Biomaterials (v.26, #20)

Calendar (I).

Biodegradable cationic PEG–PEI–PBLG hyperbranched block copolymer: synthesis and micelle characterization by HuaYu Tian; Chao Deng; Hao Lin; Jingru Sun; Mingxiao Deng; Xuesi Chen; Xiabin Jing (4209-4217).
A novel amphiphilic biodegradable cationic hyperbranched poly(ethylene glycol)–polyethylenimine–poly(γ-benzyl l-glutamate) (PEG–PEI–PBLG) block copolymer was successfully synthesized by ring-opening polymerization (ROP) of N-carboxyanhydride of γ-benzyl-l-glutamate (BLG–NCA) with PEG–PEI as a macroinitiator. PEG–PEI was firstly prepared by coupling of PEG and PEI using hexamethylene diisocyanate (HMDI). The structural properties of PEG–PEI–PBLG copolymers were confirmed by 1H NMR and GPC. The copolymers were found to be self-assembled in water with critical micelle concentration (CMC) in the range of 0.00368–0.0125 g/l and high hydrophobic micelle core. The micelle size and CMC obviously depended on the hydrophobic block content in the copolymer and the ionic state of the PEI block. The CMC decreased with the increase in the PBLG block content. The decrease of micelle size and the increase of CMC simultaneously occurred with the protonated degree of PEI block by addition of HCl solution. ESEM and Gel retardation assay showed that the cationic micelles had ability to encapsulate plasmid DNA. The copolymer has potential medical applications in drug and gene delivery.
Keywords: Polyethylenimine; Biodegradable; Hyperbranched; Amphiphilic copolymer; Micelle; Complex micelle; Drug delivery;

Uncatalyzed synthesis, thermal and mechanical properties of polyurethanes based on poly( ε -caprolactone) and 1,4-butane diisocyanate with uniform hard segment by Ralf G.J.C. Heijkants; Ralph V. van Calck; Tony G. van Tienen; Jacqueline H. de Groot; Pieter Buma; Albert J. Pennings; Reen P.H. Veth; Arend Jan Schouten (4219-4228).
Polyurethanes based on poly( ε -caprolactone) (PCL) (750–2800 g/mol) and 1,4-butane diisocyanate (BDI) with different soft segment lengths and constant uniform hard segment length were synthesized in absence of catalysts for the production of a degradable meniscus scaffold. First the polyesterdiols were endcapped with BDI yielding a macrodiisocyanate with a minimal amount of side reactions and a functionality of 2.0. Subsequently, the macrodiisocyanates were extended with 1,4-butanediol in order to obtain the corresponding polyurethane. The polyurethanes had molecular weights between 78 and 160 kg/mol.Above molar masses of 1900 g/mol of the polyesterdiol crystalline PCL was found while the hard segment showed an increase in melting point from 78 to 122 °C with increasing hard segment content. It was estimated that the percentage crystallinity of the hard segment varied between 92 and 26%. The Young's modulus varied between 30 and 264 MPa, the strain at break varied between 870 and 1200% and tear strengths varied between 97 and 237 kJ/m2.
Keywords: Polyurethane; Degradation; Polycaprolactone; Elastomer; Scaffold;

Characterization of collagen matrices crosslinked using microbial transglutaminase by Ray-Neng Chen; Hsiu-O Ho; Ming-Thau Sheu (4229-4235).
In search of a new approach for crosslinking collagen-based biomaterials, we examined the effect of microbial transglutaminase (MTGases) as a crosslinking reagent on collagenous matrices made from porcine type I collagen. As the results revealed, MTGase exhibited a crosslinking action that raised the viscosity of the collagen solution. Matrices crosslinked with MTGase at the low pH values of pH 3 and 4 exhibited higher tensile strengths than those at high pH values. In comparison with untreated matrices, the denaturation temperatures of the corresponding matrices shifted toward higher temperatures. These enzyme-catalyzed crosslinked matrices were proven by MTT assay to be non-cytotoxic. In conclusion, this enzymatic method of using MTGase provides an alternative potential way for crosslinking collagen-based matrices.
Keywords: Transglutaminase; Collagen; Matrix; Enzyme; Crosslinking; Biomaterials;

Impedance and QCM analysis of the protein resistance of self-assembled PEGylated alkanethiol layers on gold by Barbara Menz; Robert Knerr; Achim Göpferich; Claudia Steinem (4237-4243).
In this study, we describe the formation and characterization of self-assembled layers of undecanthiol with poly(ethylene glycol) (PEG) moieties of 350 Da (PEG350-undecanthiol) and 2000 Da (PEG2000-undecanthiol) on gold surfaces. The functionalized surfaces were investigated by means of electrical impedance spectroscopy, which allows calculating the surface coverage from the obtained capacitance values of the formed layers. For both, PEG350-undecanthiol and PEG2000-undecanthiol layers, a surface coverage well above 90% was obtained. Protein resistance of those layers was investigated using the quartz crystal microbalance (QCM) technique, which enables one to monitor protein adsorption label free and in a time resolved manner. The change in resonance frequency of the quartz plate was monitored upon addition of fetal bovine serum indicating that PEG-functionalized surfaces are partly protein resistant compared to hydroxyundecanthiol- and non-functionalized gold surfaces. From QCM experiments, where only a single protein component was added to the PEG-functionalized gold surface, we conclude that the surfaces are fully resistant against serum albumins, while the main protein components that adsorb are globulins. A kinetic analysis reveals that PEG modified gold surfaces do not only significantly diminish the overall amount of bound protein but also significantly slows down the adsorption process.
Keywords: Biocompatibility; Electrochemistry; Protein adsorption; Self assembly;

Purmorphamine is a new molecule with osteogenesis-inducing activity in multipotent progenitor cells. The aim of this study was to evaluate whether purmorphamine maintains its osteogenic potential on human bone marrow mesenchymal cells cultured on commercially pure titanium (cpTi). Cells were cultured either in the absence or presence of purmorphamine 3 μm on cpTi in supplemented α-MEM. At 7, 14, and 21 days, cell proliferation, viability, total protein content, collagen content, and alkaline phosphatase (ALP) activity were evaluated. Bone-like nodule formation was evaluated at 21 days. All experiments were done in quintuplicate and data were compared by ANOVA or t-test. Purmorphamine did not affect cell proliferation (p=0.619), viability (p=0.831), and collagen content (p=0.088). Total protein content (p=0.047), ALP activity (p=0.001), and bone-like nodule formation (p=0.002) were increased by purmorphamine. The present results indicate that events related to osteoblast differentiation, including increased ALP activity and bone-like nodule formation, are enhanced by purmorphamine in the presence of cpTi. It means that this molecule could be useful as an adjunct therapy to improve the osseointegration of the implants in the fields of dentistry and orthopaedics.
Keywords: Cell differentiation; Human bone marrow mesenchymal cells; Osteoblast; Purmorphamine; Titanium;

Modulation of the responses of human osteoblast-like cells to physiologic mechanical strains by biomaterial surfaces by Fabrice Di Palma; Alain Guignandon; Annette Chamson; Marie-Hélène Lafage-Proust; Norbert Laroche; Sylvie Peyroche; Laurence Vico; Aline Rattner (4249-4257).
In a previous study we demonstrated that MG-63 cells cultured on Ti-6Al-4V discs covered by alumina ceramic and submitted to intermittent mechanical strain (IMS) presented morphological alteration associated with enhanced differentiation. Here we examine how the mechanical response of osteoblasts can be modulated by the nature of the substrate. MG-63 cells were cultured on four materials: polystyrene and Ti-6Al-4V (average roughness=0.48 μm) as smooth substrates; Ti-6Al-4V (average roughness=5.76 μm) and Ti-6Al-4V covered with alumina (average roughness=5.21 μm) as rough substrates. Mechanical strains were applied for 15 min, three times a day for 1–5 days with a 600 μstrains magnitude and a 0.25 Hz frequency. IMS stimulated alkaline phosphatase activity by 25–35% on all substrates and had no effect on cell growth on either substrate. Fibronectin (FN) was chosen as representative of cell-matrix interaction. FN production was increased by 60% after 1 day of stretching only on alumina-coated discs. FN organization examined on smooth substrates was affected by 5 days of IMS, showing a thickening of the fibres. The same modifications induced by IMS were previously observed on alumina-covered discs. Vinculin expression was not affected by IMS whatever the substrate. Cell–cell interactions were determined by N-cadherin immunoblotting. N-cadherin expression was increased by IMS specifically on rough substrates. Our results suggest that the nature of the surface did not influence the up-regulation of alkaline phosphatase activity induced by IMS, but modulates specifically cell–substrate as well as cell–cell interactions in response to IMS.
Keywords: Osteoblast; Biomaterial; Roughness; Mechanical strain; Cell number; Alkaline phosphatase; Fibronectin; Cadherin; Titanium; MG-63;

Identifying enzyme activities within human saliva which are relevant to dental resin composite biodegradation by Benjamin A. Lin; Fayaaz Jaffer; Missy D. Duff; Yi Wen Tang; J. Paul Santerre (4259-4264).
Esterase activities similar to those of cholesterol and pseudocholine esterases (CE and PCE, respectively) have been detected within whole human saliva. Since commercial CE has been shown to possess distinct activity relative to PCE for select components in dental composites, it is hypothesized that esterases isolated from human saliva will also show selectivity towards specific monomer elements within the composites. The objective of this work was to carry out the isolation of these activities from whole human saliva and study their individual effects on resin monomers such as Bis-phenyl glycidyl dimethacrylate (aromatic structure) and triethylene glycol dimethacrylate (hydrophilic structure), and on cured composites containing the latter monomers. Human saliva samples were processed, fractionated on a gel filtration column and assayed for CE and PCE-like activity. Selected fractions were incubated at 37 °C with the above monomers and select commercial composites. Degradation was monitored using high-performance liquid chromatography. The fraction with the highest cholesterol esterase-like character preferentially degraded the aromatic monomer and significantly degraded more of the composite's material relative to a fraction containing low amounts of the cholesterol esterase activity but elevated pseudocholine esterase-like activity. Hence, it was concluded that select salivary esterases had preferences for distinct composite resin components.
Keywords: Saliva; Enzymes; Resin; Composites; Monomers; Biodegradation;

Ectopic osteoinduction and early degradation of recombinant human bone morphogenetic protein-2-loaded porous β-tricalcium phosphate in mice by Ge Liang; Yunzhi Yang; Sunho Oh; Joo L Ong; Changqiong Zheng; Junguo Ran; Guangfu Yin; Dali Zhou (4265-4271).
The present study investigated the ectopic osteoinduction and early degradation of recombinant human bone morphogenetic protein-2 (rhBMP-2)-loaded porous beta-tricalcium phosphate (β-TCP) in mice. The porous β-TCP with 50 μg of rhBMP-2 ( n = 25 ) and porous β-TCP (control group, n = 25 ) were implanted into muscle pouches in the right and left thigh of 28-day-old mice ( n = 25 ), respectively. At every time point (3, 7, 14, 21 and 28 days after implantation), five mice were euthanized and the histological examinations of implantation sites were performed. In addition, the alkaline phosphatase (ALP) activity was also quantitatively analyzed. For the rhBMP-2-loaded group, blood vessel formation and immature cartilage was observed within the porous β-TCP 3 days after implantation. Mature cartilage was observed 7 days after implantation of rhBMP-2-loaded porous β-TCP. Newly formed woven bone, lamellar bone as well as marrow were observed 14 and 21 days after implantation of the rhBMP-2-loaded porous β-TCP. Lamellar bone and marrow were observed 28 days after implantation of the rhBMP-2-loaded porous β-TCP. For the control group, no bone or cartilage was observed at all time points. However, multinucleated giant cells and fibrous tissues were observed in the control group at 7 and 28 days after implantation, respectively. At 21 and 28 days after implantation, porous β-TCP was observed to fragment indicating early degradation of the porous β-TCP in both groups. In addition, ALP was observed to be significantly higher in the rhBMP-2-loaded β-TCP as compared to the control β-TCP. It was concluded from this study that the rhBMP-2-loaded porous β-TCP induced blood vessel and ectopic bone formation.
Keywords: Recombinant human bone morphogenetic protein-2; β-tricalcium phosphate; Ectopic implantation; Osteoinduction; Biodegradation;

Cartilage regeneration using mesenchymal stem cells and a three-dimensional poly-lactic-glycolic acid (PLGA) scaffold by Kota Uematsu; Koji Hattori; Yoshiyuki Ishimoto; Jun Yamauchi; Takashi Habata; Yoshinori Takakura; Hajime Ohgushi; Takeshi Fukuchi; Masao Sato (4273-4279).
Cartilage engineered from mesenchymal stem cells (MSCs) requires a scaffold to keep the cells in the cartilage defect and to act as a support for inducing hyaline cartilage formation. We developed a novel three-dimensional special poly-lactic-glycolic acid (PLGA) scaffold that provided structural support and stimulated repair. Three-dimensional PLGA scaffolds seeded with cultured MSCs were transplanted into large defects in rabbit knees and analyzed histologically at 4 and 12 weeks after the operation. Our findings showed that in the engineered cartilage with the PLGA scaffold, the defects were filled with smooth, shiny white tissue macroscopically and hyaline-like cartilage histologically at 12 weeks after the transplantation. The structure of the novel PLGA scaffolds provided architectural support for the differentiation of progenitor cells and demonstrated successful induction of in vivo chondrogenesis.
Keywords: Bone marrow; Cartilage; Cartilage tissue engineering; Mesenchymal stem cell; Scaffold;

Scaffold fabrication by indirect three-dimensional printing by Min Lee; James C.Y. Dunn; Benjamin M. Wu (4281-4289).
Three-dimensional printing (3DP) has been employed to fabricate porous scaffolds by inkjet printing liquid binder droplets onto particulate matter. Direct 3DP, where the final scaffold materials are utilized during the actual 3DP process, imposes several limitations on the final scaffold structure. This study describes an indirect 3DP protocol, where molds are printed and the final materials are cast into the mold cavity to overcome the limitations of the direct technique. To evaluate the resolution available in this technique, scaffolds with villi features (500 μm diameter, 1 mm height) were produced by solvent casting into plaster molds, followed by particulate leaching. Scanning electron microscope (SEM) showed highly open, well interconnected, uniform pore architecture (∼100–150 μm). The ability of these scaffolds to support intestinal epithelial cell (IEC6) culture was investigated in vitro. IEC6 cells attached to scaffolds uniformly in vitro and grew preferentially in the villi region. To exploit the freeform nature of this technique with large pore size, anatomically shaped zygoma scaffolds with 300–500 μm interconnected pores were produced and characterized. Indirect 3DP provides an alternative method to complement other direct solid freeform fabrication methods.
Keywords: Three-dimensional printing; Scaffolds; Tissue engineering; Solid freeform fabrication;

Cell behavior is determined by intrinsic programs and complex interactions among cells, medium components and substrates. Several previous reports have demonstrated the usefulness of extrinsic signals coming from soluble growth factors and cell–cell contact for regulating the proliferation and differentiation of neural stem cells. At present, the effects of substrate on neural stem cells are not known. In this study, the behavior of neural stem cells, isolated from embryonic rat cerebral cortex, was observed and compared on the polyvinyl alcohol (PVA) and poly (ethylene-co-vinyl alcohol) (EVAL) substrates in the presence of the mitogenic effect of basic fibroblast growth factor (bFGF) in the serum-free medium. It was found that PVA and EVAL exerted different influences on the fate of neural stem cells. The behavior of neural stem cells on the EVAL was independent of cell density at the single-cell level. Single neural stem cells seemed to remain dormant on the EVAL. Conversely, the development of cell clusters, termed neurospheres, was in a density-dependent manner on the EVAL. Neurospheres continuously proliferated under high-density culture condition, but differentiated into neurons and astrocytes under low-density culture condition. However, regardless of single cells or neurospheres, cultured cells could not survive on the PVA. Therefore, it is reasonable to assume that biomaterials may stimulate or inhibit the proliferation and differentiation of neural stem cells. These in vitro results are very encouraging since this information should be useful for the development of strategies for regulating the preservation, proliferation and differentiation of neural stem cells.
Keywords: Neural stem cells; Proliferation; Differentiation; EVAL; PVA;

Enhanced repair of large osteochondral defects using a combination of artificial cartilage and basic fibroblast growth factor by Aki Fukuda; Ko Kato; Masahiro Hasegawa; Hitoshi Hirata; Akihiro Sudo; Kenshi Okazaki; Kaoru Tsuta; Yasuo Shikinami; Atsumasa Uchida (4301-4308).
The purpose of this study was to examine the efficacy of a combination of artificial cartilage and basic fibroblast growth factor (bFGF) for the repair of large osteochondral defects. The artificial cartilage was a three-dimensional fabric (3-DF) composed of an ultra-high molecular weight polyethylene fiber with a triaxial three-dimensional structure. We implanted 3-DF impregnated with type I collagen gel containing 500 ng of bFGF (bFGF-treated group) or 3-DF impregnated with type I collagen gel alone (non-treated group) into a large full-thickness osteochondral defect (6×6×3 mm) of the patellar groove of rabbits. The defect area was examined grossly, histologically and biomechanically 4–48 weeks after surgery. Bone ingrowth into and around the 3-DF was evaluated with micro-computed tomography (μ-CT). Addition of bFGF to the 3-DF greatly accelerated cartilage formation on the articular surface and subchondral bone formation into and around the 3-DF, and improved biomechanical properties. These findings suggest that a combination of artificial cartilage and bFGF is clinically useful in cases involving large osteochondral defects.
Keywords: Artificial cartilage; Cartilage repair; Fibroblast growth factor;

Acrylic bone cements modified with β -TCP particles encapsulated with poly(ethylene glycol) by Blanca Vázquez; María Pau Ginebra; Xavier Gil; Josep Antón Planell; Julio San Román (4309-4316).
β -Tricalcium phosphate ( β -TCP) has been encapsulated with poly(ethylene glycol) (PEG) to improve the filler/cement interface, and it was later incorporated to a poly(methyl methacrylate) bone cement in order to obtain cements with improved stability in the long term. Size and size distribution of the agglomerates forming the initial powder was drastically changed after its dispersion in a PEG aqueous solution. Whereas the initial β -TCP particles had a 584 μm average diameter, the treated particles (TCP-PEG) presented more than 60% of the particles in a range of 2–6 μm. The effect of adding the treated particles to an acrylic cement was evaluated in terms of curing parameters, in vitro behaviour and mechanical performance. The presence of the TCP-PEG particles did not affect either peak temperature or setting time, indicating a good homogeneity of polymerising mass in contrast to the effect observed with the plain β -TCP particles, which gave rise to higher setting times. In vitro behaviour studies revealed hydration degree values of the modified cements comparable to that of PMMA cements. Early stages of water uptake was Fickian in nature for all the experimental formulations indicating that the water absorption followed a diffusion controlled mechanism. After 3 months of storage in SBF the experimental formulations presented values of compressive strength in the range 76–78 MPa, higher than the minimum required by ISO 5833 (70 MPa) and those of tensile strength in the range 42–48 MPa, higher than the minimum reported for commercial formulations (30 MPa), but no significant differences in the strengths and elastic modulus were observed with the treatment of the filler particles. This observation was confirmed by ESEM analysis of the tensile fracture surfaces, which revealed a rather good cohesion between the bioceramic particles with some gaps around them, independently of the type of particles. The themogravimetric analysis of dry and wet specimens showed a higher dissolution rate of the plain β -TCP particles in comparison to the encapsulated ones, indicating that the PEG adsorbed on the surface of the TCP particles could be a way to control the resorbability of the bioceramic component.
Keywords: Poly(methyl methacrylate); β -TCP particles; Poly(ethylene glycol); Acrylic bone cements;

Two variants of antibiotic powder-loaded acrylic bone cements (APLBCs) are widely used in primary total joint replacements. In the United States, the antibiotic is manually blended with the powder of the cement at the start of the procedure, while, in Europe, pre-packaged commercially-available APLBCs (in which the blending is carried out using an industrial mixer) are used. Our objective was to investigate the influence of the method of blending gentamicin sulphate with the powder of the Cemex® XL formulation on a wide collection of properties of the cured cement. The blending methods used were manual mixing (the MANUAL Set), use of a small-scale, easy-to-use, commercially-available mechanical powder mixer, OmoMix®1 (the MECHANICAL Set), and use of a large-scale industrial mixer (Cemex®Genta) [the INDUSTRIAL Set]. In the MECHANICAL and MANUAL Sets, the blending time was 3 min. In preparing the test specimens for each set, the blended powder used contained 4.22 wt% of the gentamicin powder. The properties determined were the strength, modulus, and work-to-fracture (all obtained under four-point bending), plane-strain fracture toughness, Weibull mean fatigue life (fatigue conditions: ±15 MPa; 2 Hz), activation energy and frequency factor for the cement polymerization process (both determined using differential scanning calorimetry, at heating rates of 5, 10, 15, and 20 K min−1), the diffusion coefficient for the absorption of phosphate buffered saline, PBS, at 37 °C, and the rate of elution of the gentamicin into PBS, at 37 °C (E). Also determined were the particle size, particle size distribution, and morphology of the blended powders and of the gentamicin. For each of the cured cement properties (except for E), there is no statistically significant difference between the means for the 3 cements, a finding that parallels the observation that there are no significant differences in either the mean particle size or the morphology of the blended cement powders. Notwithstanding these results, it is suggested that when the powder mixture is blended in the operating room, using the OmoMix®1 is more likely to produce a more consistent and reproducible mixture than when manual mixing is used.
Keywords: Acrylic bone cement; Gentamicin sulphate; Physical properties; Mechanical properties; Thermal properties;

Effect of load and temperature on in vitro degradation of poly(glycolide-co-l-lactide) multifilament braids by M. Deng; J. Zhou; G. Chen; D. Burkley; Y. Xu; D. Jamiolkowski; T. Barbolt (4327-4336).
The effects of load and temperature on in vitro degradation behaviors of poly(glycolide-co-l-lactide) 90/10 multifilament braids were investigated in phosphate buffer solution at pH 7.4. The property changes of the braids with time were monitored by tensile test, gel permeation chromatography analysis, and scanning electron microscopy. The interrelationships between material properties, time and experimental conditions were explored. The results showed that the polymer braids gradually lost their strength and molecular weight with the increasing in vitro time. While the load levels applied had no effect on the materials, raising temperatures significantly accelerated the degradation. It was found that for a given tensile breaking strength retention (BSR), the dependence of degradation time on temperature could be illustrated by an Arrhenius-type equation, from which the activation energy could be derived. Further analysis indicated that there are well-defined relationships between molecular weight, BSR and breaking strain retention, and these relationships can be illustrated mathematically. Finally, the surface morphology of the fiber showed visible change during the degradation process.
Keywords: Degradation; In vitro test; Mechanical properties; Molecular weight; Poly(glycolide-co-lactide); Surface analysis;

Cellulose acetate butyrate microcapsules containing dextran ion-exchange resins as self-propelled drug release system by Gheorghe Fundueanu; Marieta Constantin; Elisabetta Esposito; Rita Cortesi; Claudio Nastruzzi; Enea Menegatti (4337-4347).
Sulfopropylated dextran microspheres (SP-Ms), (Dm=80 μm) loaded with a water soluble drug (Tetracycline HCl), were included in cellulose acetate butyrate (CAB) microcapsules.Spherical CAB microcapsules were obtained by oil in water (o/w) solvent evaporation method in the presence of an inert solvent as cyclohexane (CyH) or n-hexane (N-Hex), and different excipients (Phospholipon, Tween, Span, Eudragit RS 100). Chloroform was found to be the best solvent for the preparation of the microcapsules. Also, the sphericity as well as the porosity of the microcapsules was controlled by the presence of an inert solvent. The final concentration of the drug in CAB microparticles was up to 25% (w/w).The key factors for the successful preparation were also the viscosity of the polymer, while the wettability of the resulted microcapsules, the temperature of the preparation, and the porosity have modulated the release of the drug. The higher is the amount of encapsulated microspheres the thinner is the CAB wall between the compartments created by their incorporation.When these microspheres come in contact with the release medium, the pressure created by their swelling breaks the polymer film and the drug starts to be released. The more drug is released in phosphate buffer the higher is the swelling degree of the encapsulated ion exchange resins and the force created by their supplementary swelling will break the more resistants walls. In this way a self-propelled drug release is achieved, until almost all drug was eliberated.
Keywords: Dextran cation-exchange microspheres; Solvent evaporation process; Drug release;