Biomaterials (v.28, #4)

Surface-textured PEG-based hydrogels with adjustable elasticity: Synthesis and characterization by Pascal M. Pfister; Michael Wendlandt; Peter Neuenschwander; Ulrich W. Suter (567-575).
Poly(ethylene glycol)–dimethacrylate (PEGDMA)-based hydrogels with adjustable shear modulus within the range of 10 kPa to 1 MPa and precisely predefinable surface textures on a micro-scale were made. It was observed that the volume of all hydrogels after preparation almost exactly matched the volume of the precursor solution and that there were only slight volume changes upon equilibration in excess solvent. This characteristic swelling behavior enables the preparation of textures on the hydrogel's surface with precisely predefinable dimensions. The behavior can be modeled with the Flory–Huggins theory assuming a concentration-dependent polymer–solvent interaction parameter. Additionally, activation of the hydrogels by electrophilic oxirane groups creates reactive sites that will enable the later grafting of the hydrogel's surface with various specific nucleophiles, e.g. biomolecules. Thus, these hydrogels are particularly suitable as biomaterials for systematic investigations of cellular response to surface topography and elasticity of the substrate, both in vivo and in vitro.
Keywords: Hydrogel; Polyethylene glycol; Elasticity; Surface texture; Swelling;

The effect of surface chemistry on the formation of thin films of native fibrillar collagen by John T. Elliott; John T. Woodward; Anita Umarji; Ying Mei; Alessandro Tona (576-585).
In this study, we used well-defined, homogeneous, gradient and patterned substrates to explore the effects of surface chemistry on the supramolecular structure of adsorbed type I collagen. Type I collagen (320 μg/mL) was allowed to adsorb onto self-assembled CH3-, COOH-, NH2- and OH-terminated alkylthiolate monolayers at 37  °C. Atomic force microscopy, ellipsometry and phase microscopy indicated that large supramolecular collagen fibril structures (approximately 200 nm in diameter, several microns long) assembled only at the hydrophobic CH3-terminated surfaces. By varying the surface energy using a mixture of OH- and CH3-terminated thiols during monolayer formation, we found that large fibril assembly occurred at surfaces with a water contact angle above 83°, but not on surfaces with a water contact angle below 63°. Examining a surface with a linear hydrophobicity gradient revealed that the assembly of large collagen fibrils requires a hydrophobic surface with a water contact angle of at least 78°. Collagen fibril density increased over a narrow range of surface energy and reached near-maximum density on surfaces with a water contact angle of 87°. These studies confirm that the supramolecular structure of adsorbed collagen is highly dependent on the underlying substrate surface chemistry. We can take advantage of this dependency to pattern areas of fibrillar and non-fibrillar collagen on a single surface. Morphology studies with vascular smooth muscle cells indicated that only collagen films formed on hydrophobic substrates mimicked the biological properties of fibrillar collagen gels.
Keywords: Collagen structure; Protein adsorption; Surface modification; AFM; Micropatterning; Smooth muscle cells;

The influence of surface energy on competitive protein adsorption on oxidized NiTi surfaces by Alexandra Michiardi; Conrado Aparicio; Buddy D. Ratner; Josep A. Planell; Javier Gil (586-594).
NiTi shape memory alloy surfaces, untreated, and oxidized by a new oxidation treatment (OT) in order to obtain a Ni-free surface, have been compared in terms of surface energy and protein adsorption behavior. The polar and dispersive components of the surface energy have been determined. A competitive adsorption process between fibronectin and albumin has been carried out by 125I-radiolabeling. Moreover, the adhesion strength between both proteins and NiTi surfaces has been evaluated by performing an elution test. The results show that the OT treatment enhances the hydrophilic character of NiTi surfaces by significantly increasing the polar component of their surface energy. Moreover, the OT treatment increases the amount of fibronectin and albumin adsorbed. It also increases the fibronectin affinity for NiTi surfaces. The elution test results could suggest a conformational change of fibronectin as a function of chemical composition of NiTi material and of surface treatment. Finally, a linear correlation between the amount of adsorbed albumin and the polar component of the surface energy of NiTi surfaces has been demonstrated. This work indicates that the OT treatment has an influence on the surface energy value of NiTi materials, which in turn influences the protein adsorption process.
Keywords: Nickel–titanium alloys; Protein adsorption; Surface energy; Fibronectin; Albumin;

Effect of titanium carbide coating on the osseointegration response in vitro and in vivo by Marina Brama; Nicholas Rhodes; John Hunt; Andrea Ricci; Roberto Teghil; Silvia Migliaccio; Carlo Della Rocca; Silvia Leccisotti; Attilio Lioi; Marta Scandurra; Giovanni De Maria; Daniela Ferro; Fanrong Pu; Gianluca Panzini; Laura Politi; Roberto Scandurra (595-608).
Titanium has limitations in its clinical performance in dental and orthopaedic applications. This study describes a coating process using pulsed laser deposition (PLD) technology to produce surfaces of titanium carbide (TiC) on titanium substrates and evaluates the biological response both in vitro and in vivo. X-ray photoelectron spectroscopy (XPS) analysis revealed the presence of 18.6–21.5% TiC in the surface layer, accompanied by oxides of titanium 78.5–81.4% in the following concentrations: 11.1–13.0% Ti2O3, 50.8–55.8% TiO2, 14.5–14.7% TiO. Expression of genes central to osteoblast differentiation (alkaline phosphatase, A2 pro-collagen type 1, osteocalcin, BMP-4, TGFβ and Cbfa-1) were up-regulated in all cell lines (primary human osteoblasts, hFOB1.19 and ROS.MER#14) grown on TiC compared with uncoated titanium when measured by semiquantitative PCR and real time-PCR, whilst genes involved in modulation of osteoclastogenesis and osteoclast activity (IL-6 and M-CSF) were unchanged. Bone density was shown to be greater around TiC-coated implants after 2 and 4 weeks in sheep and both 4 and 8 weeks in rabbits compared to uncoated titanium. Rapid bone deposition was demonstrated after only 2 weeks in the rabbit model when visualized with intravital staining. It is concluded that coating with TiC will, in comparison to uncoated titanium, improve implant hardness, biocompatibility through surface stability and osseointegration through improved bone growth.
Keywords: Titanium; Titanium carbide; Pulsed laser deposition; Plasma coating; Osseointegration;

In vivo biocompatibility and stability of a substrate-supported polymerizable membrane-mimetic film by John T. Wilson; Wanxing Cui; Xue-Long Sun; Carol Tucker-Burden; Collin J. Weber; Elliot L. Chaikof (609-617).
The cell membrane establishes an important paradigm for the molecular engineering of coatings for implantable devices because of its intrinsic biocompatibility and ability to act as a template for the assembly of diverse membrane-associated macromolecules. A stabilized membrane-mimetic film was assembled on alginate/Ca2+ hydrogel microcapsules by in situ polymerization of an acrylate functionalized phospholipid. The phospholipid monomer was prepared as unilamellar vesicles and fused onto octadecyl chains that were components of an amphiphilic terpolymer anchored onto a polyelectrolyte multilayer (PEM) by electrostatic interactions. Microcapsules coated with a membrane-mimetic film were implanted into the peritoneal cavity of C57BL/6 mice, and the short-term biostability and biocompatibility of membrane-mimetic films assembled on two different alginate/poly(l-lysine) PEM cushions were compared. The nature of the underlying PEM support had a profound impact on the biocompatibility of the membrane-mimetic film, as the percentage of retrieved microcapsules completely overgrown with host cells shifted from 66±5.9% to less than 1% when modifications to the PEM were made. When assembled on the appropriate PEM support, biocompatibility of membrane-mimetic-coated microspheres was high wherein 87.5±5.7% of the implanted microspheres were retrieved 4 weeks after implantation and 92.6±6.4% of the retrieved capsules were free of cell adhesion or fibrotic overgrowth. Finally, 4 weeks after implantation, microspheres coated with a Texas red-labeled membrane-mimetic film were imaged with confocal microscopy and exhibited a uniform film around the periphery of the implant, indicating a high degree of film biostability. Hence, membrane-mimetic films provide a new route for generating robust, biocompatible, and biochemically heterogeneous coatings for implantable devices through molecular self-assembly.
Keywords: Biomimetics; Microcapsule; Polyelectrolyte multilayer; Thin film; Biocompatibility; Self assembly;

Plasma-sprayed carbon nanotube reinforced hydroxyapatite coatings and their interaction with human osteoblasts in vitro by Kantesh Balani; Rebecca Anderson; Tapas Laha; Melanie Andara; Jorge Tercero; Eric Crumpler; Arvind Agarwal (618-624).
Carbon nanotubes (CNT) possess excellent mechanical properties to play the role as reinforcement for imparting strength and toughness to brittle hydroxyapatite (HA) bioceramic coating. However, lack of processing technique to uniformly distribute multiwalled CNTs in HA coating and limited studies and sparse knowledge evincing toxicity of CNTs has kept researchers in dispute for long. In the current work, we have addressed these issues by (i) successfully distributing multiwalled CNT reinforcement in HA coating using plasma spraying to improve the fracture toughness (by 56%) and enhance crystallinity (by 27%), and (ii) culturing human osteoblast hFOB 1.19 cells onto CNT reinforced HA coating to elicit its biocompatibility with living cells. Unrestricted growth of human osteoblast hFOB 1.19 cells has been observed near CNT regions claiming assistance by CNT surfaces to promote cell growth and proliferation.
Keywords: Hydroxyapatite coating; Carbon nanotube (CNT); Plasma spraying; Titanium alloy; Crystallinity; Human osteoblast;

The alteration of cell membrane charge after cultured on polymer membranes by Chih-Chi Wang; Jui-Nan Lu; Tai-Horng Young (625-631).
In this work, cell electrophoresis, measuring the electrophoretic mobility of cells, was used to investigate the variation of surface charge property of cells after cultured on different polymer membranes. HepG2 cell line, derived from a well-differentiated, human hepatoma, was used as a model cell. The polymer biomaterials used in this study included polyvinyl alcohol (PVA), poly(ethylene-co-vinyl alcohol) (EVAL), and polyvinylidene fluoride (PVDF). For cells cultured in the presence of serum, cell mobility after being cultured on PVA substrates was considerably higher than that on EVAL or PVDF substrates. This effect was completely suppressed by cycloheximide (CHX) in the serum-free medium. Taken together, the cell surface charge property can be altered after cells cultured on different polymer substrates. The precise mechanism by which the variation of electrophoretic mobility of cultured cells is unknown, but it is reasonable to assume that the polymer substrates could influence the absorption of serum proteins on cell membrane surface to change cell electrophoretic mobility and, simultaneously, to regulate adhesion, growth and function of cultured cells.
Keywords: Electrophoresis; HepG2 cells; PVA; EVAL; PVDF;

Cytotoxicity of polyethyleneimine (PEI), precursor base layer of polyelectrolyte multilayer films by Céline Brunot; Laurence Ponsonnet; Christelle Lagneau; Pierre Farge; Catherine Picart; Brigitte Grosgogeat (632-640).
Polyethyleneimine (PEI) is a synthetic polymer commonly used as precursor base layer in polyelectrolyte multilayer films. However, the biological properties of this cationic macromolecule are poorly understood. The aim of this experimental investigation was to evaluate in vitro the biocompatibility of PEI towards two different human cell lines. The experimental investigation was undertaken on pure titanium (Ti) and nickel–titanium (NiTi) alloy samples with an average surface roughness of Ra = 0.3 μ m . A biological study was undertaken at day 0 (2 h after seeding), day 2, day 4 and day 7 to observe the cellular response of fibroblasts and osteoblasts cell lines in terms of morphology, adhesion (as observed by scanning electron microscopy), and viability (Mosmann's test). The results showed that PEI can be successfully deposited onto Ti or NiTi alloy, but generates a detrimental cellular response on both substrates as illustrated by a decrease of both fibroblast and osteoblast adhesion and proliferation over a 7-day culture period. These results suggest that PEI is potentially cytotoxic and may not be biocompatible enough in clinical applications using high molecular weight. As a consequence, polyelectrolyte multilayer films, which are promising in prosthesis and implantology fields, could not be coated with PEI at a high molecular weight. A lower molecular weight should be considered or a more biocompatible molecular base as precursor layer of polyelectrolyte multilayer films would be better to use for a good human bio-integration.
Keywords: Polyethyleneimine; Titanium; Nickel–titanium alloy; Polyelectrolyte multilayer films; Fibroblasts; Osteoblasts;

Effects of cardiac patches engineered with bone marrow-derived mononuclear cells and PGCL scaffolds in a rat myocardial infarction model by Hainan Piao; Jin-Sook Kwon; Shuguang Piao; Ju-Hee Sohn; Yeong-Shin Lee; Jang-Whan Bae; Kyung-Kuk Hwang; Dong-Woon Kim; Oju Jeon; Byung-Soo Kim; Young-Bae Park; Myeong-Chan Cho (641-649).
Little is known about the cardioprotective effects against heart failure (HF), the effects on differentiation of bone marrow-derived mononuclear cell (BMMNC), and the biocompatibility of BMMNC-seeded biodegradable poly-glycolide-co-caprolactone (PGCL) scaffolds in a myocardial infarction (MI) animal model. This study hypothesized that implantation of a BMMNC-seeded PGCL scaffold into the epicardial surface in a rat MI model would be biocompatible, induce BMMNC migration into infarcted myocardium, and effectively improve left ventricular (LV) systolic dysfunction. One week after the implantation of a BMMNC-seeded PGCL scaffold, BMMMC showed migration into the epicardial region. Four weeks after implantation, augmented neovascularization was observed in infarcted areas and in infarct border zones. Some BMMNCs exhibited the presence of α-MHC and troponin I, markers of differentiation into cardiomyocytes. In echocardiographic examinations, BMMNC-seeded PGCL scaffold and non-cell-seeded simple PGCL scaffold groups effectively reduced progressive LV dilatation and preserved LV systolic function as compared to control rat MI groups. Thus, BMMNC-seeded PGCL scaffolding influences BMMNC migration, differentiation to cardiomyocytes, and induction of neovascularization, ultimately effectively lessening LV remodeling and progressive LV systolic dysfunction. PGCL scaffolding can be considered as an effective treatment alternative in MI-induced advanced HF.
Keywords: Myocardial infarction; Heart failure; Stem cell; Polymer;

A Nd:YAG laser-microperforated poly(3-hydroxybutyrate-co-3-hydroxyvalerate)-basal membrane matrix composite film as substrate for keratinocytes by Fernando Serrano; Laura López-G; Maria Jadraque; Mariëlle Koper; Gary Ellis; Pilar Cano; Margarita Martín; Leoncio Garrido (650-660).
Epithelia cultured for the treatment of ulcers, burns and for gene therapy applications require a flexible biomaterial for growth and transplantation that is adaptable to body contours. We tested several materials and found that a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) polyester provided support for keratinocytes, although adhesion to this material proved to be suboptimal. Since epithelia adhere to the mesoderm through basal membranes, we engineered a basal membrane surrogate by preparing composites of PHBHV with basal membrane matrix (BMM). To allow cell migration into injuried areas the polyester film was micromachined to insert high-density micropores through a Nd:YAG laser ablation process. These flexible composites provided firm attachment for keratinocytes from the outer root sheath of human hair allowing keratinocyte migration through micropores. Films of microperforated PHBHV-BMM may be of use for the replacement of diseased or injured skin epithelia.
Keywords: PHBHV; Basal membrane matrix; Epithelium; Microperforated biomaterial;

Amphiphilic block terpolymer conetworks composed of butyl methacrylate (BMA), 2,3 propandiol-1-methacrylate (GMMA) and ethandiol dimethacrylate (EDMA) were synthesized. Telechelic oligomers with the carboxylic acid end groups were made via ozonolysis of poly(BMA-co-butadiene) and then these were reacted with glycidyl methacrylate to obtain cross-linkable vinyl groups at both chain ends. Networks were then formed via free radical copolymerization with EDMA and GMMA or 2-methyl-acrylic acid 2,2-dimethyl-[1,3]dioxolan-4-ylmethyl ester (GMAc). The acetonide groups of the GMAc units were then removed, by reaction with selenium dioxide and hydrogen peroxide, to give networks with the same molecular structure as the GMMA terpolymers but different cell adhesion and protein adsorption properties. Protein adsorption was maximised in networks prepared with GMMA rather than with GMAc followed by removal of the acetonide. Block conetworks that were synthesised with GMAc were poor substrates for cell proliferation whilst the GMMA class support good levels of both cell viability and proliferation. It is suggested that the difference in behaviour is derived from changes in the surface composition.
Keywords: Amphiphilic conetwork; Protein adsorption; Cell culture; Fibroblast;

Cell adaptation to a physiologically relevant ECM mimic with different viscoelastic properties by Kaustabh Ghosh; Zhi Pan; E Guan; Shouren Ge; Yajie Liu; Toshio Nakamura; Xiang-Dong Ren; Miriam Rafailovich; Richard A.F. Clark (671-679).
To successfully induce tissue repair or regeneration in vivo, bioengineered constructs must possess both optimal bioactivity and mechanical strength. This is because cell interaction with the extracellular matrix (ECM) produces two different but concurrent signaling mechanisms: ligation-induced signaling, which depends on ECM biological stimuli, and traction-induced signaling, which depends on ECM mechanical stimuli. In this report, we provide a fundamental understanding of how alterations in mechanical stimuli alone, produced by varying the viscoelastic properties of our bioengineered construct, modulate phenotypic behavior at the whole-cell level. Using a physiologically relevant ECM mimic composed of hyaluronan and fibronectin, we found that adult human dermal fibroblasts modify their mechanical response in order to match substrate stiffness. More specifically, the cells on stiffer substrates had higher modulus and a more stretched and organized actin cytoskeleton (and vice versa), which translated into larger traction forces exerted on the substrate. This modulation of cellular mechanics had contrasting effects on migration and proliferation, where cells migrated faster on softer substrates while proliferating preferentially on the stiffer ones. These findings implicate substrate rigidity as a critical design parameter in the development of bioengineered constructs aimed at eliciting maximal cell and tissue function.
Keywords: Dermal fibroblasts; Cell mechanics; Hydrogel stiffness; Hyaluronan; Fibronectin;

The sequential seeding of epithelial and mesenchymal cells for tissue-engineered tooth regeneration by Masaki J. Honda; Shuhei Tsuchiya; Yoshinori Sumita; Hiroshi Sagara; Minoru Ueda (680-689).
Progress is being made toward regenerating teeth by seeding dissociated postnatal odontogenic cells onto scaffolds and implanting them in vivo, but tooth morphology remains difficult to control. In this study, we aimed to facilitate tooth regeneration using a novel technique to sequentially seed epithelial cells and mesenchymal cells so that they formed appropriate interactions in the scaffold. Dental epithelium and mesenchyme from porcine third molar teeth were enzymatically separated and dissociated into single cells. Mesenchymal cells were seeded onto the surface of the scaffold and epithelial cells were then plated on top so that the two cell types were in direct contact. The cell–scaffold constructs were evaluated in vitro and also implanted into immunocompromised rats for in vivo analysis. Control groups included constructs where direct contact between the two cell types was prevented. In scaffolds seed using the novel technique, alkaline phosphatase activity was significantly greater than controls, the tooth morphology in vivo was developed in similar to that of natural tooth, and only one tooth structure formed in each scaffold. These results suggest that the novel cell-seeding technique could be useful for regulating the morphology of regenerated teeth.
Keywords: Sequential seeding; Cell–cell interaction; Postnatal tooth cells; Tooth tissue engineering; Tooth morphology; TEM (transmission electron microscopy);

Mechanisms of the in vivo inhibition of calcification of bioprosthetic porcine aortic valve cusps and aortic wall with triglycidylamine/mercapto bisphosphonate by H. Scott Rapoport; Jeanne M. Connolly; James Fulmer; Ning Dai; Brandon H. Murti; Robert C. Gorman; Joseph H. Gorman; Ivan Alferiev; Robert J. Levy (690-699).
Heart valve replacements fabricated from glutaraldehyde (Glut)-crosslinked heterograft materials, porcine aortic valves or bovine pericardium, have been widely used in cardiac surgery to treat heart valve disease. However, these bioprosthetic heart valves often fail in long-term clinical implants due to pathologic calcification of the bioprosthetic leaflets, and for stentless porcine aortic valve bioprostheses, bioprosthetic aortic wall calcification also typically occurs. Previous use of the epoxide-based crosslinker, triglycidyl amine (TGA), on cardiac bioprosthetic valve materials demonstrated superior biocompatibility, mechanics, and calcification resistance for porcine aortic valve cusps (but not porcine aortic wall) and bovine pericardium, vs. Glut-prepared controls. However, TGA preparation did not completely prevent long-term calcification of cusps or pericardium. Herein we report further mechanistic investigations of an added therapeutic component to this system, 2-mercaptoethylidene-1,1-bisphosphonic acid (MABP), a custom synthesized thiol bisphosphonate, which has previously been shown in a preliminary report to prevent bioprosthetic heterograft biomaterial calcification when used in combination with initial TGA crosslinking for 7 days. In the present studies, we have further investigated the effectiveness of MABP in experiments that examined: (1) The use of MABP after optimal TGA crosslinking, in order to avoid any competitive interference of MABP-reactions with TGA during crosslinking; (2) Furthermore, recognizing the importance of alkaline phosphatase (ALP) in the formation of dystrophic calcific nodules, we have investigated the hypothesis that the mechanism by which MABP primarily functions is through the reduction of ALP activity. Results from cell-free model systems, cell culture studies, and rat subcutaneous implants, show that materials functionalized with MABP after TGA crosslinking have reduced ALP activity, and in vivo have no significant calcification in long-term implant studies. It is concluded that bioprosthetic heart valves prepared in this fashion are compelling alternatives for Glut-prepared bioprostheses.
Keywords: Calcification; Biomineralization; Bioprostheses; Heart valve; Cross-linking; Alkaline phosphate;

Endovascular stents made of the superelastic nickel-titanium alloy Nitinol are subjected in service to tens of millions of loading cycles and even “single-event” overloads, both of which can potentially result in fracture and/or complete failure of the device. A fracture-mechanics-based methodology can provide a means to quantify relevant material parameters critical to the design against such failures. However, there is a dearth of relevant experimental data in the literature on such fracture-mechanics-based approaches to fatigue in Nitinol; furthermore, that which does exist invariably pertains to product forms that are not appropriate for stent manufacture, e.g., bulk Nitinol bar and strip. Consequently, the current work is focused on characterizing in vitro both subcritical and critical crack growth (fatigue–crack growth and R-curve fracture toughness) behavior in thin-walled (∼400 μm thick) Nitinol tubing similar to that used for medical device manufacture (following shape-setting procedures to flatten the material), with a resultant austenite finish temperature of A f∼25–30 °C, identical to self-expanding Nitinol stents. Fatigue–crack growth behavior, measured in Hanks’ Balanced Saline Solution over a wide spectrum of growth rates (down to 10−10  m/cycle) and at a range of positive load ratios ( R = 0.1 – 0.7 ), revealed significantly higher fatigue thresholds than had been previously reported for bulk Nitinol material. In addition, we examine the critical effect of test frequency, as most fatigue experiments on Nitinol have been performed at 30 Hz or above, despite the fact that this is far in excess of the frequency of physiological loading. Finally, the fracture toughness properties are characterized in thin-section Nitinol and show marked crack-resistance (R-curve) behavior with a dependence on crack-growth angle (with respect to the tube drawing axis); additionally, measured toughnesses are found to be lower than has been previously reported for bulk Nitinol.
Keywords: Fatigue; Fracture toughness; Fracture mechanism; Nitinol; Nickel-titanium alloy; Stent;

Surface modification of magnetite nanoparticles using lactobionic acid and their interaction with hepatocytes by K.M. Kamruzzaman Selim; Yong-Soo Ha; Sun-Jung Kim; Yongmin Chang; Tae-Jeong Kim; Gang Ho Lee; Inn-Kyu Kang (710-716).
In the current study, superparamagnetic magnetite nanoparticles were surface-modified with lactobionic acid (LA) to improve their intracellular uptake and ability to target hepatocytes. Maltotrionic acid (MA)-modified nanoparticles were also synthesized as a control. Cell culture experiment showed that LA-modified nanoparticles were internalized into hepatocytes and atomic absorption spectrometer (AAS) measurement indicated that the uptake amount of LA-modified magnetite into hepatocytes was higher than that of unmodified and MA-modified nanoparticles. LA-modified nanoparticle solution was injected in rabbit and the magnetic resonance (MR) images obtained showed that LA-coated nanoparticles were selectively accumulated onto the hepatocytes. This result demonstrates that the LA-modified magnetite nanoparticles have a great potential to be used as contrast agent for liver diagnosis.
Keywords: Magnetite nanoparticles; Lactobionic acid; Maltotrionic acid; Intracellular uptake; Hepatocytes; Particle internalization;

Biomimetic hydrogels for enhanced loading and extended release of ocular therapeutics by Siddarth Venkatesh; Stephen P. Sizemore; Mark E. Byrne (717-724).
We have applied the principles of biomimesis by incorporating a natural receptor-based rational design strategy in the synthesis of novel recognitive soft contact lenses. We have demonstrated the potential of biomimetic carriers to load significant amounts of ocular medication such as H1-antihistamines, as well as to release a therapeutic dosage of drug in vitro in a controlled fashion for 5 days, with an even further extension in the presence of protein. Gels of multiple complexation points with varying functionalities outperformed gels formed with less diverse functional monomers and showed superior loading with a six-fold difference over control gels and a three-fold difference over less biomimetic gels. Moreover, mechanical and optical properties of these hydrogels agreed with conventional lenses, and increased loading was reflected in a reduced propagation of polymer chains. This approach can be extended to a wider biological spectrum in the design of novel, controlled and modulated delivery devices to alleviate ocular disorders and provide an alternative to topical therapy.
Keywords: Biomimetic hydrogel; Ophthalmic drug delivery; Extended release; Molecular imprinting; Differential scanning calorimetry (DSC); Therapeutic contact tenses;

Shell-crosslinked Pluronic L121 micelles as a drug delivery vehicle by Ting-Fan Yang; Chun-Nan Chen; Mei-Chin Chen; Chien-Hsun Lai; Hsiang-Fa Liang; Hsing-Wen Sung (725-734).
Pluronic block copolymers (PBCs) have been shown to reverse multidrug resistance (MDR) by inhibiting the P-glycoprotein (P-gp) pump in cancer cells. One of the problems encountered with the use of PBCs is that the micelles disassociate at low concentrations. The study focused on the stabilization of PBC L121 micelles by the formation of crosslinks within their outer shells. To form crosslinks, the two terminal alcohols on L121 were first chemically converted into aldehydes (L121-CHO) using the Dess–Martin periodinane. Diamine compounds were then used to bridge the converted aldehyde termini on L121-CHO via conjugated Schiff bases. After crosslinking, the morphology of the L121 micelles remained spherical in shape and the mean particle sizes of the micelles before and after crosslinking were comparable (100 nm). After exposure of MDR KBv cells to free rhodamine-123 (R123), the accumulation of R123 in cells was limited due to the function of P-gp. In contrast, crosslinking of L121 micelles within their outer shells significantly reduced their critical micelle concentration and greatly enhanced their stability, while maintaining their ability to inhibit P-gp function in resistant cells. The results indicated that the L121 micelles with shell crosslinks may be useful as a drug delivery vehicle for cancer chemotherapy.
Keywords: Pluronics; Micelle; Crosslinking; Drug delivery; Critical micelle concentration;

A biodegradable poly(ester amine) based on polycaprolactone and polyethylenimine as a gene carrier by Rohidas Arote; Tae-Hee Kim; You-Kyoung Kim; Soon-Kyung Hwang; Hu-Lin Jiang; Ho-Hyun Song; Jae-Woon Nah; Myung-Haing Cho; Chong-Su Cho (735-744).
The aim of research was to develop and optimize delivery systems for plasmid DNA (pDNA) based on biodegradable polymers, in particular, poly(ester amine)s (PEAs), suitable for non-viral gene therapy. Poly(ester amine)s were successfully synthesized by Michael addition reaction between polycaprolactone (PCL) diacrylate and low molecular weight polyethylenimine (PEI). PEA/DNA complexes showed effective and stable DNA condensation with the particle sizes below 200 nm, implicating its potential for intracellular delivery. PEAs showed controlled degradation and were essentially non-toxic in all three cells (293 T: Human kidney carcinoma, HepG2: Human hepatoblastoma and HeLa: Human cervix epithelial carcinoma cell lines) at higher doses in contrast to PEI 25 K. PEAs also revealed much higher transfection efficiencies in three cell lines as compared to PEI 25 K. The highest reporter gene expression was observed for PCL/PEI-1.2 (MW 1200) complex having transfection efficiency 15–25 folds higher than PEI 25 K in vitro. Also PEA/DNA complexes successfully transfected cells in vivo after aerosol administration than PEI 25 K. These PEAs can be used as most efficient polymeric vectors which provide a versatile platform for further investigation of structure property relationship along with the controlled degradation, significant low cytotoxicity and high transfection efficiency.
Keywords: Gene delivery; Degradable polymers; Poly(ester amine)s; Polycaprolactone; Polyethylenimine;

Development of transplantable genetically modified corneal epithelial cell sheets for gene therapy by Katsuhiko Watanabe; Masayuki Yamato; Yasutaka Hayashida; Joseph Yang; Akihiko Kikuchi; Teruo Okano; Yasuo Tano; Kohji Nishida (745-749).
The purpose of this study was to establish a method for the fabrication of exogenous gene-transferred, transplantable corneal epithelial cell sheets. Corneo-limbal epithelial cells collected from USA eye bank eyes were transduced with an EGFP-expressing lentiviral vector at differential MOI. Multi-layered corneal epithelial cell sheets were fabricated by co-cultivation of transduced cells and mitomycin C-treated 3T3 feeder layers on temperature-responsive culture dishes. These cultured epithelial cells could be harvested as intact sheets by simply lowering the temperature. The number of EGFP-positive cells was increased as the MOI raised, and at an MOI of 100, nearly 100% of the superficial cells showed strong EGFP expression. Histological analysis revealed that EGFP was expressed in all layers of the cell sheet of which cell source was transduced with the lentiviral vector at an MOI of 100. Immunofluorescence data showed that p63 was also expressed in the basal layer of the same cell sheet. These results suggest that this technique will likely be applicable to ex vivo gene therapies for various corneal disorders.
Keywords: Cornea; Epithelium; Gene therapy; Stem cell; Thermally responsive material; Ophthalmology;

To investigate the effects of the microstructure of collagenous carriers on the in vivo function of bone sialoprotein (BSP) in mineralization and osteoblast differentiation, we examined the ultrastructure of reconstituted type I collagen (collagen) and heat-denatured collagen (gelatin) and the in vivo responses to purified bone-derived BSP that was implanted with collagen or gelatin into surgically created 8-mm rat calvarial bone defects. Scanning and transmission electron microscopies revealed that the collagen displayed a fine fibrillar structure with interconnecting spaces between the fibrils/fibers, while the gelatin completely lost this unique three-dimensional structure after denaturation. The rates of in vivo release of BSP from the collagen scaffold were significantly lower than those from the gelatin. Collagen-BSP, but not gelatin-BSP, induced early mineral deposition in the matrix of proliferating repair cells in the calvarial defects at ∼4–7 days after implantation. Expression levels of osteoblast markers, alkaline phosphatase activity and amounts of new bone synthesized in the collagen-BSP treated defects were significantly greater than that in the gelatin-BSP treated defects (p<0.001). The data suggest that the fibrillar microstructure of reconstituted collagen is essential for retaining BSP at a higher concentration within the defects, which enhances BSP-mediated matrix mineralization and osteoblast differentiation during the repair of rat calvarial defects.
Keywords: Collagen; Gelatin; Osteogenesis; Osteoblast; Bone repair;