Biomaterials (v.26, #6)

Calendar (I).

The bisphosphonate pamidronate on the surface of titanium stimulates bone formation around tibial implants in rats by Hiroshi Kajiwara; Takayoshi Yamaza; Masao Yoshinari; Tetsuya Goto; Shinji Iyama; Ikiru Atsuta; Mizuho A Kido; Teruo Tanaka (581-587).
Many materials with differing surfaces have been developed for clinical implant therapy in dentistry and orthopedics. We analyzed the quantity of new bone formed in vivo around calcium-immobilized titanium implants with surfaces modified using pamidronate (PAM), a nitrogen-containing bisphosphonate (N-BP), implants of pure titanium, and titanium implants immobilized with calcium ions. New bone formation was visualized using fluorescent labeling (calcein blue and alizarin complexone) with intravenous injection at 1 and 3 weeks after implantation. After 4 weeks, undecalcified sections were prepared, and new bone formation around the implants was examined by morphometry using confocal laser scanning microscopy images. After 1 week, more new bone formed around the PAM-immobilized implant than around the calcium-immobilized and pure titanium implants. This was also seen with the new bone formation after 3 weeks. After 4 weeks, significantly more new bones were formed around the BP-immobilized implant than around the calcium ion-implanted and pure titanium implants. The new N-BP-modified titanium surface stimulates new bone formation around the implant, which might contribute to the success of implant therapy.
Keywords: Bisphosphonate; Titanium; Implant; Bone formation;

To improve the surface biocompatibility, asymmetric membranes fabricated from poly(acrylonitrile-co-maleic acid)s (PANCMAs) synthesized by water-phase precipitation copolymerization were tethered (or immobilized) with poly(ethylene glycol)s (PEGs) by esterification reaction. Chemical changes on the membrane surface were characterized by Fourier transform infrared spectroscopy and elemental analysis to confirm the immobilization of PEG onto the PANCMA membranes. The hydrophilicity and blood compatibility of the PEG-tethered PANCMA membrane were investigated by water contact angle, water absorption, protein adsorption, plasma platelets adhesion and cell adhesion measurements, and the results were compared with the corresponding PANCMA membranes. It was found that, after the tethering of PEG, the hydrophilicity of the membrane can be improved significantly, and the protein adsorption, platelets adhesion and macrophage attachment on the membrane surface are obviously suppressed. Furthermore, not only the content of maleic acid in PANCMA, which influences the tethering density of PEG, but also the molecular weight of PEG has great effect on the surface modification of PANCMA membranes for biocompatibility.
Keywords: Polyacrylonitrile; Membrane; Surface modification; Biocompatibility; Platelets adhesion; Cell adhesion;

A three-dimensional nanofibrous scaffold for cartilage tissue engineering using human mesenchymal stem cells by Wan-Ju Li; Richard Tuli; Chukwuka Okafor; Assia Derfoul; Keith G Danielson; David J Hall; Rocky S Tuan (599-609).
The utilization of adult stem cells in tissue engineering is a promising solution to the problem of tissue or organ shortage. Adult bone marrow derived mesenchymal stem cells (MSCs) are undifferentiated, multipotential cells which are capable of giving rise to chondrocytes when maintained in a three-dimensional culture and treated with members of the transforming growth factor-β (TGF-β) family of growth factors. In this study, we fabricated a nanofibrous scaffold (NFS) made of a synthetic biodegradable polymer, poly(ε-caprolactone) (PCL), and examined its ability to support in vitro chondrogenesis of MSCs. The electrospun PCL porous scaffold was constructed of uniform, randomly oriented nanofibers with a diameter of 700 nm, and structural integrity of this scaffold was maintained over a 21-day culture period. MSCs cultured in NFSs in the presence of TGF-β1 differentiated to a chondrocytic phenotype, as evidenced by chondrocyte-specific gene expression and synthesis of cartilage-associated extracellular matrix (ECM) proteins. The level of chondrogenesis observed in MSCs seeded within NFSs was comparable to that observed for MSCs maintained as cell aggregates or pellets, a widely used culture protocol for studying chondrogenesis of MSCs in vitro. Due to the physical nature and improved mechanical properties of NFSs, particularly in comparison to cell pellets, the findings reported here suggest that the PCL NFS is a practical carrier for MSC transplantation, and represents a candidate scaffold for cell-based tissue engineering approaches to cartilage repair.
Keywords: Electrospinning; Nanofiber; Mesenchymal stem cell; Cartilage tissue engineering; Transforming growth factor-beta1;

Feasibility of chitosan-based hyaluronic acid hybrid biomaterial for a novel scaffold in cartilage tissue engineering by Shintaro Yamane; Norimasa Iwasaki; Tokifumi Majima; Tadanao Funakoshi; Tatsuya Masuko; Kazuo Harada; Akio Minami; Kenji Monde; Shin-ichiro Nishimura (611-619).
In this study, we hypothesized that hyaluronic acid could provide superior biological effects on the chondrocytes in a three-dimensional culture system. To test this hypothesis, we investigated the in vitro behavior of rabbit chondrocytes on a novel chitosan-based hyaluronic acid hybrid polymer fiber. The goal of the current study was to show the superiority of this novel fiber as a scaffold biomaterial for cartilage tissue engineering. Chitosan polymer fibers (chitosan group) and chitosan-based hyaluronic acid hybrid polymer fibers (HA 0.04% and HA 0.07% groups, chitosan coated with hyaluronic acid 0.04% and 0.07%, respectively) were originally developed by the wetspinning method. Articular chondrocytes were isolated from Japanese white rabbits and cultured in the sheets consisting of each polymer fiber. The effects of each polymer fiber on cell adhesivity, proliferation, morphological changes, and synthesis of the extracellular matrix were analyzed by quantitative a cell attachment test, DNA quantification, light and scanning electron microscopy, semi-quantitative RT-PCR, and immunohistochemical analysis. Cell adhesivity, proliferation and the synthesis of aggrecan were significantly higher in the hybrid fiber (HA 0.04% and 0.07%) groups than in the chitosan group. On the cultured hybrid polymer materials, scanning electron microscopic observation showed that chondrocytes proliferated while maintaining their morphological phenotype and with a rich extracellular matrix synthesis around the cells. Immunohistochemical staining with an anti-type II collagen antibody demonstrated rich production of the type II collagen in the pericellular matrix from the chondrocytes. The chitosan-based hyaluronic acid hybrid polymer fibers show great potential as a desirable biomaterial for cartilaginous tissue scaffolds.
Keywords: Chitin/chitosan; Chondrocyte; Hyaluronic acid/hyaluronan; Cell culture; Cell adhesion;

The polyurethane elastomer (PU) Corethane 80A (Corvita) is being considered as the acetabular bearing material in a novel total replacement hip joint. The biostability of Corethane 80A was investigated in vitro (this work) and in vivo (reported separately) in a fully functioning ovine total hip arthroplasty (THA) model, with the PU as the bearing layer in a prototype compliant layer acetabular cup. The in vitro studies assessed the resistance of Corethane 80A to the main degradation mechanisms observed in PUs: hydrolysis, environmental stress cracking (ESC), metal ion oxidation (MIO) and calcification. The performance of the polycarbonate PU Corethane 80A was assessed alongside three other commercially available biomedical PUs: polyether PUs Pellethane 2363-80A (DOW Chemical) and PHMO-PU (CSIRO, not supplied as a commercial material) as well as polycarbonate PU ChronoFlex AL-80A (CardioTech). Chemical and structural variables that affect the properties of the materials were analysed with particular attention to the nature of the material's hard and soft segments. PU degradation was probed using a range of analytical tools and physical-testing methods, including mechanical testing, differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR) and environmental scanning microscopy (ESEM). Corethane 80A displayed the best overall resistance to hydrolysis, ESC, MIO and calcification, followed by ChronoFlex 80A and PHMO-PU. Pellethane 80A was the least stable. This study provides compelling evidence for the biostability and effectiveness of Corethane 80A and points to its suitability for use as a compliant bearing layer in hip arthroplasty, and possibly also other joints.
Keywords: Hip replacement prosthesis; Polyurethane; Elastomer; Compliance; Biocompatibility; Animal model;

The polyurethane (PU) elastomer Corethane 80A (Corvita) is being considered as the acetabular bearing material in a novel total replacement hip joint. Its biostability was investigated in vitro (Analysis and evaluation of a biomedical polycarbonate urethane tested in an in vitro study and an ovine arthroplasty model. Part I: material selection and evaluation, Biomaterials, in press) together with three other commercially available biomedical PUs: Pellethane 2363-80A (DOW Chemical), a polyhexamethylene oxide based PU, PHMO-PU (CSIRO, not supplied as a commercial product) and ChronoFlex AL-80A (CardioTech). From the in vitro studies, Corethane 80A displayed the best overall resistance to hydrolysis, ESC, MIO and calcification, followed by ChronoFlex 80A and PHMO-PU, with Pellethane 80A being the least stable. Building on the in vitro investigation, the follow-up in vivo study (reported here) assessed Corethane 80A as the bearing layer in a prototype compliant layer acetabular cup, in a fully functioning ovine total hip arthoplasty (THA) model. PU degradation in the retrieved cups was analysed using a range of analytical and physical-testing methods including mechanical testing, differential scanning calorimetry, Fourier transform infrared spectroscopy and environmental scanning electron microscopy. The Corethane 80A functioned well in the THA model, with the bearing surfaces of the retrieved hip cups showing no significant evidence of biodegradation or wear damage after 3 years in vivo. The findings in this study provide compelling evidence for the biostability and effectiveness of acetabular cups incorporating a Corethane 80A compliant bearing layer.
Keywords: Hip replacement prostheses; Polyurethane; Elstomer; Compliance; Biocompatibility; Animal model;

Fixation of distal femoral osteotomies with self-reinforced polymer/bioactive glass rods: an experimental study on rabbits by Tuomo Pyhältö; Matti Lapinsuo; Hannu Pätiälä; Mika Pelto; Pertti Törmälä; Pentti Rokkanen (645-654).
Two self-reinforced poly(desamino tyrosyl–tyrosine ethyl ester carbonate), poly(DTE carbonate) or self-reinforced poly(DTE carbonate)/bioactive glass rods, (2 mm by 40 mm) were implanted into the dorsal subcutaneous tissue and osteotomies of the distal femur were fixed with these rods (2 mm by 26 mm) in 36 rabbits. The follow-up times varied from three to 100 weeks. After sacrifice, three-point bending and shear tests and molecular weight measurements were performed for subcutaneously placed rods. Radiological, histological, histomorphometrical, microradiographic, and oxytetracycline-fluorescence studies of the osteotomized and intact control femora were performed. The initial mechanical properties were higher with the SR-poly(DTE carbonate) rods, but the SR-poly(DTE carbonate)/bioactive glass rods lost their mechanical properties slower. At 100 weeks the bending strength had decreased to 21% of the initial value with the SR-poly(DTE carbonate) rods and to 49% with the SR-poly(DTE carbonate)/bioactive glass rods. The shear strength had decreased to 10% with the SR-poly(DTE carbonate) rods and to 23% of the initial value with the SR-poly(DTE carbonate)/bioactive glass rods. Two slight displacements and one delayed union and one failure of fixation were seen in the SR-poly(DTE carbonate) group. In the SR-poly(DTE carbonate)/bioactive glass group five delayed unions and seven slight displacements were seen. No signs of osteolysis or foreign body reactions were observed. Signs of resorption of the implants were seen at 100 weeks in the SR-poly(DTE carbonate)/bioactive glass group. The present investigation showed that the mechanical strength and fixation properties of SR-poly(DTE carbonate) and SR-poly(DTE carbonate)/bioactive glass rods are suitable for fixation of cancellous bone osteotomies in rabbits.
Keywords: Bioabsorbable rods; Self-reinforced; SR-poly(DTE carbonate); SR-poly(DTE carbonate)/bioactive glass; Osteotomy fixation; Experimental study;

Poly(N-isopropylacrylamide)-grafted hyaluronan (PNIPAM–HA) and PNIPAM-grafted gelatin (PNIPAM–gelatin), which exhibit sol-to-gel transformation at physiological temperature, were applied as control of tissue adhesions: tissue adhesion prevention material and hemostatic aid, respectively. The rat cecum, which was abraded using surgical gauze, was coated with PNIPAM–HA-containing PBS (concentration: 0.5 w/v%). The coated solution was immediately converted to an opaque precipitate at body temperature, which weakly adhered to and covered the injured rat cecum. One week after coating, tissue adhesion between the PNIPAM–HA-treated cecum and adjacent tissues was significantly reduced as compared with that between non-treated tissue and adjacent tissues. On the other hand, the coating of bleeding spots of a canine liver with PNIPAM–gelatin-containing PBS (concentration: 20 w/v%) resulted in spontaneous gel formation on the tissues and subsequently suppressed bleeding. Although these thermoresponsive tissue adhesion prevention and hemostatic materials are still prototypes at this time, both thermoresponsive biomacromolecules bioconjugated with PNIPAM, PNIPAM–HA and PNIPAM–gelatin, may serve as a tissue adhesion prevention material and hemostatic aid, respectively.
Keywords: Poly(N-isopropylacrylamide)-grafted biomacromolecules; Gel; Thermoresponsiveness; Tissue adhesion prevention; Hemostasis;

The site-specific expression of selectins (P- and E-selectin) on endothelial cells of blood vessels during inflammation provides an opportunity for the targeted delivery of anti-inflammatory drugs to sites of chronic inflammation. It is well documented that the selectins mediate the initial interaction (rolling) of leukocytes in an inflamed vessel by binding to carbohydrate-presenting counter-receptors displayed on leukocytes. Previous work in our laboratory has shown that artificial capsules with the adhesive properties of leukocytes can be made by attaching leukocyte adhesive ligands to polymer microspheres (Biomaterials 23(10) (2002) 2167). Specifically, we showed that drug-loaded poly (lactic-co-glycolic-acid) (PLGA) microspheres coated with biotinylated-Sialyl LewisX (sLeX), a carbohydrate that serves as a ligand to selectins, mimic the adhesive behavior of leukocytes on selectins in flow chambers, displaying slow rolling under flow, suggesting that these drug-loaded particles can potentially target inflammatory sites in vivo. Since the effectiveness of this delivery system might depend on the degradation of polymer microspheres as well as the degradation of sLeX molecules, we measured the effect of polymer and ligand degradation on the adhesiveness of microspheres over time. We show that degrading sLeX microspheres maintain the ability to recognize selectin surfaces under flow for at least 2 weeks and that the ability to sustain recognition depends upon the extent at which microspheres are loaded. We also show that microsphere rolling velocity increases as microsphere degrade and that this increase is due to a combination of increase in average microsphere size and loss of sLeX molecules on microsphere surface—a result of microsphere degradation confirmed by flow cytometry. Control experiments show that microsphere, not sLeX, degradation limits the lifetime of our targeted delivery system; therefore, factors affecting degradation such as type of polymer, type of drug, extent of drug loading and microsphere size, provide an opportunity for engineering the time-scale of activity for the delivery system.
Keywords: Selectins; Drug targeting; Poly (lactic-co-glycolic acid) (PLGA); Inflammation; Leukocytes; Degradation;

Tissue anti-adhesion potential of ibuprofen-loaded PLLA–PEG diblock copolymer films by Jin Ho Lee; Ae Kyung Go; Se Heang Oh; Ka Eul Lee; Soon Hong Yuk (671-678).
This study was designed to evaluate the effect of polyethylene glycol (PEG) and nonsteroidal anti-inflammatory drug (ibuprofen) on the prevention of postsurgical tissue adhesion. For this, poly(l-lactic acid) (PLLA)–PEG diblock copolymers were synthesized by ring opening polymerization of l-lactide and methoxy polyethylene glycol (Mw 5000) of different compositions. The synthesized copolymers were characterized by gel permeation chromatography and 1H-nuclear magnetic resonance spectroscopy. PLLA–PEG copolymer films were prepared by solvent casting. The prepared copolymer films were more flexible and hydrophilic than the control PLLA film, as investigated by the measurements of glass transition temperature, water absorption content, and water contact angle. The drug release behavior from the ibuprofen (10 wt%)-loaded copolymer films was examined by high performance liquid chromatography. It was observed that the drug was released gradually up to about 40% of total loading amount after 20 days, depending on PEG composition; more drug release from the films with higher PEG compositions. In vitro cell adhesions on the copolymer films with/without drug were compared by the culture of NIH/3T3 mouse embryo fibroblasts on the surfaces. For in vivo evaluation of tissue anti-adhesion potential, the copolymer films with/without drug were implanted between the cecum and peritoneal wall defects of rats and their tissue adhesion extents were compared. It was observed that the ibuprofen-containing PLLA–PEG films with high PEG composition (particularly PLLA113–PEG113 film with PEG composition, 50 mol%) were very effective in preventing cell or tissue adhesion on the film surfaces, probably owing to the synergistic effects of highly mobile, hydrophilic PEG and anti-inflammatory drug, ibuprofen.
Keywords: Poly(l-lactic acid); Poly(ethylene glycol); Block copolymer; Anti-inflammatory drug; Tissue anti-adhesion;

Targeted gene delivery to diseased subtypes of neurons will be beneficial to the success of gene therapy of neurological disorders. We designed a recombinant cationic polypeptide to facilitate gene delivery to neuronal-like PC12 cells that express the nerve growth factor (NGF) receptors. The recombinant polypeptide was composed of a targeting moiety derived from loop 4-containing hairpin motif of NGF and a DNA-binding moiety of 10-lysine sequence and expressed in Escherichia coli. It activated NGF receptor, TrkA and its downstream signaling pathways in PC12 and promoted the survival of neuronally differentiated PC12 cells deprived of serum. The polypeptide could also bind plasmid DNA and enhance polycation-mediated gene delivery in NGF receptor-expressing PC12 cells, but not in COS7 cells lacking NGF receptors. The enhancement of gene transfer in PC12 was inhibited by pretreatment of free, unbound polypeptides, suggesting a NGF-receptor-specific effect of the polypeptide. These observations demonstrated the concept of using receptor-mediated mechanism for targeted gene delivery to neurons.
Keywords: Nerve growth factor; Recombinant cationic polypeptide; Gene delivery; Nonviral;

Antibody microarray for correlating cell phenotype with surface marker by In Kap Ko; Koichi Kato; Hiroo Iwata (687-696).
To correlate cell surface markers with the cell phenotype, an antibody microarray prepared by covalently immobilizing antibodies onto a cellulose membrane and subsequent immunocytochemical staining were employed. The direct binding assay of a lymphoblastic leukemia cell line on the microarray showed that the immobilized antibody served to capture cells expressing the specific antigen. The density of bound cells increased linearly with an increasing content of antigen-expressing cells in suspension. The method was further applied to the analysis of surface antigens expressed on neural stem cells. A binding assay was performed with neural cells obtained from the neurosphere culture of the rat fetal striatum on a microarray spotted with eight kinds of antibodies and four different proteins, followed by immunocytochemical staining of cells bound to the microarray using antibodies to the intracellular markers of immature (nestin and vimentin) and mature (β-tubulin III and glial fibrillary acidic protein) neural cells. As a result, the phenotype of bound cells could be correlated to surface antigen expression, which illustrated the potential of the solid-phase cytometry developed here for the identification of surface markers.
Keywords: Cellulose; Micropatterning; Neural cell; Immunochemistry; Stem cell; Transplantation;