Biomaterials (v.26, #32)

Calendar (I).

A novel thermotropic liquid-crystalline biocopolymer, poly{trans-4-hydroxycinnamic acid (4HCA: trans-coumaric acid)-co-lithocholic acid (LCA)}, was synthesized by a thermal polycondensation of 4HCA and LCA. When the LCA composition of P(4HCA-co-LCA) was 0, 5, 7, 23, 27, and 45 mol%, the copolymers showed a nematic liquid-crystalline phase. The melting point of the copolymers was 120–250 °C depending on the LCA composition, and showed a maximum at 7 mol%. Wide angle X-ray diffraction (WAXD) analyses showed a typical diffraction pattern of a hexagonal arrangement for 0, 7, 23, and 27 mol% LCA composition, which were cooled from a nematic melt. The other polymers showed no distinct diffraction. In particular, the copolymer of 7 mol% LCA composition showed four distinct diffractions corresponding to spacings with a reciprocal ratio of 1, √3, 2, and √7, indicating the highest structural ordering of all copolymers prepared here. The results of the cell adhesion and static contact angle tests suggest that the 7 mol% LCA composition copolymer had the highest hydrophobicity and cell adhesion ability, which was easily controlled by altering in feed. We conclude that the structural ordering may have a significant correlation with cell adhesion activity.
Keywords: Biocompatibility; Cell adhesion; Copolymer; Liquid crystal;

Interactions between endothelial cells and a poly(carbonate-silsesquioxane-bridge-urea)urethane by Geoffrey Punshon; Dina S. Vara; Kevin M. Sales; Asmeret G. Kidane; Henryk J. Salacinski; Alexander M. Seifalian (6271-6279).
We have recently developed a polymer which contains silsesquioxane in the form of nano-bridges poly(carbonate-silsesquioxane-bridge-urea)urethane (PCBSU) for cardiovascular device applications. The polymer has been characterised and the durability has been confirmed with long-term in vivo tests. The aim of this study was to test the cytocompatibility of the new polymer and to investigate any potential cytotoxic effects. To assess the effect of direct contact with PCBSU sections of polymer material were cut and placed into a 24-well plate. Six discs were seeded with 2×105 human umbilical vein cells (HUVEC). As a positive control, six wells were seeded with the same number of HUVEC. In a further experiment to assess indirect contact with PCBSU a sample of the polymer was powdered using a Micro-Dismembrator. Cell culture medium was exposed to powdered polymer (1–100 mg/ml) for a period of 7 days. HUVEC seeded as above were then exposed to the treated cell culture medium for 24 and 96 h. Finally, cell proliferation was studied over 16 days by seeding 2×105 HUVEC on films of PCBSU cast in glass Petri dishes. Cell viability and growth were assessed using Alamar blue™, lactate dehydrogenase and Pico green assays and morphology was studied by Toluidine blue staining and scanning electron microscopy. Viable cells were demonstrated to be present after 16 days seeded on PCBSU. Exposing cells to PCBSU-treated cell culture medium resulted in no apparent damage to the cells at concentrations of 1 or 10 mg/ml, and only a slight reduction at 100 mg/ml after 96 h exposure. This study demonstrates that PCBSU can support the growth of endothelial cells for a prolonged period and does not demonstrate any significant toxic effects to cells. Thus it has the potential to be used both as a medical device and as scaffolding in tissue engineering applications.
Keywords: Cell viability; Copolymer; Cytotoxicity; Bypass graft; Stent; Polymer; Silsesquioxane; Silsesquioxane nano-bridges; Medical device;

Ultrastructural localization of laminin-5 ( γ 2 chain) in the rat peri-implant oral mucosa around a titanium-dental implant by immuno-electron microscopy by Ikiru Atsuta; Takayoshi Yamaza; Masao Yoshinari; Tetsuya Goto; Mizuho A. Kido; Tadayoshi Kagiya; Satoya Mino; Masaki Shimono; Teruo Tanaka (6280-6287).
Laminin-5 (Ln-5) is an important molecule associated with epithelial cell adhesion and migration. In the gingiva around the tooth, Ln-5 localizes within basement membranes between the junctional epithelium (JE) and the tooth or connective tissue. Recently, we reported that in the oral mucosa around a dental implant, Ln-5 is expressed within the basement membranes at the implant-peri-implant epithelium (PIE) interface, and at the PIE–connective tissue interface. However, the ultrastructural localization of Ln-5 within or along the PIE has not yet been reported. Therefore, peri-implant oral mucosa was treated with anti-Ln-5 ( γ 2 chain) antibody and examined using immuno-electron microscopy. Ln-5 was localized in the cells of the innermost-third layer and basal layer of the PIE. A 100-nm-wide Ln-5-positive internal basal lamina (basement membrane) and hemidesmosomes as adhesion structures were formed at the apical portion of the implant–PIE interface. However, at the upper-middle portion of the interface, these adhesion structures were not observed. Furthermore, at the PIE–connective tissue interface, the Ln-5-positive external basal lamina (basement membrane) and hemidesmosomes were partially deficient. Judging from these findings, we concluded that Ln-5 contributes to the attachment of the PIE to the titanium surface, and that PIE attached to titanium at the apical portion of the dental implant–PIE interface.
Keywords: Laminin-5; Peri-implant epithelium; Junctional epithelium; Internal and external basal laminae; Titanium dental implant; Immuno-electron microscopy;

In vitro behavior of osteoblast-like cells on fluoridated hydroxyapatite coatings by Kui Cheng; Wenjian Weng; Huiming Wang; Sam Zhang (6288-6295).
In this work, fluoridated hydroxyapatite (Ca10(PO4)6F x (OH)2− x or FHA) coatings are prepared by sol–gel method for study of the influence of F content on the behavior of osteoblast-like cells. The results show that the cells well attach and proliferate on the FHA coatings studied (Ca10(PO4)6F0.67–2.00(OH)0–1.33). With increasing F content in the FHA coatings, percentage of cell in S period increases, indicating F in the coating favors the proliferation process of the cells. On the other hand, the proliferation rate increases inversely with zeta potential of the coating surface. As tested from the MTT of the cells cultured in the leaching out solution, increase of F content in the FHA coatings results in a slight decrease in cell proliferation rate, which is most probably due to reduction in release of Ca2+ ions. As a compromise among cell attachment, cell proliferation, apatite deposition and ability to resist dissolution, it is suggested that FHA coatings (Ca10(PO4)6F x (OH)2− x ) with x in the range of 0.67–1.48, from the results of this study, may be most suitable for real case implantation.
Keywords: Fluoridated hydroxyapatite; Surface fluorine content; Osteoblast-like cells;

Nano-composite of poly(l-lactide) and surface grafted hydroxyapatite: Mechanical properties and biocompatibility by Zhongkui Hong; Peibiao Zhang; Chaoliang He; Xueyu Qiu; Aixue Liu; Li Chen; Xuesi Chen; Xiabin Jing (6296-6304).
In order to improve the bonding between hydroxyapatite (HAP) particles and poly(l-lactide) (PLLA), and hence to increase mechanical properties of the PLLA/HAP composite as potential bone substitute material, the HAP nano-particles were surface-grafted with PLLA and further blended with PLLA. The structure and properties of the composites were subsequently investigated by the mechanical property testing, the differential scanning calorimeter measurements (DSC), the scanning electron microscopy (SEM), the polarized optical microscopy (POM), and the cell culture. The PLLA molecules grafted on the HAP surfaces, as inter-tying molecules, played an important role in improving the adhesive strength between the particles and the polymer matrix. At a low content (∼4 wt%) of surface grafted-HAP (g-HAP), the PLLA/g-HAP nano-composites exhibited higher bending strength and impact energy than the pristine PLLA, and at a higher g-HAP content (e.g., 20 wt%), the modulus was remarkably increased. It implied that PLLA could be strengthened as well as toughened by g-HAP nano-particles. The results of biocompatibility test showed that the g-HAP existing in the PLLA composite facilitated both adhesion and proliferation of chondrocytes on the PLLA/g-HAP composite film.
Keywords: Polylactide; Hydroxyapatite; Composite; Mechanical properties; Biocompotibility; Cell culture;

Collagen-coated polylactide microspheres as chondrocyte microcarriers by Yi Hong; Changyou Gao; Ying Xie; Yihong Gong; Jiacong Shen (6305-6313).
Polylactide (PLA) microspheres were coated with collagen for cell culture and injectable cell carriers. Utilizing a method of emulsion-solvent evaporation, PLA microspheres with diameter ranging from 180 to 280 μm were prepared, followed with aminolysis in hexanediamine/n-propanol solution to introduce free amino groups on their surfaces. After the amino groups were transferred into aldehyde groups by a treatment of glutaraldehyde, collagen type I was covalently coupled via Schiff base formation between the aldehyde groups and the amino groups on collagen molecules. Meanwhile, physically entangled collagen molecules were retained following a grafting-coating protocol to yield microspheres coated with larger amount of collagen. Aminolysis resulted in weight loss of the microspheres following a linear relationship with the aminolysis time. The NH2 and collagen contents existed on the microsphere surface were quantitatively determined by ninhydrin and hydroproline (Hyp) analyses, respectively. Larger amount of collagen was immobilized on the microspheres with higher content of NH2. In vitro chondrocyte culture revealed that the cells could attach, proliferate and spread on these PLA microspheres, in particular on the ones having higher content of collagen. These results show that the collagen-coated PLA microspheres are promising candidate as cell microcarriers.
Keywords: Microspheres; Polylactide; Chondrocytes; Collagen; Cell microcarriers;

A poly(lactic acid)/calcium metaphosphate composite for bone tissue engineering by Youngmee Jung; Sang-Soo Kim; Young Ha Kim; Sang-Heon Kim; Byung-Soo Kim; Sukyoung Kim; Cha Yong Choi; Soo Hyun Kim (6314-6322).
A new method to prepare PLA/CMP (poly-l-lactide/calcium metaphosphate) composite scaffolds was developed for effective bone tissue engineering. This novel sintering method is composed of pressing the mixture of PLA, CMP, and salt particles at 150 MPa for 3 min followed by heat treatment at 210 °C for 30 min. The scaffolds had a homogeneously interconnected porous structure without a skin layer, and they exhibited a narrower pore size distribution and higher mechanical strength in comparison with scaffolds made by a solvent casting method. The scaffolds were seeded by osteoblasts and cultured in vitro or implanted into nude mice subcutaneously for up to 5 weeks. The number of cells attached to and proliferated on the scaffolds at both in vitro and in vivo was in the order of; PLA by novel sintering<PLA/CMP by solvent casting<PLA/CMP by novel sintering. In addition, the alkaline phosphatase activity of and calcium deposition in the scaffolds explanted from mice were enhanced significantly for the scaffolds by novel sintering compared to them by solvent casting. The in vitro results agreed well with the in vivo data. Such a superior characteristic of the novel sintering method should have resulted from the fact that the CMP particles could contact directly with cells/tissues to stimulate the cell proliferation and osteogenic differentiation, while the CMP particles would be coated by polymers and hindered to interact with cells/tissues in the case of a solvent casting method. As the novel sintering method does not use any solvents it offers another advantage to avoid problems associated with solvent residue.
Keywords: Bone regeneration; Tissue engineering; Composites; Poly-l-lactide; Calcium metaphosphate;

Biodegradable polymer-ceramic composites are attractive systems for bone tissue engineering applications. These composites have the combined advantages of the component phases, as well as the inherent ease in optimization where desired material properties can be tailored in a well-controlled manner. This study focuses on the optimization of a polylactide-co-glycolide (PLAGA) and 45S5 bioactive glass (BG) composite for bone tissue engineering. The first objective is to examine the effects of composition or overall BG content on the formation of a Ca–P layer on the PLAGA–BG composite. It is expected that with increasing BG content (0%, 10%, 25%, 50% by weight), the required incubation time in a simulated body fluid (SBF) for the composite to form a detectable surface Ca–P layer will decrease. Both the kinetics and the chemistry will be determined using SEM+EDAX, FTIR, and μ -CT methods. Solution phosphorous and calcium concentrations will also be measured. The second objective of the study is to determine the effects of BG content on the maturation of osteoblast-like cells on the PLAGA–BG composite. It is hypothesized that mineralization will increase with increasing BG content, and the composite will support the proliferation and differentiation of osteoblasts. Specifically, cell proliferation, alkaline phosphatase activity and mineralization will be monitored as a function of BG content (0%, 10%, 50% by weight) and culturing time. It was found that the kinetics of Ca–P layer formation and the resulting Ca–P chemistry were dependent on BG content. The response of human osteoblast-like cells to the PLAGA–BG composite was also a function of BG content. The 10% and 25% BG composite supported greater osteoblast growth and differentiation compared to the 50% BG group. The results of this study suggest that there is a threshold BG content which is optimal for osteoblast growth, and the interactions between PLAGA and BG may modulate the kinetics of Ca–P formation and the overall cellular response.
Keywords: Polymer-ceramic composite; Bioactivity; Degradable polymers; Bioactive glass; Osteoblasts; Mineralization;

Evaluation of an in situ forming hydrogel wound dressing based on oxidized alginate and gelatin by Biji Balakrishnan; M. Mohanty; P.R. Umashankar; A. Jayakrishnan (6335-6342).
Wound dressings that can be formed in situ offer several advantages over the use of preformed dressings such as conformability without wrinkling or fluting in the wound bed, ease of application and improved patient compliance and comfort. Here we describe such an in situ forming hydrogel wound dressing from gelatin, oxidized alginate and borax. Periodate oxidized alginate rapidly cross-links proteins such as gelatin in the presence of borax to give in situ forming hydrogels that are both non-toxic and biodegradable. The composite matrix has the haemostatic effect of gelatin, the wound healing-promoting feature of alginate and the antiseptic property of borax to make it a potential wound dressing material. The hydrogel was found to have a fluid uptake of 90% of its weight which would prevent the wound bed from accumulation of exudates. The water vapour transmission rate (WVTR) of the hydrogel was found to be 2686±124 g/m2/day indicating that the hydrogel can maintain a moist environment over wound bed in moderate to heavily exuding wound which would enhance epithelial cell migration during the healing process. The wound healing efficacy of hydrogel was evaluated in experimental full thickness wounds using a rat model which demonstrated that within 2 weeks, the wound covered with gel was completely filled with new epithelium without any significant adverse reactions. These in situ forming hydrogels fulfil many critical elements desirable in a wound dressing material.
Keywords: Alginate; Gelatin; Cross-linking; Hydrogel; Wound dressing; Wound healing; In vivo test;

Synthesis, characterization and cytotoxicity of poly(ethylene glycol)-graft-trimethyl chitosan block copolymers by Shirui Mao; Xintao Shuai; Florian Unger; Matthias Wittmar; Xiulan Xie; Thomas Kissel (6343-6356).
PEGylated trimethyl chitosan (TMC) copolymers were synthesized in an attempt to both increase the solubility of chitosan in water, and improve the biocompatibility of TMC. A series of copolymers with different degrees of substitution were obtained by grafting activated poly(ethylene glycol)s (PEG) of different MW onto TMC via primary amino groups. Structure of the copolymers was characterized using 1H, 13C NMR spectroscopy and GPC. Solubility experiments demonstrated that PEG-g-TMC copolymers were completely water-soluble over the entire pH range of 1–14 regardless of the PEG MW, even when the graft density was as low as 10%. Using the methyl tetrazolium (MTT) assay, the effect of TMC molecular weight, PEGylation ratio, PEG and TMC molecular weight in the copolymers, and complexation with insulin on the cytotoxicity of TMC was examined, and IC50 values were calculated with L929 cell line. All polymers exhibited a time- and dose-dependent cytotoxic response that increased with molecular weight. PEGylation can decrease the cytotoxicity of TMC to a great extent in the case of low molecular weight TMCs. According to the cytotoxicity results, PEG 5 kDa is superior for PEGylation when compared to PEG 550 Da at similar graft ratios. Complexation with insulin further increased cell viability. In addition, Lactate dehydrogenase (LDH) assays were performed to quantify the membrane-damaging effects of the copolymers, which is in line with the conclusion drawn from MTT assay. Moreover, the safety of the copolymers was corroborated by observing the morphological change of the cells with inverted phase contrast microscopy. Based upon these results PEG-g-TMC merits further investigations as a drug delivery vehicle.
Keywords: Trimethyl chitosan; PEGylation; Copolymer; In vitro cytotoxicity; MTT assay; LDH assay;

A pH responsive pseudopeptide, poly(l-lysine iso-phthalamide), has been modified with a hydrophilic poly(ethylene glycol) analogue, Jeffamine M-1000® and the effect of grafting ratio on the pH responsive behaviour of the grafted polymers in aqueous solution investigated using fluorescence and 1H NMR spectroscopy. It was demonstrated that at below 35.1 wt% grafting, the modified polymers retained the pH-driven conformational transition of the parent polymer from an expanded structure at high degrees of ionisation to a compact hydrophobically stabilised structure at low degrees of ionisation. The onset of pH response and the pH range over which the conformational transition occurred varied significantly with degree of grafting. At Jeffamine M-1000® ratios in excess of 48.0 wt%, the graft polymer existed in a micellular form over the whole pH studied. Potential applications in drug delivery of both the linear and micellular forms are discussed.
Keywords: Peptide; Polyethylene oxide; Hydrophilicity; Fluorescence; Drug delivery;

Based on inclusion character of β-cyclodextrin (β-CD) with drug molecule and low glass transition temperature of poly(2-hydroxyethyl acrylate) (PHEA), a series of hydrogels with different compositions were synthesized by the copolymerization of a monovinyl cyclodexrin monomer with 2-hydroxyethyl acrylate (HEA). The structure and properties of the hydrogels were characterized by FTIR, DSC, TGA and swelling measurements. It is found that swelling ratios of these β-CD hydrogels can keep a relative stability in the range of pH from 1.4 to 7.4, and are not sensitive to change in NaCl concentration. Using drug N-acety-5-methoxytryptamine (melatonin, MEL) as a model molecule, the controlled drug release behaviors of these hydrogels were investigated. The results indicate that the diffusion and permeation of MEL from the hydrogels may be a dominant factor for its release. Owing to the formation of MEL/β-CD retarding diffusion rate of MEL, a sustained release of MEL from hydrogel with high content of β-CD can be obtained compared with hydrogel PHEA without β-CD.
Keywords: Hydrogel; Cyclodextrin; Inclusion complex; The controlled release;

Covalent conjugation of polyethyleneimine on biodegradable microparticles for delivery of plasmid DNA vaccines by Sudhir P Kasturi; Karishma Sachaphibulkij; Krishnendu Roy (6375-6385).
Microparticle-based delivery of nucleic acids has gained particular attention in recent years in view of improving the potency of DNA vaccination. Such improvement has been reported by encapsulation of pDNA within biodegradable microparticles or through surface adsorption on cationic microparticles. However, the intrinsic intracellular barriers for gene delivery to antigen presenting cells (APCs) have not been adequately addressed in the rational design of delivery systems for DNA vaccines. Here we report synthesis and characterization of biodegradable microparticles that (a) can passively target phagocytic APCs, (b) have intrinsic buffering ability that might allow for enhanced phagosomal escape, (c) are not cytotoxic and (d) have improved APC transfection efficiency. Branched polyethyleneimine (b-PEI) was covalently conjugated using carbodiimide chemistry to the surface of poly(lactide-coglycolide) (PLGA) microparticles to create cationic microparticles capable of simultaneously delivering both DNA vaccines as well as other immunomodulatory agents (cytokines or nucleic acids) within a single injectable delivery vehicle. Our results indicate that covalent conjugation of b-PEI allows efficient surface loading of nucleic acids, introduces intrinsic buffering properties to PLGA particles and enhances transfection of phagocytic cells without affecting the cytocompatibility of PLGA carriers.
Keywords: DNA; Microsphere; Gene transfer; Immunomodulation; Polyethyleneimine;

Real-time 3-D dark-field microscopy for the validation of the cross-linking process of alginate microcapsules by R. Wolf; D. Zimmermann; M. Weber; P. Feilen; F. Ehrhart; M. Salinas Jungjohann; A. Katsen; M. Behringer; P. Geßner; L. Pließ; A. Steinbach; J. Spitz; J.A. Vásquez; S. Schneider; E. Bamberg; M.M. Weber; U. Zimmermann; H. Zimmermann (6386-6393).
Alginate-based microencapsulation is a promising method for long-term maintenance of cellular and membrane function of the cells and tissue fragments required for in vitro and in vivo biosensors, for tissue engineering and particularly for immunoisolation of non-autologous transplants. Microcapsules of high mechanical strength and optimum permeability can be produced by injection of BaCl2 crystals into alginate droplets before they come into contact with external Ba2+. A key requirement is that the system parameters (number of crystals, speed of the crystal stream etc.) are properly adjusted according to the mannuronic and guluronic acid ratio and the average molecular mass of the alginate as well as to the diameter of the microcapsules. Robust, reliable, rapid and low-cost validation tools are, therefore, needed for assurance of the microcapsule quality. Here, we describe a novel three-dimensional (3-D) dark-field microscopy that allows the real-time measurement of the number and spatial distribution of the injected Ba2+ ions throughout the microcapsules after treatment with sulphate. This novel method requires only a conventional microscope equipped with three polarising filters and a double aperture stop. In contrast to confocal laser scanning microscopy images, peripherally attached BaSO4 precipitates can clearly be distinguished from internal ones. The data also demonstrate that several steps of the alginate gelling process must be improved before such immunoisolation can be used in patients.
Keywords: Alginate; Crystal gun method; Cross-linking; Ba2+; Dark-field microscopy;

Chitosan-tethered poly(acrylonitrile-co-maleic acid) hollow fiber membrane for lipase immobilization by Peng Ye; Zhi-Kang Xu; Ai-Fu Che; Jian Wu; Patrick Seta (6394-6403).
A protocol was used to prepare a dual-layer biomimetic membrane as support for enzyme immobilization by tethering chitosan on the surface of poly(acrylonitrile-co-maleic acid) (PANCMA) ultrafiltration hollow fiber membrane in the presence of 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxylsuccin-imide (NHS). The chemical change of the chitosan-modified PANCMA membrane surface was confirmed with Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Lipase from Candida rugosa was immobilized on this dual-layer biomimetic membrane using glutaraldehyde (GA), and on the nascent PANCMA membrane using EDC/NHS as coupling agent. The properties of the immobilized enzymes were assayed and compared with those of the free one. It was found that both the activity retention of the immobilized lipase and the amount of bound protein on the dual-layer biomimetic membrane (44.5% and 66.5 mg/m2) were higher than those on the nascent PANCMA membrane (33.9% and 53.7 mg/m2). The kinetic parameters of the free and immobilized lipases, K m and V max, were also assayed. The K m values were similar for the immobilized lipases, while the V max value of the immobilized lipase on the dual-layer biomimetic membrane was higher than that on the nascent PANCMA membrane. Results indicated that the pH and thermal stabilities of lipase increased upon immobilization. The residual activity of the immobilized lipase after 10 uses was 53% on the dual-layer biomimetic membrane and 62% on the nascent PANCMA membrane.
Keywords: Dual-layer biomimetic membrane; Poly(acrylonitrile-co-maleic acid) membrane; Chitosan; Lipase; Enzyme immobilization;

The nature and formation of calcitic columnar prismatic shell layers in pteriomorphian bivalves by Antonio Gerardo Checa; Alejandro B. Rodríguez-Navarro; Fransico J. Esteban-Delgado (6404-6414).
The calcitic columnar prismatic (CCP) microstructure constitutes the external shell layer of pterioid and ostreoid bivalves. It consists of calcite monocrystals, which extend perpendicular to the growth surface and are surrounded by thick interprismatic membranes (<1–8 μm). The exceptional biomechanical properties of CCP layers make them interesting for biomechanical–biomimetic studies. Their potential as biomedical implants still requires further testing. Previous studies attributed the mineral phase a crucial role in the organization of the microstructure. Conversely, our observations on the morphology of prisms and abnormally calcified shells reveal that the organic web may form in the absence of the mineral infilling and that the boundaries between prismatic units are shaped by interfacial tension. Accordingly, we consider interprismatic organic membranes as the main shaping agent. We propose that the prismatic layer initiates as an emulsion formed by the fluid precursor of organic membranes and the extrapallial fluid, which causes formation of empty organic cavities. Prismatic crystals began to form within the organic cavities a little later, but their secretion surface soon levelled that of organic membranes.
Keywords: Biomineralisation; Microstructure; Shell; Calcite; Prismatic layer; Bivalves;