Biomaterials (v.26, #18)

Calendar (I).

Chemosynthesis of bioresorbable poly(γ-butyrolactone) by ring-opening polymerisation: a review by Tim Moore; Raju Adhikari; Pathiraja Gunatillake (3771-3782).
Recent advances in the synthesis of poly(γ-butyrolactone) have yielded homopolymers of up to 50,000Mw from the low-cost monomer γ-butyrolactone. This monomer has for the better part of a century been thought impossible to polymerise. Poly(γ-butyrolactone) displays properties that are ideal for tissue-engineering applications and the bacterially derived equivalent, poly(4-hydroxybutyrate) (P4HB), has been evaluated for such uses. The glass transition temperature (−48 to −51 °C), melting point (53–60 °C), tensile strength (50 MPa), Young's modulus (70 MPa) and elongation at break (1000%) of P4HB make it a very useful biomaterial. Poly(γ-butyrolactone) degrades to give γ-hydroxybutyric acid which is a naturally occurring metabolite in the body and it has been shown to be bioresorbable.Investigation into the synthesis of poly(γ-butyrolactone) has recently produced homo-oligomeric diols 400–1000Mw that are suitable for reacting with diisocyanates to form polyurethanes. Biodegradable polyurethanes made from diols of polyglycolide (PGA) and poly(ε-caprolactone) (PCL) have the disadvantage of high glass transition and slow degradation, respectively. Poly(γ-butyrolactone) can be thought of as being the missing link in the biodegradable polyester family immediately between PGA and PCL and displaying intermediate properties.
Keywords: Polyhydroxybutyric acid; Polymerisation; Scaffold; Degradation;

A potentiostatic study of oxygen transport through poly(2-ethoxyethyl methacrylate-co-2,3-dihydroxypropylmethacrylate) hydrogel membranes by Vicente Compañ; Pilar Tiemblo; F. García; J.M. García; Julio Guzmán; Evaristo Riande (3783-3791).
The oxygen permeability and diffusion coefficients of hydrogel membranes prepared with copolymers of 2-ethoxyethyl methacrylate (EEMA)/2,3-dihydroxypropylmethacrylate (MAG) with mole fraction of the second monomer in the range between 0 and 0.75 are described. Values of the permeability and diffusion coefficients of oxygen are determined by using electrochemical procedures involving the measurement of the steady-state current in membranes prepared by radical polymerization of the monomers. The results obtained for the transport properties were analyzed taking into account the fractional free volumes, the cohesive energy densities and the glass transition temperatures of the hydrogels.
Keywords: Oxygen permeability; Hydrogel membranes; Oxygen diffusion; Fractional free volume;

The most commonly used anti-adhesion device for separation and isolation of wounded tissues after surgery is the polymeric film. In this study, a new anti-adhesion membrane based on polygalacturonic acid (PGA) has been synthesized, and its biocompatibility and anti-adhesion capabilities evaluated. The PGA film was reacted with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to obtain a cross-linked PGA film with an 86% gel content and a 47% water content when immersed in aqueous solution. This PGA–EDC film did not show any evidence of cytotoxic effects since it did not induce any significant increase in cytoplasmic LDH release from the L929 cells in contact with it. When implanted into rats, the PGA–EDC film exhibited a most promising anti-adhesion potential with only 1 out of 21 rats operated not forming any tissue adhesion. This anti-adhesion potency is significantly higher than that found for Seprafilm™ and untreated rats where 11 out of 21 and 18 out of 21 operated rats, respectively, formed tissue adhesions. The implanted PGA–EDC film did not elicit any acute inflammatory reaction based on the results of histological examination and peritoneal fluid leukocytes analysis. The newly developed PGA–EDC film thus has a great potential for future use in clinical applications.
Keywords: Polygalacturonic acid; Anti-adhesion; EDC; Cross-linking film;

The effect of hydrophilic adhesive monomers on the stability of type I collagen by Takashi Nezu; Norihiro Nishiyama; Kimiya Nemoto; Yoshihiro Terada (3801-3808).
The adsorption effects of adhesive monomers on the structural stability of type I collagen were studied at an acid pH condition for two monomers: 2-hydroxyethyl methacrylate (HEMA), a neutral monomer and N-methacryloyl glycine (NMGly), an acidic monomer. Differential scanning calorimetry (DSC) measurements were done to assess the denaturation temperature (T d), which is a measure of the structural stability of the proteins, including the bovine tendon collagen (BTC). While HEMA lowered the T d of the BTC linearly with HEMA concentrations, NMGly exhibited a two-step decrease of the T d. The rate of decrease in the T d by the NMGly was by far greater than the rate of decrease with the HEMA. The first step had a larger slope than the second step in the T d vs. C NMGly plot. The degree of adsorption of these two monomers to the BTC was estimated from infrared absorption measurements on the monomer solutions of various concentrations, before and after the immersion of the BTC. Both the adsorption of HEMA to the BTC and the T d of the BTC were linearly dependent on HEMA concentrations. Conversely, NMGly was adsorbed to the BTC, again, in a two-step decrease similar to the T d vs. C NMGLy profile. An enhanced adsorption of NMGly, which might be attributed to a strong electrostatic interaction, was observed below 0.013 mol%. Circular dichroism measurements of the collagen of the same type as the BTC, in the absence and in the presence of the monomers, revealed that the native collagen helix structure was scarcely affected by the monomers. From these observations, it was concluded that (1) both of the monomers were adsorbed onto the BTC, which thus destabilized the triple helical collagen structure, and that (2) the effect was higher for NMGly in which the electrostatic attraction with the oppositely charged collagen might be effective at a pH of 3. If compared to HEMA, an acidic NMGly is a potential monomer that binds strongly to collagen and one that is hardly hydrolyzed.
Keywords: Collagen; HEMA; N-methacryloyl glycine; Adsorption; Structural stability; Electrostatic attraction;

Influence of electron-beam radiation on the hydrolytic degradation behaviour of poly(lactide-co-glycolide) (PLGA) by Say Chye Joachim Loo; Chui Ping Ooi; Yin Chiang Freddy Boey (3809-3817).
The purpose of this study is to examine the effect of electron-beam (e-beam) radiation on the hydrolytic degradation of poly(lactide-co-glycolide) (PLGA) films. PLGA films were irradiated and observed to undergo radiation-induced degradation through chain scission, as observed from a drop in its average molecular weight with radiation dose. Irradiated (5, 10 and 20 Mrad) and non-irradiated (0 Mrad) samples of PLGA were subsequently hydrolytically degraded in phosphate-buffered saline solution at 37.0 °C over a span of 12 weeks. It was observed that the natural logarithmic molecular weight (ln  M n) of PLGA decreases linearly with hydrolytic degradation time. The rate of water uptake is higher for samples irradiated at higher radiation dose (e.g. 20 Mrad) and subsequently causing an earlier onset of mass loss. It is postulated that the increase in water uptake is due to the presence of more hydrophilic end groups, which results in the formation of microcavities because of an increase in osmotic pressure. A relationship between radiation dose and the rate of hydrolytic degradation of PLGA films, through its molecular weight was also established. This relationship allows a more accurate and precise control of the life span of PLGA through the use of e-beam radiation.
Keywords: Poly(lactide-co-glycolide; PLGA; Electron-beam irradiation; Chain scission; Hydrolytic degradation; Microcavities; Osmotic pressure;

Electron spin resonance in silicon substituted apatite and tricalcium phosphate by Alexis M. Pietak; Joel W. Reid; Michael Sayer (3819-3830).
Impurity centers associated with silicon have been observed in the phase mixture of silicon substituted apatite (Si-Ap) and silicon stabilized tricalcium phosphate (Si-TCP) using electron spin resonance (ESR). Two unique centers occur upon addition of SiO2 to the calcium phosphate system: an orthorhombic center with g-values 2.0072±0.0001, 2.0024±0.0001 and 2.0003±0.0001 (Si-h1) and a center with tetrahedral symmetry having g-values components 2.0054±0.0001 and 1.9992±0.0003 (Si-h2). Both centers are hypothesized to be characteristic of defects associated with silicon in the Si-Ap phase. Through comparison of the intensity of F-OH centers in undoped calcium hydroxyapatite (HA) prepared with various levels of OH occupancy, a relationship is demonstrated between the ESR intensity of an F-center signal with g=2.0019+0.0004 (F-OH) and the OH occupation of HA. Relative changes in the intensity of ESR signals Si-h1 and F-OH are consistent with a chemical model describing the substitution of SiO4 4− for PO4 3− in HA with the creation of OH vacancies as charge compensation, resulting in a mixed phase composition of Si-Ap and Si-TCP that results when a hydroxyapatite precipitate (HA) is heated in the presence of added SiO2.
Keywords: Calcium phosphate bioceramic; Silica; Ceramic structure; Electron spin resonance;

Stable plasma-deposited acrylic acid surfaces for cell culture applications by Loredana Detomaso; Roberto Gristina; Giorgio S. Senesi; Riccardo d’Agostino; Pietro Favia (3831-3841).
Continuous and modulated glow discharges were used to deposit thin films from acrylic acid vapors. Different deposition regimes were investigated, and their effect on chemical composition, morphology and homogeneity of the coatings, as well as on their stability in water and resistance to sterilization. Stable films were utilized in cell adhesion experiments with human fibroblasts.
Keywords: Acrylic acid; Plasma deposition; Sterilization; Cell adhesion; Fibroblasts;

Fluoridated apatite coatings on titanium obtained by electron-beam deposition by Eung-Je Lee; Su-Hee Lee; Hae-Won Kim; Young-Min Kong; Hyoun-Ee Kim (3843-3851).
In this report, a series of fluoridated apatite coatings were obtained by the electron-beam deposition method. The fluoridation of the apatite was aimed to improve the stability of the coating and elicit the fluorine effect, which is useful in the dental restoration area. Apatites fluoridated at different levels were used as initial evaporants for the coatings. The as-deposited coatings were amorphous, but after heat treatment at 500 °C for 1 h, the coatings crystallized well to an apatite phase without forming any cracks. The adhesion strengths of the as-deposited coatings were about 40 MPa. After heat treatment at 500 °C, the strengths of the pure HA and FA coatings decreased to about 20 MPa, however, the partially fluoridated coatings maintained their initial strength. The dissolution rate of the fluoridated coatings was lower than that of the pure HA coating, and the rate was the lowest in the coatings with 25% and 50% fluorine substitutions. The osteoblast-like cells responded to the coatings in a similar manner to the dissolution behavior. The cells on the fluoridated coatings showed a lower (p<0.05) proliferation level compared to those on the pure HA coating. The alkaline phosphatase activity of the cells was slightly lower than that on the pure HA coating, but this difference was not statistically significant.
Keywords: Fluoridated apatite; E-beam deposition; Dissolution rate; Cellular responses;

This article reports an electrochemical method to activate titanium surface for biomimetic calcium phosphate (Ca–P) coatings. Titanium serving as cathode was treated in an electrochemical cell with a supersaturated calcium and phosphate solution serving as electrolyte. This treatment generated a gel-like film with thickness of about 100 nm on the titanium surface. The amorphous film was composed by calcium and phosphate ions and contained a large number of crystal nuclei of octacalcium phosphate (OCP). The effectiveness of this novel treatment was demonstrated by comparing the behavior of treated and untreated titanium when used for biomimetic coating. A uniform Ca–P coating was formed on the treated titanium after immersion for several hours in aqueous solution. This work explored a new method to activate surfaces of metal implants for osseointegration, which is considerably faster than treatments currently in use, such as alkaline treatment.
Keywords: Calcium phosphate; Electrochemistry; Surface treatment; Titanium;

Evaluation of mechanical properties and biological response of an alumina-forming Ni-free ferritic alloy by J.L. González-Carrasco; G. Ciapetti; M.A. Montealegre; S. Pagani; J. Chao; N. Baldini (3861-3871).
PM 2000 is a Ni-free oxide dispersion strengthened Fe–20Cr–5Al alloy able to develop a fine, dense and tightly adherent α-alumina scale during high-temperature oxidation. Despite the high temperature involved during thermal oxidation (1100 °C), microstructural changes in the candidate material, a hot rolled product, hardly occurs. Consequently, the good mechanical properties of the as-received material are not significantly affected. Moreover, due to the high compressive residual stresses at the alumina scale, an increase in the fatigue limit from 500 to 530 MPa is observed. Such stresses also account for the high capability of the coating/metal system to withstand more than 1% tensile deformation without cracking. The biocompatibility of the alloy was assessed in comparison to commercial alumina. Saos-2 osteoblast-like cells were either challenged with PM 2000 particles, or seeded onto PM 2000 (with and without scale) solid samples. Viability, growth, and ALP release from cells were assessed after 3 or 7 days, while mineralization was checked at 18 days. This study has demonstrated that PM 2000 with and without scale are capable of supporting in vitro growth and function of osteoblast-like cells over a period of 18 days. Results from this study suggest that the resulting alumina/alloy system combines the good mechanical properties of the alloy with the superior biocompatibility of the α-alumina, for which there is very good clinical experience.
Keywords: Steel; In vitro tests; Mechanical properties; Biocompatibility; Oxidation; Alumina; Cell culture; Surface modification;

Bone marrow is a mixture of hematopoietic, vascular, stromal and mesenchymal cells capable of skeletal repair/regeneration thanks to the ability of bone marrow cells to differentiate into osteoblasts and osteoclasts. This ability is important in tissue regeneration during fracture healing, or for successful osteointegration of implanted prostheses, and in bone remodelling. Therefore, bone marrow cell culture systems seem to be useful and relatively close to in vivo conditions models to study interactions occurring at the cell–material interface of implants directed to hard tissue engineering.The purpose of this study was to investigate the ability of three bioactive glasses (45S, 58S and 77S) to induce osteogenic differentiation and cell mineralisation.A significant effect of the 45S and 77S bioactive materials was seen on early differentiation of the marrow stromal cells into osteoblast-like cells. 45S bioglass® evidenced also the highest effect on cell mineralisation at the same level as cells treated with dexametasone, used as positive control. 77S treated cells evidenced also a significant inhibition in the number of multinucleated TRAP-positive cells (ostoclast-like cells) in comparison with the control untreated cells and in marrow cells treated with 45S and 58S bioactive glasses.These findings have potential implications and applications for tissue engineering where three-dimensional bioactive glass substrates could be used as scaffolds for in vitro production of bioengineered bone.
Keywords: Bioactive glass; Bone marrow; Stromal cells differentiation;

Phospholipase A2 pathway association with macrophage-mediated polycarbonate-urethane biodegradation by Donna Lee M. Dinnes; J. Paul Santerre; Rosalind S. Labow (3881-3889).
Activation of the phospholipase A2 (PLA2) pathway is a key cell signaling event in the inflammatory response. The PLA2 family consists of a group of enzymes that hydrolyze membrane phospholipids, resulting in the liberation of arachidonic acid (AA), a precursor to pro-inflammatory molecules. Given the well-documented activating role of biomaterials in the inflammatory response to medical implants, the present study investigated the link between PLA2 and polycarbonate-based polyurethane (PCNU) biodegradation, and the effect that material surface had on PLA2 activation in the U937 cell line. PCNUs were synthesized with poly(1,6-hexyl 1,2-ethyl carbonate)diol, 1,4-butanediol and one of two diisocyanates (hexane 1,6-diisocyanate or 4,4′-methylene bisphenyl diisocyanate) in varying stoichiometries and incubated with adherent U937 cells. PLA2 inhibiting agents resulted in significantly decreased PCNU biodegradation ( p < 0.05 ). Moreover, when activation of PLA2 was assessed (3H-AA release), significantly more 3H-AA was released from PCNU-adherent U937 cells than polystyrene-adherent U937 cells ( p < 0.05 ) which was significantly decreased in the presence of PLA2 inhibitors. The pattern of inhibition of U937 cell-mediated biodegradation and 3H-AA release that was modulated by PCNU surface differences, suggests a role for secretory PLA2 along with cytosolic PLA2. Understanding PCNU activation of intracellular pathways, such as PLA2, will allow the design of materials optimized for their intended use.
Keywords: Biodegradation; Polyurethane; Phospholipase A2; U937 cell line; Inflammation;

Protein adsorption on 18-alkyl chains immobilized on hydroxyl-terminated self-assembled monolayers by Inês Castro Gonçalves; Ma Cristina L. Martins; Mário A. Barbosa; Buddy D. Ratner (3891-3899).
Surfaces of devices that contact blood accumulate adsorbed and denatured proteins perhaps triggering activation of the coagulation system. A renewable layer of albumin would biologically “passivate” the surface and prevent thrombus formation. Based on the approach of selectively binding albumin to fatty acids, different percentages of a compound with 18 carbons (C18) were immobilized on OH-terminated self-assembled monolayers (SAMs). Fourier transform infrared reflection absorption spectroscopy (IRAS), ellipsometry, contact angle (and surface free energy) and X-ray photoelectron spectroscopy (XPS) measurements were used to characterize these surfaces and proved that there is an efficient immobilization of C18. There is an increase of the thickness and hydrophobicity of SAMs with an increasing percentage of C18. Adsorption of human serum albumin (HSA) was evaluated using radiolabelled 125I-HSA and IRAS. This study showed a gradual increase of HSA adsorption with the increase of surface hydrophobicity. Regarding competitive binding and exchangeability of albumin towards fibrinogen, it was proved, by radiolabelling, that SAMs prepared from solutions with 2.5% C18 presented considerable adsorption in a selective and reversible way.
Keywords: Albumin; Competitive protein adsorption; Self-assembled monolayers; Surface modification; Fatty acids; Selective adsorption;

Tissue reactions of in situ formed dextran hydrogels crosslinked by stereocomplex formation after subcutaneous implantation in rats by Gert W. Bos; Wim E. Hennink; Linda A. Brouwer; Wim den Otter; Theo F.J. Veldhuis; Cornelus F. van Nostrum; Marja J.A. van Luyn (3901-3909).
In this study, the in vivo biocompatibility of physically crosslinked dextran hydrogels was investigated. These hydrogels were obtained by mixing aqueous solutions of dextran grafted with l-lactic acid oligomers and dextran grafted with d-lactic acid oligomers. Gelation occurs due to stereocomplex formation of the lactic acid oligomers of opposite chirality. Since gelation takes some time, in situ gel formation is possible with this system.A number of sterilization methods was evaluated for their effect on the chemical and physical properties of the hydrogel. It was shown that of the investigated options (filtration, gamma irradiation, dry-heat and autoclaving) dry-heat sterilization was the preferred method to prepare sterile gels suitable for in vivo evaluations.Two types of stereocomplex gels were prepared and implanted subcutaneously in rats. The tissue reaction was evaluated over a period of 30 days. A mild ongoing foreign body reaction was observed characterized by infiltration of macrophages. Giant cells were only scarcely formed and the low numbers of lymphocytes showed that priming of the immune system is hardly involved. Importantly, the gels fully degraded in vivo within 15 days, which is in good agreement with the in vitro degradation behaviour of these gels. In conclusion, stereocomplexed dextran-oligolactic gels showed good biocompatibility which makes them suitable candidates for the design of controlled release devices for pharmaceutically active proteins.
Keywords: Hydrogel; In situ gelation; Tissue reactions; Biocompatibility; Drug delivery; Stereocomplex; Dextran;

An in vivo evaluation of a biodegradable genipin-cross-linked gelatin peripheral nerve guide conduit material by Yueh-Sheng Chen; Ju-Ying Chang; Chun-Yuan Cheng; Fuu-Jen Tsai; Chun-Hsu Yao; Bai-Shuan Liu (3911-3918).
We evaluated peripheral nerve regeneration using a biodegradable nerve conduit, which was made of genipin-cross-linked gelatin. The genipin-cross-linked gelatin conduit (GGC) was dark blue in appearance, which was concentric and round with a rough outer surface whereas its inner lumen was smooth. After subcutaneous implantation on the dorsal side of the rat, the GGC only evoked a mild tissue response, forming a thin tissue capsule surrounding the conduit. Biodegradability of the GGC and its effectiveness as a guidance channel were examined as it was used to repair a 10 mm gap in the rat sciatic nerve. As a result, tube fragmentation was not obvious until 6 weeks post-implantation and successful regeneration through the gap occurred in all the conduits at the three experimental periods of 4, 6, and 8 weeks. Histological observation showed that numerous regenerated nerve fibers, mostly unmyelinated and surrounded by Schwann cells, crossed through and beyond the gap region 6 weeks after operation. Peak amplitude and area under the muscle action potential curve both showed an increase as a function of the recovery period, indicating that the nerve had undergone adequate regeneration. Thus, the GGC can not only be an effective aids for regenerating nerves but can also lead to favorable nerve functional recovery.
Keywords: Peripheral nerve regeneration; Genipin; Gelatin;

Chitosan–alginate hybrid scaffolds for bone tissue engineering by Zhensheng Li; Hassna R. Ramay; Kip D. Hauch; Demin Xiao; Miqin Zhang (3919-3928).
A biodegradable scaffold in tissue engineering serves as a temporary skeleton to accommodate and stimulate new tissue growth. Here we report on the development of a biodegradable porous scaffold made from naturally derived chitosan and alginate polymers with significantly improved mechanical and biological properties as compared to its chitosan counterpart. Enhanced mechanical properties were attributable to the formation of a complex structure of chitosan and alginate. Bone-forming osteoblasts readily attached to the chitosan–alginate scaffold, proliferated well, and deposited calcified matrix. The in vivo study showed that the hybrid scaffold had a high degree of tissue compatibility. Calcium deposition occurred as early as the fourth week after implantation. The chitosan–alginate scaffold can be prepared from solutions of physiological pH, which may provide a favorable environment for incorporating proteins with less risk of denaturation. Coacervation of chitosan and alginate combined with liquid–solid separation provides a scaffold with high porosity, and mechanical and biological properties suitable for rapid advancement into clinical trials.
Keywords: Scaffold; Bone regeneration; Chitosan; Alginate; Tissue engineering;

Nano- to micro-structured biodegradable poly(l-lactide-co- ε -caprolactone) (PLCL) fabrics were prepared by electrospinning. Electrospun microfiber fabrics with different compositions of PLCL (mol% in feed; 70/30, 50/50, and 30/70), poly(l-lactide) (PLL) and poly( ε -caprolactone) (PCL) were obtained using methylene chloride (MC) as a solvent. The PLL microfiber exhibited a nanoscale-pore structure with a pore diameter of approximately 200–800 nm at the surface and subsurface regions, whereas such a surface structure was hardly observed in other polymers containing CL. The microfiber fabric made of PLCL 50/50 was elastomeric. Nanoscale-fiber fabrics with PLCL 50/50 (approx. 0.3 or 1.2 μm in diameter) were electrospun using 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) as a solvent. Mercury porosimetry showed that the decrease in the fiber diameter of the fabric decreased porosity, but increased fiber density and mechanical strength. Human umbilical vein endothelial cells (HUVECs) were adhered well and proliferated on the small-diameter-fiber fabrics (0.3 and 1.2 μm in diameter), both of which are dense fabrics, whereas markedly reduced cell adhesion, restricted cell spreading and no signs of proliferation were observed on the large-diameter-fiber fabric (7.0 μm in diameter). The potential biomedical application of electrospun PLCL 50/50 was discussed.
Keywords: Electrospinning; Nanofibers; Microfibers; Dielectric constant; Human umbilical vein endothelial cell (HUVEC);

The injectable polymer scaffolds which are biocompatible and biodegradable are important biomaterials for tissue engineering and drug delivery. Hydrogels derived from natural proteins and polysaccharides are ideal scaffolds for tissue engineering since they resemble the extracellular matrices of the tissue comprised of various amino acids and sugar-based macromolecules. Here, we report a new class of hydrogels derived from oxidized alginate and gelatin. We show that periodate-oxidized sodium alginate having appropriate molecular weight and degree of oxidation rapidly cross-links proteins such as gelatin in the presence of small concentrations of sodium tetraborate (borax) to give injectable systems for tissue engineering, drug delivery and other medical applications. The rapid gelation in the presence of borax is attributed to the slightly alkaline pH of the medium as well as the ability of borax to complex with hydroxyl groups of polysaccharides. The effect of degree of oxidation and concentration of alginate dialdehyde, gelatin and borax on the speed of gelation was examined. As a general rule, the gelling time decreased with increase in concentration of oxidized alginate, gelatin and borax and increase in the degree of oxidation of alginate. Cross-linking parameters of the gel matrix were studied by swelling measurements and trinitrobenzene sulphonic acid (TNBS) assay. In general, the degree of cross-linking was found to increase with increase in the degree of oxidation of alginate, whereas the swelling ratio and the degree of swelling decreased. The gel was found to be biocompatible and biodegradable. The potential of the system as an injectable drug delivery vehicle and as a tissue-engineering scaffold is demonstrated by using primaquine as a model drug and by encapsulation of hepatocytes inside the gel matrix, respectively.
Keywords: Hydrogel; Alginate; Gelatin; Oxidation; Drug delivery; Cell encapsulation;

The role of muscle-derived stem cells in bone tissue engineering by Jui-Sheng Sun; Steven Yueh-Hsiu Wu; Feng-Huei Lin (3953-3960).
The formation of bone and repair of bone defect requires a source of pluripotential mesenchymal stem cells. However, the capacity of the human body to generate bone components is limited. In this report, we show that the highly purified myogenic cells by the preplate technique have the capacity to differentiate into osteogenic lineage in vitro.The recombinant human bone morphogenic protein (rh-BMP-2) was immobilized on the molded gelatin composite. Primary muscle cells were isolated from newborn Wistar-rats calf muscle. The cells were then preplated in collagen-coated flasks. After six serial platings, the culture was enriched with small, round cells [pp6]. The effects of immobilized rhBMP-2 on the gelatin scaffold were evaluated by the analysis of alkaline phosphatase (ALP) and osteocalcin in culture medium after seedings of muscle-derived cells [pp6].The results showed that the cells isolated from pp6 slow adhering cells possessed round mononuclear phenotype, marked ALP stain and matrix mineralization. The synthesis and secretion of ALP from pp6 muscle-derived cells were persistent higher than that of pp1–pp5 groups. The efficacy of rhBMP-2 immobilization on the gelatin scaffolds as manifested as the synthesis and secretion of ALP and osteocalcin from muscle-derived cells was always significantly higher than that of the control samples.In summary, our results suggest that the muscle-derived pp6 cells were capable of inducing and participating in bone formation. These results suggest that muscle tissue is a valuable resource for osteoprogenitor cells to be used in clinical practice to improve bone healing.
Keywords: Muscle-derived cells; Gelatin; Bone morphogenetic protein 2; Osteocalcin; Bone tissue engineering;

Preparation and characterization of highly porous, biodegradable polyurethane scaffolds for soft tissue applications by Jianjun Guan; Kazuro L. Fujimoto; Michael S. Sacks; William R. Wagner (3961-3971).
In the engineering of soft tissues, scaffolds with high elastance and strength coupled with controllable biodegradable properties are necessary. To fulfill such design criteria we have previously synthesized two kinds of biodegradable polyurethaneureas, namely poly(ester urethane)urea (PEUU) and poly(ether ester urethane)urea (PEEUU) from polycaprolactone, polycaprolactone-b-polyethylene glycol-b-polycaprolactone, 1,4-diisocyanatobutane and putrescine. PEUU and PEEUU were further fabricated into scaffolds by thermally induced phase separation using dimethyl sulfoxide (DMSO) as a solvent. The effect of polymer solution concentration, quenching temperature and polymer type on pore morphology and porosity was investigated. Scaffolds were obtained with open and interconnected pores having sizes ranging from several μm to more than 150 μm and porosities of 80–97%. By changing the polymer solution concentration or quenching temperature, scaffolds with random or oriented tubular pores could be obtained. The PEUU scaffolds were flexible with breaking strains of 214% and higher, and tensile strengths of approximately 1.0 MPa, whereas the PEEUU scaffolds generally had lower strengths and breaking strains. Scaffold degradation in aqueous buffer was related to the porosity and polymer hydrophilicity. Smooth muscle cells were filtration seeded in the scaffolds and it was shown that both scaffolds supported cell adhesion and growth, with smooth muscle cells growing more extensively in the PEEUU scaffold. These biodegradable and flexible scaffolds demonstrate potential for future application as cell scaffolds in cardiovascular tissue engineering or other soft tissue applications.
Keywords: Biodegradation; Polyurethane; Scaffold; Thermally induced phase separation;

Shear bond strength of experimental methacrylated beta-cyclodextrin-based formulations by Latiff A. Hussain; Sabine H. Dickens; Rafael L. Bowen (3973-3979).
Previous studies have shown that methacrylated beta-cyclodextrins (MCDs) can be used as comonomers in resin-based dental composites. These MCDs by virtue of having several polymerizable methacrylate groups and hydrophilic hydroxyl groups, may also promote bonding of dental composites to dentin. This study evaluated MCDs as adhesive comonomers, and optimized comonomer and polymerization initiator concentrations for maximum shear bond strength (SBS). Experimental MCD-based bonding formulations in acetone were prepared by mixing 33 mass fraction % MCDs with (10, 20, 30, 40, or 50) mass fraction % of 2-hydroxyethyl methacrylate (HEMA). The MCD/HEMA-based solutions were activated with varied amounts of camphorquinone (CQ) and ethyl 4-dimethylamino benzoate (4E). Samples for SBS were prepared by bonding a composite resin to acid-etched dentin surfaces of extracted human molars with the experimental bonding solutions. The specimens were immersed in 37 °C water for 24 h and bond strengths were determined in shear mode. With increasing HEMA concentration, the SBS values of MCD-bonding solutions increased to 16 MPa at a composition of 33% MCD, 30% HEMA, and 37% acetone by mass. Also, SBS values of MCD-bonding solutions varied as a function of the CQ and 4E concentrations and passed through a maximum SBS at 21 MPa, which was comparable to that of a commercial control. This preliminary study indicated that nonacidic MCD monomers could be used as an adhesion-promoting comonomer. Additional modification of MCDs having both polymerizable groups and anionic ligand groups, e.g., polymerizable acidic cyclodextrin derivatives should increase the SBS even further.
Keywords: Adhesion; Bond strength; Collagen; Dental adhesive; Dentine bonding agent; Surface treatment; Cyclodextrin methacrylates; Polymerization initiators;

To determine the effect of Light emitting diode (LED) curing on dental resins, microleakage, shear bond strength and surface hardness of a dental composite cured with different LEDs were determined and compared with conventional halogen curing. For microleakage, Class V cavities were restored with Esthet-X, divided into groups, and exposed to one of the curing protocols (Elipar Freelight in soft start and standard modes; Ultra-Lume 2; Spectrum 800). Standard dye penetration tests were performed and the data summarised in a 2-way contingency table of observed frequencies. The Chi-square test was used ( p < 0.05 ) to test for significant differences between the lights. For surface hardness, samples of Esthet-X were exposed to the light-curing units (LCUs). Vickers hardness was determined on the upper and the bottom surfaces. Data was subjected to statistical analysis using ANOVA ( p < 0.05 ). Shear bond strength was determined using a push out method. Comparisons (ANOVA, p < 0.05 ) were made between the different curing protocols. No significant difference in microleakage could be demonstrated between the different LCUs at the enamel side ( p = 0.60 ). At the dentin side only the Elipar Freelight (soft start), could significantly reduce microleakage ( p < 0.01 ). The hardness score for the halogen light was significantly lower than for the LED lights ( p < 0.01 ). The Spectrum 800 and the Elipar Freelight (soft start) have significantly higher shear bond strengths than the others ( p < 0.01 ). It was concluded that the LED source is more efficient for a comparable overall power output.
Keywords: LED; Microleakage; Surface hardness; Shear bond strength; Dental composite;

Development of an RNA isolation procedure for the characterisation of human endothelial cell interactions with polyurethane cardiovascular bypass grafts by Dina S. Vara; Geoffrey Punshon; Kevin M. Sales; Henryk J. Salacinski; Sas Dijk; Robert A. Brown; George Hamilton; Alexander M. Seifalian (3987-3993).
To date no reliable method has been developed for the isolation of RNA from cells seeded onto cylindrical vascular grafts. This study was performed in order to develop a reliable methodology for isolating RNA from cylindrical conduits made from poly(carbonate-urea)urethane (PU).Human umbilical vein EC were seeded onto PU vascular grafts and an Alamar blue™ assay performed to assess cell viability. Cells were prepared for RNA extraction by trypsinisation, cell scraping and direct application of cell lysis buffer. In all cases RNA was extracted using a “Qiagen RNeasy™” kit.Alamar blue™ showed viable cells were present on all of the seeded PU vascular grafts. Levels of RNA extracted from the cells removed from the graft by the trypsinisation yielded 0.130 μg/μl, by scraping 0.078 μg/μl and by direct lysing 0.093 μg/μl of RNA, respectively. RTPCR was conducted successfully for GAPDH and TGF-β1. Trypsinisation prior to RNA extraction provided the highest RNA yield and attained near complete cell removal ensuring that gene expression obtained was representative.
Keywords: Alamar blue™ assay; Vascular graft; Poly(carbonate-urea) urethane; RNA extraction; Gene expression; Human umbilical vein endothelial cells;

Superparamagnetic iron oxide nanoparticles (SPION) with appropriate surface chemistry have been widely used experimentally for numerous in vivo applications such as magnetic resonance imaging contrast enhancement, tissue repair, immunoassay, detoxification of biological fluids, hyperthermia, drug delivery and in cell separation, etc. All these biomedical and bioengineering applications require that these nanoparticles have high magnetization values and size smaller than 100 nm with overall narrow particle size distribution, so that the particles have uniform physical and chemical properties. In addition, these applications need special surface coating of the magnetic particles, which has to be not only non-toxic and biocompatible but also allow a targetable delivery with particle localization in a specific area. To this end, most work in this field has been done in improving the biocompatibility of the materials, but only a few scientific investigations and developments have been carried out in improving the quality of magnetic particles, their size distribution, their shape and surface in addition to characterizing them to get a protocol for the quality control of these particles. Nature of surface coatings and their subsequent geometric arrangement on the nanoparticles determine not only the overall size of the colloid but also play a significant role in biokinetics and biodistribution of nanoparticles in the body. The types of specific coating, or derivatization, for these nanoparticles depend on the end application and should be chosen by keeping a particular application in mind, whether it be aimed at inflammation response or anti-cancer agents. Magnetic nanoparticles can bind to drugs, proteins, enzymes, antibodies, or nucleotides and can be directed to an organ, tissue, or tumour using an external magnetic field or can be heated in alternating magnetic fields for use in hyperthermia. This review discusses the synthetic chemistry, fluid stabilization and surface modification of superparamagnetic iron oxide nanoparticles, as well as their use for above biomedical applications.
Keywords: Magnetic nanoparticles; MRI; Drug delivery; Surface modification; Hyperthermia; Cell labelling; Iron oxide;

Collagen–chondroitin sulfate-based PLLA–SAIB-coated rhBMP-2 delivery system for bone repair by Dilek Sendil Keskin; Aysen Tezcaner; Petek Korkusuz; Feza Korkusuz; Vasif Hasirci (4023-4034).
Bone morphogenetic proteins (BMPs) are osteoinductive proteins used intensively in clinical investigations involving various bone-related treatments. Owing to their high potential in new bone formation they require local application at the treatment site. For this purpose various controlled delivery systems with BMPs as the excipients have been prepared in recent years. Focusing on this clinical need a disc-shaped BMP carrier was designed as a local delivery system using soluble collagen and chondroitin sulfate. In situ release studies carried out with a model protein (FITC-labeled Protein A) presented a very high rate of release; with most of the protein content being released within 24 h. This rate could be decreased by providing a poly(l-lactide) (PLLA) and sucrose acetate isobutyrate-based (SAIB-based) coat around the release system, applied after BMP loading. In this way, it was possible to extend the release period from 24 h to about 12 days. In situ release of BMP from the same carriers, as quantitated using an ELISA kit, was even slower, with 50% of the protein being released in 15 days.In order to be able to secure the BMP delivery system at the bone defect site and to provide support a mesh knitted using Vicryl sutures and bonded with poly(l-lactide-co-glycolide) (PLGA) was tested in in vivo. Two time periods, 1 and 3 weeks, were used to evaluate the healing process. Osteoinduction by the BMP carrier system was assessed by histology-based bone scoring and X-ray examinations. PLLA–SAIB-coated collagen discs containing BMP presented good biocompatibility and optimum osteogenic stimulation. Structural changes in histological micrographs at week 1 indicated dose-dependent periosteal ossification. At the end of week 3 histological findings with both BMP (1 and 2 μg) doses were almost the same.
Keywords: Collagen; Chondroitin sulfate; PLLA; SAIB; rhBMP-2; Controlled drug delivery;

Short term in vivo biocompatibility testing of biodegradable poly(D,L-lactide)—growth factor coating for orthopaedic implants by Britt Wildemann; Andre Sander; Philipp Schwabe; Martin Lucke; Ulrich Stöckle; Michael Raschke; Norbert P. Haas; Gerhard Schmidmaier (4035-4040).
Fracture healing can be stimulated by exogenous application of growth factors. Using porcine and rat models the efficacy of locally delivered IGF-I and TGF-β1 from an implant coating has been demonstrated. A thin and biomechanical stable biodegradable poly(D,L-lactide) was used to coat implants and serve as a drug carrier. Due to reports of possible foreign body reactions caused by polymer materials in orthopedic surgery, this study investigated the biocompatibility of the polylactide implant coating and the locally released growth factors during the time course of rat tibial fracture healing (days 5, 10, 15, and 28 after fracture). Monocytes/macrophages and osteoclast were detected using an monoclonal antibody against ED1 (comparable to CD68 in mice and human). The antibody ED1 stains monocytes, macrophages and osteoclast in the bone marrow and in the newly formed fracture callus. A moderate density of the monocytes/macrophages was seen in the proximal part of the medullary canal, but almost no cells were detectable in the region distal to the fracture. The amount of stained cells increased during the observation time with a maximum at days 10 and 15 followed by a decrease at day 28. No differences were detectable between the investigated groups from day 5 to 15 post fracture indicating, that the used poly(D,L-lactide) or the incorporated growth factors do not evoke an elevated immunological response compared to the uncoated titanium implant at the investigated time points. A significantly higher amount of ED1 positive cells was measured 28 days after fracture in the control group compared to the groups with the coated implants.In conclusion, no indication of a foreign body reaction due to the use of the polylactide or the growth factors was found indicating a good short-term biocompatibility of this bioactive coating.
Keywords: Drug delivery system; Monocytes; Macrophages; ED1; Growth factors; Polylactide; Immunohistochemistry;

Photomediated crosslinking of C6-cinnamate derivatized type I collagen by Chang-Ming Dong; Xiaoyi Wu; Jeffrey Caves; Shyam S. Rele; Benjamin S. Thomas; Elliot L. Chaikof (4041-4049).
Synthesis and characterization of cinnamated Type I collagen and its related mechanical properties after photomediated crosslinking were investigated in detail. Using an EDC/NHS conjugation method, collagen was chemically modified to incorporate a photosensitive cinnamate moiety. The cinnamated collagen was fully characterized by 1H NMR, UV–vis, and circular dichroism (CD) spectroscopy, as well as by rheological and mechanical analyses. Cinnamated collagens with varying degrees of derivatization retained collagen triple helical structure. The rheological spectra of collagen solutions demonstrate that the storage modulus decreases with increasing cinnamate content, owing to a decrease in physical crosslinking. The kinetics of the crosslinking process in both hydrated gels and dry films were monitored by UV–vis spectroscopy and confirmed that crosslinking was complete within 60 min of irradiation. The uniaxial stress–strain behavior of crosslinked collagen films, including Young's modulus and ultimate tensile strength, was comparable to values reported for glutaraldehyde-crosslinked monomeric collagen films. These data demonstrate that derivatization of collagen with photosensitive cinnamate moieties provides a facile route for solid-state crosslinking, thereby improving the mechanical properties of collagen and enhancing the potential applicability of collagen-based materials in tissue engineering and drug delivery.
Keywords: Collagen; Crosslinking; Photocrosslinkable; Photodimerizable; Cinnamate; Blood vessel;

Effects of aging on the mechanical behavior of human dentin by D. Arola; R.K. Reprogel (4051-4061).
An experimental study on the mechanical behavior of human dentin and the influence of age was conducted. Beams with rectangular cross-section were sectioned from the coronal dentin of virgin extracted molars (N=76) that were obtained from (N=70) patients between 17 and 80 years of age. The beams were loaded in either quasi-static 4-point flexure or 4-point flexural fatigue to failure and the stiffness, strength and fatigue properties were evaluated. In characterizing the fatigue response the beams were divided into two age groups that were regarded as young (17⩽age⩽30, mean±std. dev.=25±5 years) and old (50⩽age⩽80, mean±std. dev.=64±9 years) dentin. Results from monotonic loading showed that both the flexural strength and strain to fracture of dentin decreased significantly with age. The fatigue life of dentin increased with a reduction in cyclic stress amplitude and the fatigue strength of young dentin was greater than that of old dentin at all cyclic stress amplitudes. The endurance strength of young dentin (at 107 cycles) was approximately 44 MPa, whereas the old dentin exhibited an endurance strength of approximately 23 MPa.Based on differences in the mechanical behavior and microscopic features of the fracture surfaces from the young and old specimens, aging appears to result in an increase in both the rate of damage initiation and propagation in dentin.
Keywords: Age; Dentin; Fatigue; Fracture;