Biomaterials (v.27, #1)

Calendar (I).

Performance of decellularized xenogeneic tissue in heart valve replacement by Ulrich A. Stock; Katja Schenke-Layland (1-2).
Due to human valve (allograft) scarcity, the concept of decellularization of xenogeneic valves is currently pursued. The two current concepts of guided tissue regeneration and tissue engineering are critically discussed. While guided tissue regeneration shows promising results in animal experiments, there is no scientific evidence that the obtained results might be transferred to the human. The approach of tissue engineering using decellularized xenogeneic heart valves bears enormous potential, however, numerous issues need to be studied and clarified prior potential clinical application.
Keywords: Heart valves; Tissue engineering; Tissue regeneration; Decellularized xenogeneic tissue;

Resorbable silicon stabilized tricalcium phosphate (Si-TCP)-based bioceramics are characterized from a biological perspective by measuring the intermolecular interaction force between osteopontin (OPN) protein and the material surface using atomic force microscopy (AFM). OPN protein was covalently bound to silicon nitride AFM tips and adsorption and adhesion forces were measured in an electrolyte with a composition similar to that of physiological fluids. A strong relationship exists between the adhesion force of OPN on the material surface, the number of adherent osteoclasts (OC) and the resorption of the material. OPN adhesion is strongest on hydroxyapatite (HA) surfaces, or in samples that induce a HA-like surface through a precipitation reaction in electrolytic media. It is proposed that the increased biological response of the Si-TCP phase can be attributed in part to its reactivity in a physiological electrolyte, which involves a rapid conversion to a calcium deficient HA phase with a corresponding increase in the adhesion strength of OPN to the material, with a consequentially higher OC resorption response.
Keywords: Atomic force microscopy; Osteopontin; Osteoclast;

Electron beam irradiation modification of collagen membrane by Bo Jiang; Zhihong Wu; Huichuan Zhao; Fangyuan Tang; Jian Lu; Qingrong Wei; Xingdong Zhang (15-23).
A critical observation of reconstituted collagen membrane radiated by electron beam (EB) indicated that these collagenous fibers become cross-linked network when the irradiation is carried out in greater than melt temperature and nitrogen atmosphere. Studies on the membrane properties showed that glass transformation temperature ( T g ) and melt point ( T m ) of reconstituted collagen have no changes, but thermal gravity curves and infrared (IR) spectra become obviously different before and after irradiation. Cross-linking density calculated by the equation based on the theory of Flory–Rehner proved further that the densities increase with radiation doses increasing. Resistance to enzymatic digestions in vitro and implantation in vivo were determined to evaluate the physicochemical properties of cross-linked matrices. Based on the above results, it was concluded that EB radiation inducing cross-linking in greater than melt temperature and nitrogen atmosphere condition is an attractive, effective method, which introduce into intermolecular covalent cross-linkings.
Keywords: Reconstituted collagen; Fiber; Membrane; Electron beam irradiation; Cross-linking; Biodegradation;

Solid-state deformation processing is a promising technique for modifying the physical and mechanical properties of highly crosslinked ultra-high molecular weight polyethylene (UHMWPE) beyond simple thermal treatment cycles that have been employed previously. This study evaluates anisotropy and oxidative resistance in a novel, radiation crosslinked (50 kGy) UHMWPE material (ArComXL: Biomet, Inc., Warsaw, IN), incorporating solid-state, deformation processing by extrusion below the melt transition for application in total hip arthroplasty. Tensile, compression, and small punch tests were conducted to evaluate the material properties in the three principal axes of the resulting material. Furthermore, short-term oxidative resistance was evaluated using Fourier transform infrared spectroscopy and the small punch test in conjunction with accelerated shelf aging protocols. The results of this testing indicate that the material is anisotropic, with significantly enhanced strength oriented along the long axis of the rod. For certain other properties, the magnitude of the anisotropy was relatively slight, especially in the elastic regime, in which only a 20% difference was noted between the long axis of the rod and the orthogonal, radial direction. The highly crosslinked material contains detectable free radicals, at a concentration that is 90% less than control, gamma inert sterilized UHMWPE. An unexpected finding of this study was evidence of oxidative stability of the deformation-processed material, even after 4 weeks of accelerated aging in a pressure vessel containing five atmospheres of oxygen (ASTM F2003), which resulted in macroscopic embrittlement of the control material. The oxidative stability observed in ArComXL suggests that the deformation-processed material may be suitable for air-permeable packaging and gas sterilization, which has thus far been reserved for remelted highly crosslinked UHMWPE.
Keywords: UHMWPE; Ultra-high molecular weight polyethylene; Radiation crosslinking; Gamma radiation; Mechanical properties; Oxidation; Anisotropy; Solid-state extrusion; Deformation processing;

Two-dimensional patterning of thin coatings for the control of tissue outgrowth by Helmut Thissen; Graham Johnson; Patrick G. Hartley; Peter Kingshott; Hans J. Griesser (35-43).
Control of the precise location and extent of cellular attachment and proliferation, and of tissue outgrowth is important in a number of biomedical applications, including biomaterials and tissue engineered medical devices. Here we describe a method to control and direct the location and define boundaries of tissue growth on surfaces in two dimensions. The method relies on the generation of a spatially defined surface chemistry comprising protein adsorbing and non-adsorbing areas that allow control over the adsorption of cell-adhesive glycoproteins. Surface modification was carried out by deposition of thin acetaldehyde and allylamine plasma polymer coatings on silicon wafer and FEP substrates, followed by grafting of a protein resistant layer of poly(ethylene oxide). Spatially controlled patterning of the surface chemistry was achieved by masking during plasma polymerization. XPS and AFM were used to provide evidence of successful surface modifications. Adsorption of the extracellular matrix protein collagen I followed by tissue outgrowth experiments with bovine corneal epithelial tissue for up to 21 days showed that two-dimensional control over tissue outgrowth is achievable with our patterning method over extended time frames. The method promises to be an effective tool for use in a number of in vitro and in vivo applications.
Keywords: Surface modification; Plasma polymerization; Polyethylene oxide; Epithelial cell; Surface analysis; In vitro test;

Plasma surface modification of poly vinyl chloride for improvement of antibacterial properties by Wei Zhang; Paul K. Chu; Junhui Ji; Yihe Zhang; Xuanyong Liu; Ricky K.Y. Fu; Peter C.T. Ha; Qi Yan (44-51).
Plasma immersion ion implantation (PIII) was used to modify medical-grade PVC coated by triclosan and bronopol to enhance the antibacterial properties. The surface was first activated by O2 plasma to produce more hydrophilic groups so that triclosan and bronopol could be coated more effectively on the surface. Subsequently, an argon plasma treatment was conducted under optimal conditions to improve the antibacterial properties of the triclosan and bronopol-coated PVC samples. The modified surfaces were characterized by XPS, ATR-FTIR, SEM, and contact angle measurements. The antibacterial properties were evaluated utilizing the method of plate-counting of Staphylococcus aureus (gram positive) and Escherichia coli (gram negative). Our experimental results show that the plasma-modified PVC with bronopol exhibits good antibacterial properties while the favorable bulk properties of PVC are retained. The plasma-modified PVC with triclosan has better antibacterial performance against E. coli than bronopol. The change in the antibacterial effect on the modified PVC with time was also investigated and the antibacterial effect was observed to decrease with time.
Keywords: Plasma immersion ion implantation; Antibacterial; Medical poly vinyl chloride; Triclosan; Bronopol;

Electrochemical processing of fibrinogen modified-graphite surfaces: Effect on plasmin generation from adsorbed plasminogen by Séverine Alfonsi-Hourdin; Sylvie Longchamp; Olivier Gallet; Jean-Maxime Nigretto (52-60).
With the aim to improve the fibrinolytic properties of carbons by different biological and electrochemical treatments, we modified graphite surfaces by fibrinogen adsorption and subsequent application of various constant potentials before submitting them to plasminogen adsorption. First, we verified that plasminogen (purified or present in human plasma) could adsorb onto these modified surfaces and that adsorbed plasminogen could be converted by t-PA (the principal physiological activator of plasminogen) to adsorbed plasmin. The catalytic properties of the generated enzyme were characterized in assay solutions containing t-PA, fibrinogen and the chromogenic substrate S-2403 (pyroGlu-Phe-Lys-p-nitroaniline, HCl). Experiments showed that the application of electrical potentials to the fibrinogen coating could indirectly affect the properties of the material. In the case of anodic potentials, the amidolytic activity of the generated plasmin was significantly enhanced. Especially, this activity was 10 times higher at a particular potential value.
Keywords: Carbon; Fibrinogen; Plasminogen; Surface modification; Electrochemistry;

Preparation of hybrid scaffold from fibrin and biodegradable polymer fiber by Akishige Hokugo; Tomoaki Takamoto; Yasuhiko Tabata (61-67).
A biodegradable hybrid scaffold was prepared from fibrin and poly(glycolic acid) (PGA) fiber. Mixed fibrinogen and thrombin solution homogeneously dispersed in the presence of various amounts (0, 1.5, 3.0, and 6.0 mg) of PGA fiber was freeze-dried to obtain fibrin sponges with or without PGA fiber incorporation. By scanning electron microscopy observation, the fibrin sponges had an interconnected pore structure, irrespective of the amount of PGA fiber incorporated. PGA fiber incorporation enabled the fibrin sponges to significantly enhance their compression strength. In vitro cell culture studies revealed that the number of L929 fibroblasts initially attached was significantly larger for any fibrin sponge with PGA fiber incorporation than for the fibrin sponge without PGA fiber. The shrinkage of sponges after cell seeding was suppressed by fiber incorporation. It is possible that the shrinkage suppression of sponges maintains their intraspace, resulting in the superior cell attachment of a sponge with PGA fiber incorporation. After subcutaneous implantation into the backs of mice, the residual volume of a fibrin sponge with PGA fiber incorporation was significant compared with that of a fibrin sponge without PGA fiber. Larger number of cells infiltrated deep inside the fibrin sponges with PGA fiber incorporation implanted subcutaneously. It is concluded that the fibrin sponge reinforced by fiber incorporation is a promising three-dimensional scaffold of cells for tissue engineering.
Keywords: Hybrid scaffold; Fibrin; Poly(glycolic acid) (PGA);

Protein bonding on biodegradable poly(l-lactide-co-caprolactone) membrane for esophageal tissue engineering by Yabin Zhu; Kerm Sin Chian; Mary B. Chan-Park; Priyadarshini S. Mhaisalkar; Buddy D. Ratner (68-78).
A biodegradable and flexible poly(l-lactide-co-caprolactone) (PLLC) copolymer was synthesized and surface modification has been performed aiming at application as a scaffold in esophageal tissue engineering. The PLLC membrane surface was aminolyzed by 1,6-hexanediamine to introduce free amino groups. Using these amino groups as bridges, fibronectin and collagen were subsequently bonded with glutaraldehyde as a coupling agent. The presence of free amino groups on the aminolyzed PLLC surface was quantified using fluorescamine analysis method, which revealed that the surface NH2 density increased and eventually saturated with increasing 1,6-hexanediamine concentration or reaction time. X-ray photoelectron spectroscopy (XPS) confirmed the presence of both proteins separately on the modified PLLC surface. Water contact angle measurements evaluate the wettability of modified and unmodified PLLC surfaces. Protein-bonded surface presented more hydrophilic and homogeneous, yet PLLC can also adsorb some protein molecules. In vitro long-term (12 d) culture of porcine esophageal cells proved that fibronectin- and collagen-modified PLLC surface (denoted PLLC–Fn and PLLC–Col, respectively) can more effectively support the growth of smooth muscle cells and epithelial cells; both modified and unmodified PLLC support fibroblasts growth. Mitochondrial activity assay and cell morphology observation indicate that the PLLC–Fn surface is more favorable to epithelium regeneration than PLLC–Col. These culture results provide much valuable information for our subsequent research on the construction of artificial scaffolds with esophageal function. Fibronectin-integrated PLLC will be a good candidate scaffold to support the growth of all types of esophageal cells.
Keywords: Poly(l-lactide-co-caprolactone) copolymer; Fibronectin; Collagen; Surface modification; Esophageal tissue engineering;

Bovine chondrocyte behaviour in three-dimensional type I collagen gel in terms of gel contraction, proliferation and gene expression by Laurent Galois; Sandrine Hutasse; Delphine Cortial; Cécile F. Rousseau; Laurent Grossin; Marie-Clarie Ronziere; Daniel Herbage; Anne-Marie Freyria (79-90).
This study evaluated the in vitro behaviour of bovine chondrocytes seeded in collagen gels, promising recently reported scaffolds for the treatment of full-thickness cartilage defects. To determine how chondrocytes respond to a collagen gel environment, 2×106 chondrocytes isolated from fetal, calf and adult bovine cartilage were seeded within type I collagen gels and grown for 12 days in both attached and floating (detached from the culture dish after polymerisation) conditions. Monolayer cultures were performed in parallel. All chondrocytes contracted floating gels to 55% of the initial size, by day 12. Contraction was dependent on initial cell density and inhibited by the presence of dihydrocytochalasin B as previously observed with fibroblasts. Gene expression was determined using conventional and real-time PCR. The chondrocyte phenotype was better maintained in floating gels compared to attached gels and monolayers. This was demonstrated by comparing the ratio of COL2A1/ COL1A2 mRNA and also of alpha10/alpha11 integrin mRNA. A strong up-regulation of MMP13 expression was measured at day 12 in floating gels. The composition of cartilage-like tissue obtained by growing chondrocytes in a collagen gel varied depending on the floating or attached conditions and initial cell density. It is thus important to consider these parameters when using this culture system in order to prepare a well-defined implant for cartilage repair.
Keywords: Bovine chondrocyte; Collagen gel; Cartilage repair; Tissue engineering; MMPs; Integrins;

Chondrocytic differentiation of human adipose-derived adult stem cells in elastin-like polypeptide by Helawe Betre; Shin R. Ong; Farshid Guilak; Ashutosh Chilkoti; Beverley Fermor; Lori A. Setton (91-99).
Human adipose derived adult stem (hADAS) cells have the ability to differentiate into a chondrogenic phenotype in three-dimensional culture and media containing dexamethasone and TGF-β. The current study examined the potential of a genetically engineered elastin-like polypeptide (ELP) to promote the chondrocytic differentiation of hADAS cells without exogenous chondrogenic supplements. hADAS cells were cultured in ELP hydrogels in either chondrogenic or standard medium at 5% O2 for up to 2 weeks. By day 14, constructs cultured in either medium exhibited significant increases in sulfated glycosaminoglycan (up to 100%) and collagen contents (up to 420%). Immunolabeling confirmed that the matrix formed consisted mainly of type II and not type I collagen. The composition of the constructs cultured in either medium did not differ significantly. To assess the effect of oxygen tension on the differentiation of the above constructs, samples were cultured in standard medium at either 5% or 20% O2 for 7 days and their gene expression profile was evaluated using real time RT-PCR. In both cases, the hADAS–ELP constructs upregulated SOX9 and type II collagen gene expression, while type I collagen was downregulated. However, constructs cultured in 20% O2 highly upregulated type X collagen, which was not detected in the 5% O2 cultures. The study suggests that ELP can promote chondrogenesis for hADAS cells in the absence of exogenous TGF-β1 and dexamethasone, especially under low oxygen tension conditions.
Keywords: Stem cells; Biomaterials; Tissue engineering; Cartilage; Elastin-like polypeptide; Scaffold;

Preparation of acrylic bone cements for vertebroplasty with bismuth salicylate as radiopaque agent by Lidia Hernández; Mar Fernández; Francisco Collía; Mariló Gurruchaga; Isabel Goñi (100-107).
One of the problems of percutaneous vertebroplasty attributed to the use of acrylic cements is related to the radiopacity of the formulation. The use of bismuth salicylate as the radiopaque agent is proposed in this work, taking into account the high radiopacity of organobismuth compounds used in dental applications and the possible analgesic effect of salicylic acid. Various cements formulated with this compound (some of them modified with polyethylene oxide) were examined. Setting parameters, mechanical properties, rheological behaviour, injectability, radiopacity and biocompatibility were studied for a variety of formulations, showing that the cement formulations containing bismuth salicylate have a higher radiopacity and better injection properties than commercial bone cement preparations and present good mechanical properties.
Keywords: Acrylic bone cements; Rheology; Injectability; Radiopacity;

Influence of polysaccharide coating on the interactions of nanoparticles with biological systems by Caroline Lemarchand; Ruxandra Gref; Catherine Passirani; Emmanuel Garcion; Boris Petri; Rainer Müller; Dominique Costantini; Patrick Couvreur (108-118).
Since dextran (DEX) grafted with poly(ε-caprolacton) (PCL) side chains (PCL–DEX) copolymers could form nanoparticles with a well defined core-shell structure, we investigated the ability of the DEX coating to modify the interactions with the biological media. We first studied the influence of the DEX coating on the phagocytosis of the nanoparticles by human TPH-1 and J774 murine macrophage-like cell lines. Then, the activation of the complement system (CH50 measurement) at the surface of the particles and the adsorption of plasma proteins (2D-PAGE) were investigated, too. It was found that the modification of the surface with DEX significantly reduced the cytotoxicity towards J774 macrophages: the IC50 was increased from 10 to 600 μg/ml. However, the DEX coating could activate complement, probably due to a loop-like conformation of DEX similar to that of cross-linked DEX in Sephadex (a strong complement activator). In addition, depending on whether the DEX loops were large or compact, preferential adsorption, apolipoproteins or immunoglobulins, was observed.
Keywords: Nanoparticle; Dextran; Poly(epsilon-caprolactone); Polysaccharide coating; Biological media;

Self-assembled nanoparticles based on glycol chitosan bearing hydrophobic moieties as carriers for doxorubicin: In vivo biodistribution and anti-tumor activity by Jae Hyung Park; Seunglee Kwon; Minsu Lee; Hesson Chung; Ji-Hyun Kim; Yoo-Shin Kim; Rang-Woon Park; In-San Kim; Sang Bong Seo; Ick Chan Kwon; Seo Young Jeong (119-126).
Self-assembled nanoparticles, formed by polymeric amphiphiles, have been demonstrated to accumulate in solid tumors by the enhanced permeability and retention effect, following intravenous administration. In this study, hydrophobically modified glycol chitosans capable of forming nano-sized self-aggregates were prepared by chemical conjugation of fluorescein isothiocyanate or doxorubicin to the backbone of glycol chitosan. Biodistribution of self-aggregates (300 nm in diameter) was evaluated using tissues obtained from tumor-bearing mice, to which self-aggregates were systemically administered via the tail vein. Irrespective of the dose, a negligible quantity of self-aggregates was found in heart and lung, whereas a small amount (3.6–3.8% of dose) was detected in liver for 3 days after intravenous injection of self-aggregates. The distributed amount of self-aggregates gradually increased in tumor as blood circulation time increased. The concentration of self-aggregates in blood was as high as 14% of dose at 1 day after intravenous injection and was still higher than 8% even at 3 days. When self-aggregates loaded with doxorubicin were administered into the tumor-bearing mice via the tail vein, they exhibited lower toxicity than but comparable anti-tumor activity to free doxorubicin. These results revealed the promising potential of self-aggregates on the basis of glycol chitosan as a carrier for hydrophobic anti-tumor agents.
Keywords: Drug delivery; Glycol chitosan; Nanoparticle; Self-assembly;

Improved synthesis and in vitro characterization of chitosan–thioethylamidine conjugate by Krum Kafedjiiski; Martin Hoffer; Martin Werle; Andreas Bernkop-Schnürch (127-135).
The aim of this study was to establish improved reaction conditions for the synthesis of chitosan–thioethylamidine (Ch-TEA) conjugate and to evaluate the properties of the obtained Ch-TEA conjugate. The influence of different factors on the coupling reaction, such as concentration of chitosan solution, employment of reducing agent and deprotection of S-acetyl groups, was evaluated. The cohesive properties and stability of the obtained conjugate were evaluated by disintegration test and by oxidation experiments, respectively. The adhesive properties of Ch-TEA conjugate were evaluated in vitro on freshly excised porcine mucosa via tensile studies and the rotating cylinder method. The permeation-enhancing effect of Ch-TEA conjugate was evaluated in Ussing-type chambers by using rhodamine 123 as model compound.The resulting conjugate displayed 225 μmol immobilized free thiol groups and 102 μmol disulfide bonds per gram polymer. The degree of modification depends mostly on the chitosan concentration employed and the deprotection of S-acetyl groups with hydroxylamine. During oxidation studies the amount of thiol groups decreased by 61%. Disintegration studies of tablets comprising Ch-TEA demonstrated stability for 48 h. In tensile studies, the total work of adhesion of the conjugate was determined to be 5.1-fold increased in comparison to unmodified chitosan. Results from the rotating cylinder method showed more than a 13-fold increase in the adhesion time of thiolated chitosan versus unmodified chitosan. The apparent permeability coefficient (P app) of the system 0.5% (w/v) Ch-TEA conjugate with 5% (w/v) glutathione was calculated to be 5.35×10−8  cm/s, while the P app value of the system 0.5% (w/v) unmodified chitosan was 1.73×10−8  cm/s. These features should render Ch-TEA useful as an excipient for various drug delivery systems.
Keywords: Thiolated chitosan; Chitosan- thioethylamidine conjugate; Isopropyl-S-acetylthioacetimidate hydrochloride; Disintegration; Oxidation; Permeation enhancement.;

The rate of drainage and lymphatic distribution of subcutaneously injected liposomes is controlled by inclusion of methoxypoly(ethyleneglycol), mPEG-phospholipid into the liposomal bilayer. The effect is most dramatic with liposomes containing 15 mol% mPEG-lipid, with an average PEG molecular mass of 350 Da. These vesicles are drained rapidly from the injection site into the initial lymphatics when compared to unmodified liposomes, and are retained more favourably by the scavengers of the regional lymph node. Liposomes decorated with longer surface mPEG chains (6.7 mol% of mPEG2000-lipid) exhibit faster drainage rates than vesicles having 15 mol% mPEG350-lipid in their lipid bilayer, but their lymph node retention is very poor. The lymph node retention of rapidly drained PEG-bearing vesicles was increased dramatically following conjugation of a non-specific IgG to the distal end of PEG, using a functionalized PEG2000 lipid. Adjusting the molecular architecture of surface mPEG and IgG-PEG chains to a “nearly overlapped mushroom” regime further enhanced target recognition of immuno-PEG2000 liposomes without compromising their drainage rate from the interstitium. The lymph node retention of these vesicles was further optimized by enriching their lipid bilayer with 20 mol% phosphatidylserine. These approaches have established important compositional and structural variables that control lymphatic targeting of immuno-PEG liposomes and their application in experimental medicine and biology is discussed.
Keywords: Steric stabilization; Stealth liposomes; Macrophage; Antibody conjugation; Fc receptor; Lymphatic system; Phosphatidylserine; Poly(ethyleneglycol);