application. A dominant factor in bioencapsulation is selection and characterization of suitable polymers. We present the adequacy of using high-resolution NMR for characterizing polymers. These polymers are applied for producing semipermeable membranes. We present the pitfalls of the currently applied methods and provide recommendations for standardization to avoid lab-to-lab variations. Also, we compare and present methodologies to produce biocompatible biocapsules for specific fields of applications and we demonstrate how physico-chemical technologies such as FT-IR, XPS, and TOF-SIMS contribute to reproducibility and standardization of the bioencapsulation process. During recent years it has become more and more clear that bioencapsulation requires a multidisciplinary approach in which biomedical, physical, and chemical technologies are combined. For adequate reproducibility and for understanding variations in outcome of biocapsules it is advisable if not mandatory to include the characterization processes presented in this review in future studies.
Keywords: Microencapsulation; Biocompatibility; Surface analysis; Bioartificial pancreas; XPS (X-ray photoelectron spectroscopy); Microbiology;

Microporous silk fibroin scaffolds embedding PLGA microparticles for controlled growth factor delivery in tissue engineering by Esther Wenk; Anne J. Meinel; Sarah Wildy; Hans P. Merkle; Lorenz Meinel (2571-2581).
The development of prototype scaffolds for either direct implantation or tissue engineering purposes and featuring spatiotemporal control of growth factor release is highly desirable. Silk fibroin (SF) scaffolds with interconnective pores, carrying embedded microparticles that were loaded with insulin-like growth factor I (IGF-I), were prepared by a porogen leaching protocol. Treatments with methanol or water vapor induced water insolubility of SF based on an increase in β-sheet content as analyzed by FTIR. Pore interconnectivity was demonstrated by SEM. Porosities were in the range of 70–90%, depending on the treatment applied, and were better preserved when methanol or water vapor treatments were prior to porogen leaching. IGF-I was encapsulated into two different types of poly(lactide-co-glycolide) microparticles (PLGA MP) using uncapped PLGA (50:50) with molecular weights of either 14 or 35 kDa to control IGF-I release kinetics from the SF scaffold. Embedded PLGA MP were located in the walls or intersections of the SF scaffold. Embedment of the PLGA MP into the scaffolds led to more sustained release rates as compared to the free PLGA MP, whereas the hydrolytic degradation of the two PLGA MP types was not affected. The PLGA types used had distinct effects on IGF-I release kinetics. Particularly the supernatants of the lower molecular weight PLGA formulations turned out to release bioactive IGF-I. Our studies justify future investigations of the developed constructs for tissue engineering applications.
Keywords: Silk fibroin; Controlled drug delivery; Insulin-like growth factor I; Scaffold; Microparticles;

Nanoparticle-mediated local delivery of methylprednisolone after spinal cord injury by Young-tae Kim; Jon-Michael Caldwell; Ravi V. Bellamkonda (2582-2590).
Systemic administration of a high-dose of Methylprednisolone (MP) can reduce neurological deficits after acute spinal cord injury (SCI). However, the use of high-dose MP in treating acute SCI is controversial due to significant dose related side effects and relatively modest improvements in neurological function. Here, using a rat model of SCI, we compare the efficacy of controlled, nanoparticle-enabled local delivery of MP to the injured spinal cord with systemic delivery of MP, and a single local injection of MP without nanoparticles. Based on histological and behavioral data, we report that local, sustained delivery of MP via nanoparticles is significantly more effective than systemic delivery. Relative to systemic delivery, MP-nanoparticle therapy significantly reduced lesion volume and improved behavioral outcomes. Nanoparticle-enabled delivery of MP presents an effective method for introducing MP locally after SCI and significantly enhances therapeutic effectiveness compared to bare MP administered either systemically or locally.
Keywords: Nerve regeneration; Inflammation; Hydrogel; Drug delivery;

The penetration of fresh undiluted sputum expectorated by cystic fibrosis patients by non-adhesive polymer nanoparticles by Jung Soo Suk; Samuel K. Lai; Ying-Ying Wang; Laura M. Ensign; Pamela L. Zeitlin; Michael P. Boyle; Justin Hanes (2591-2597).
Highly viscoelastic and adhesive sputum has precluded efficient nanoparticle-based drug and gene delivery to the lungs of patients with cystic fibrosis (CF). We sought to determine whether nanoparticles coated with non-mucoadhesive polymers could penetrate CF sputum, and to use these “muco-inert particles” (MIPs) as non-destructive nanoprobes to characterize the sputum microstructure. Particles as large as 200 nm in diameter that were densely coated with low MW polyethylene glycol (PEG) moved through undiluted CF sputum with average speeds up to 90-fold faster than similarly-sized uncoated particles. On the other hand, the transport of both coated and uncoated 500 nm particles was strongly hindered. The local viscosity of sputum, encountered by the fastest 10% of 200 nm MIPs, was only 5-fold higher than that of water, whereas the bulk viscosity was 10,000-fold higher at low shear rates. Using measured transport rates of various sized MIPs combined with an obstruction-scaling model, we determined that the average 3D mesh spacing of CF sputum is ∼140 ± 50 nm (range: 60–300 nm). Taken together, these results demonstrate that nanoparticles up to 200 nm in diameter that do not adhere to CF sputum can move rapidly through this critical barrier by accessing pores that are filled with a low viscosity fluid. The results also offer hope that desperately needed sputum-penetrating drug- and gene-carrier nanoparticles can be developed for CF.
Keywords: Lung disease; Nanoparticle therapeutics; Mucus; Mucus penetrating particles; Particle tracking; Rheology;

Pharmacokinetics and biodistribution of N-isopropylacrylamide copolymers for the design of pH-sensitive liposomes by Nicolas Bertrand; Jackie G. Fleischer; Kishor M. Wasan; Jean-Christophe Leroux (2598-2605).
The purpose of this work was to characterize the pharmacokinetics (PK) and biodistribution of pH-responsive N-isopropylacrylamide (NIPAAm) copolymers, and to determine the impact of some physicochemical parameters on their biological profiles. Radiolabeled copolymers of NIPAAm and methacrylic acid (MAA) of different molecular weight, amphiphilicity and lower critical solution temperature (LCST) were synthesized and injected intravenously to rats. The PK and excretion profiles were monitored over 48 h. It was found that elimination occurred mainly through urinary excretion, which was principally governed by molecular weight. Above a threshold of 32,000, the polymer chains avoided glomerular filtration and presented prolonged circulation times. Moreover, the presence of alkyl moieties at the chain extremity influenced circulation time and tissue distribution of polymer chains, hypothetically through formation of micellar structures. The polymers with an LCST situated below the physiological temperature did not circulate for prolonged periods in the bloodstream and were highly captured by the organs of the mononuclear phagocyte system. Finally, the complexation of an alkylated pH-sensitive polymer with a molecular weight of 10,000 to the bilayer of PEGylated liposomes produced a drastic change in the PK parameters, indicating that the polymer remained anchored in the phospholipid bilayer in the bloodstream. These data indicate that stable pH-sensitive liposomes can be produced using excretable NIPAAm copolymers.
Keywords: Poly(N-isopropylacrylamide); Pharmacokinetics; Biodistribution; pH-sensitivity; pH sensitive liposomes; Drug targeting;

Dual-drug delivery system based on hydrogel/micelle composites by Lan Wei; Chunhua Cai; Jiaping Lin; Tao Chen (2606-2613).
We present a dual-drug delivery system (DDDS) of hydrogel/polypeptide micelle composites in this work. The DDDS was constructed from aspirin (Asp) dispersed poly(vinyl alcohol) (PVA) or Chitosan (CS)/PVA hydrogel and doxorubicin (DOX) loaded poly(l-glutamic acid)-b-poly(propylene oxide)-b-poly(l-glutamic acid) (GPG) micelles. Independent release behaviors of the two drugs are observed. Asp has a short-term release while DOX has a long-term and sustained release behavior in all the DDDSs. The release of DOX from all the DDDSs is environmentally controlled due to the pH and temperature sensitivity of the GPG micelle. Asp shows the pH controlled release behavior in CS/PVA/micelle DDDS due to the pH sensitivity of CS hydrogel. The releasing profiles were analyzed using a power law equation proposed by Peppas. It reveals that the release of Asp is anomalous transport in all the hydrogel/micelle DDDSs. The release of DOX is Fickian type in PVA/micelle system, and changes to anomalous transport in CS/PVA/micelle system according to the release exponent n.
Keywords: Drug release; Dual-drug delivery; Hydrogel; Micelle; Polypeptide;

Polypyrrole-coated electrodes for the delivery of charge and neurotrophins to cochlear neurons by Rachael T. Richardson; Andrew K. Wise; Brianna C. Thompson; Brianna O. Flynn; Patrick J. Atkinson; Nicole J. Fretwell; James B. Fallon; Gordon G. Wallace; Rob K. Shepherd; Graeme M. Clark; Stephen J. O'Leary (2614-2624).
Sensorineural hearing loss is associated with gradual degeneration of spiral ganglion neurons (SGNs), compromising hearing outcomes with cochlear implant use. Combination of neurotrophin delivery to the cochlea and electrical stimulation from a cochlear implant protects SGNs, prompting research into neurotrophin-eluting polymer electrode coatings. The electrically conducting polypyrrole/para-toluene sulfonate containing neurotrophin-3 (Ppy/pTS/NT3) was applied to 1.7 mm2 cochlear implant electrodes. Ppy/pTS/NT3-coated electrode arrays stored 2 ng NT3 and released 0.1 ng/day with electrical stimulation. Guinea pigs were implanted with Ppy/pTS or Ppy/pTS/NT3 electrode arrays two weeks after deafening via aminoglycosides. The electrodes of a subgroup of these guinea pigs were electrically stimulated for 8 h/day for 2 weeks. There was a loss of SGNs in the implanted cochleae of guinea pigs with Ppy/pTS-coated electrodes indicative of electrode insertion damage. However, guinea pigs implanted with electrically stimulated Ppy/pTS/NT3-coated electrodes had lower electrically-evoked auditory brainstem response thresholds and greater SGN densities in implanted cochleae compared to non-implanted cochleae and compared to animals implanted with Ppy/pTS-coated electrodes (p  < 0.05). Ppy/pTS/NT3 did not exacerbate fibrous tissue formation and did not affect electrode impedance. Drug-eluting conducting polymer coatings on cochlear implant electrodes present a clinically viable method to promote preservation of SGNs without adversely affecting the function of the cochlear implant.
Keywords: Controlled drug release; Electroactive polymer; Electrode; Electrical stimulation; Neurotrophin; Cochlear implant;

The present work describes nanoparticles made of ultra low molecular weight chitosan (ULMWCh)/hyaluronic acid (HA) as novel potential carriers for gene delivery. Small and monodispersed nanoparticles with high in vitro transfection capabilities have been obtained by the complexation of these two polyelectrolytes. ULMWCh (<10 kDa) presents more advantageous characteristics over the higher molecular weight chitosan for clinical applications, namely increased solubility at physiological pH and improved DNA release. The ULMWCh:HA ratio and the HA molecular weights were varied with the aim of obtaining particles in the 100 nm range. Using chitosan (Ch) with a molecular weight of 5 kDa, HA with a molecular weight of 64 kDa, and a weight ratio of 4:1, nanoparticles with a Z-average size of 146 ± 1 nm and narrow size distribution (polydispersity index: 0.073 ± 0.030) were obtained. Nanoparticle images taken in dry conditions by SEM and AFM showed spherical particles. The optimal pH for transfection ranged from 6.4 to 6.8 for 0.25 μg of EGFP plasmid per well, with an incubation time of 4 h. Using these optimized parameters, DNA/ULMWCh:HA nanoparticles successfully transfected 25 ± 1% of the 293T cells with pEGFP, compared to 0.7% obtained for DNA/ULMWCh under the same conditions. This high transfection efficiency of our non-viral gene delivery system could be attributed to the synergic effect of ULMWCh and low charge density of the HA chain for easy release of DNA which makes the system suitable for targeted gene delivery.
Keywords: Ultra low molecular weight chitosan; Hyaluronic acid; Nanoparticles; Gene delivery; Non-viral vector; Nanocarrier;

Sustained transgene expression via citric acid-based polyester elastomers by Xue-Qing Zhang; Huanghui Tang; Ryan Hoshi; Laura De Laporte; Hongjin Qiu; Xiaoyang Xu; Lonnie D. Shea; Guillermo A. Ameer (2632-2641).
Polymeric scaffolds are an important tool in tissue engineering and gene delivery using porous scaffolds can be a viable approach to control tissue response. Herein we describe the use of a biodegradable polyester elastomer, poly(1,8-octanediol-co-citrate) (POC), as a substrate for plasmid immobilization and cellular transfection of colonizing cells. Plasmid (pDNA), either complexed with poly(ethyleneimine) (PEI) forming polyplexes or in its native state, was surface-immobilized onto POC scaffolds via adsorption. Polyplex-containing scaffolds showed higher loading and slower initial rates of release than naked pDNA-containing scaffolds. Seeding of HEK293 cells and porcine aortic smooth muscle cells (PASMC) onto polyplex loaded-scaffolds demonstrated cell proliferation and transfection in vitro up to 12 days, significantly longer relative to bolus transfection. In vivo, transfection was evaluated using the mouse intraperitoneal (IP) fat model. In contrast to the in vitro study, successful long-term transgene delivery was only achieved with the naked pDNA-containing scaffolds. In particular, naked pDNA-containing scaffolds promoted high levels of both luciferase and green fluorescent protein (GFP) expression in vivo for 2 weeks. The results demonstrate that POC scaffolds are a suitable material for substrate-mediated gene delivery. POC scaffolds can potentially support long-term biological cues to mediate tissue formation through non-viral gene delivery.
Keywords: Surface-mediated transfection; Poly(diol citrates); Scaffolds; Biodegradable polyesters;

All-trans-retinoic acid (ATRA)-grafted polymeric gene carriers for nuclear translocation and cell growth control by Kyong Mi Park; Han Chang Kang; Jung Kyo Cho; Ik-Joo Chung; Sang-Hee Cho; You Han Bae; Kun Na (2642-2652).
Polyethyleneimine (PEI)-g-All-trans-retinoic acid (ATRA) (designated as PRA) was synthesized as a gene carrier. ATRA at its low concentration is known to be linked to nuclear translocation and cell cycle control (either proliferation or growth arrest) depending on its binding protein in cells. The cytotoxicity of PRA conjugates was lower than that of PEI and was gradually reduced as increasing ATRA graft ratios. The resulting nanosized and positively charged PRA/pDNA complexes showed lower transfection efficiency than the PEI/pDNA complexes (N/P = 10) against NIH3T3 which is less sensitive to ATRA in cell growth and more sensitive HeLa cells. However, when a mixed gene complex of PEI and PRA was applied in an effort to reduce the ATRA contents, their NIH3T3 transfection evidenced effective nuclear translocation and induced 2- to 4-fold better transfection efficiency as compared with the PEI/pDNA complexes. When the PEI/pDNA complexes were utilized to transfect HeLa cells, free ATRA treatment reduced their cellular uptake and transfection efficiency. These findings show that the NIH3T3 cells against ATRA-mediated growth arrest would not damage the PRA-mediated transfection enhancement resulting from the facilitated nuclear translocation of polyplexes or pDNA. The more ATRA-sensitivity in growth arrest of HeLa cells would reduce the transfection efficiency of ATRA-incorporated polyplexes. The transfection capability of gene by newly synthesized PRA conjugates to cells is differentiated by their ATRA-sensitivity to nuclear translocation and cell growth control.
Keywords: All-trans-retinoic acid (ATRA); Polyethyleneimine (PEI); Nuclear translocation; Cell cycle control;

Inhibition of complement activation on a model biomaterial surface by streptococcal M protein-derived peptides by Anna E. Engberg; Kerstin Sandholm; Fredrik Bexborn; Jenny Persson; Bo Nilsson; Gunnar Lindahl; Kristina N. Ekdahl (2653-2659).
The aim of this study was to evaluate a new approach to inhibit complement activation triggered by biomaterial surfaces in contact with blood. In order to inhibit complement activation initiated by the classical pathway (CP), we used streptococcal M protein-derived peptides that specifically bind human C4BP, an inhibitor of the CP. The peptides were used to coat polystyrene microtiter wells which served as a model biomaterial. The ability of coated peptides to bind C4BP and to attenuate complement activation via the CP (monitored as generation of fluid-phase C3a and binding of fragments of C3 and C4 to the surface) was investigated using diluted normal human serum, where complement activation by the AP is minimal, as well as serum from a patient lacking alternative pathway activation. Complement activation (all parameters) was significantly decreased in serum incubated in well surfaces coated with peptides. Total inhibition of complement activation was obtained at peptide coating concentrations as low as 1–5 μg/mL. Successful use of Streptococcus-derived peptides shows that it is feasible to control complement activation at a model biomaterial surface by capturing autologous complement regulatory molecules from plasma.
Keywords: Blood compatibility; Complement; C4b-binding protein (C4BP); In vitro test; Regulator of complement activation (RCA); Streptococcal M proteins;