Biomaterials (v.26, #12)

Calendar (I).

Development of highly reactive mono-(meth)acrylates as reactive diluents for dimethacrylate-based dental resin systems by Hui Lu; Jeffrey W. Stansbury; Jun Nie; Kathryn A. Berchtold; Christopher N. Bowman (1329-1336).
Reactive diluents such as triethyleneglycol-dimethacrylate (TEGDMA) have been widely used with bisphenol-A-glycidyl-dimethacrylate (Bis-GMA) to achieve restorative resins with appropriate viscosity and higher conversion. However, additional water sorption and polymerization shrinkage were also introduced. The aim of this work is to investigate whether the cure and material properties can be improved in dental resins containing novel mono-(meth)acrylates as reactive diluents so that these Bis-GMA-based copolymers have reduced polymerization shrinkage but higher overall double bond conversion. Several ultra-high-reactivity mono-(meth)acrylates that contain secondary functionalities have been synthesized and investigated. The polymerization rate and double bond conversion were monitored using photo-FTIR. Polymerization shrinkage, dynamic mechanical analysis, and flexural strength were characterized. Compared with the Bis-GMA/TEGDMA control, the Bis-GMA/mono-methacrylate systems studied showed higher final conversions, faster curing rates, and decreased polymerization shrinkage. Our optimum system Bis-GMA/morpholine carbamate methacrylate achieved 86% final conversion (vs. 65%), a polymerization rate 3.5 times faster, and a 30% reduction in polymerization volumetric shrinkage. These results indicate that certain highly reactive, novel mono-(meth)acrylates possess very promising potential to replace TEGDMA as reactive diluents and can readily be applied to develop superior dental resins.
Keywords: Dental resins; Photopolymerization; Reactive diluent; Volumetric shrinkage;

Calcium phosphate cement (CPC) sets in situ to form hydroxyapatite and is highly promising for a wide range of clinical applications. However, its low strength limits its use to only non-stress applications, and its lack of macroporosity hinders cell infiltration, bone ingrowth and implant fixation. The aim of this study was to develop strong and macroporous CPC scaffolds by incorporating chitosan and water-soluble mannitol, and to examine the biocompatibility of the new graft with an osteoblast cell line and an enzymatic assay. Two-way ANOVA identified significant effects on mechanical properties from chitosan reinforcement and powder:liquid ratio (p<0.001). The flexural strength of CPC–chitosan composite at a powder:liquid ratio of 2 was (13.6±1.2) MPa, which was significantly higher than (3.2±0.6) MPa for CPC control without chitosan (Tukey's at 0.95). At a powder:liquid ratio of 3.5, CPC–chitosan had a strength of (25.3±2.9) MPa, which was significantly higher than (10.4±1.7) MPa for CPC control. The scaffolds possessed total pore volume fractions ranging from 42.0% to 80.0%, and macroporosity up to 65.5%. At total porosities of 52.2–75.2%, the scaffold had strength and elastic modulus values similar to those of sintered porous hydroxyapatite and cancellous bone. Osteoblast mouse cells (MC3T3-E1) were able to adhere, spread and proliferate on CPC–chitosan specimens. The cells, which ranged from about 20 to 50 μm including the cytoplasmic extensions, infiltrated into the 165–271 μm macropores of the scaffold. In summary, substantial reinforcement and macroporosity were imparted to a moldable, fast-setting, biocompatible, and resorbable hydroxyapatite graft. The highly porous scaffold may facilitate bone ingrowth and implant fixation in vivo. In addition, the two to three times increase in strength may help extend the use of CPC to larger repairs in moderately stress-bearing locations.
Keywords: Calcium phosphate scaffold; Hydroxyapatite; Chitosan; Strength; Bone tissue engineering; Biocompatibility;

Sol–gel processing was used to coat titanium substrates with hydroxyapatite (HA), TiO2, and poly(dl-lactic-glycolic acid). Coating surface characteristics were analyzed with XRD, EDS, AFM, SEM, and water contact angle measurements which indicated that the coatings had a high degree of crystallinity and good resistance to cracking. Coatings were also evaluated by cytocompatibility testing with osteoblast-like cells (or bone-forming cells). The cytocompatibility of the HA composite coatings prepared in the present in vitro study was compared to that of a traditional plasma-sprayed HA coating. Results showed that osteoblast-like cell adhesion was promoted on the novel HA sol–gel coating compared to the traditional plasma-sprayed HA coating. In addition, hydrothermal treatment of the sol–gel coating improved osteoblast-like cell adhesion. Since osteoblast adhesion is a necessary prerequisite for subsequent formation of bone, these results provided evidence that hydrothermally sol–gel processed HA may improve bonding of titanium implants to juxtaposed bone and, thus, warrants further investigation.
Keywords: Hydroxyapatite; Coating; Osteoblast-like cells; Sol–gel processing; Plasma spraying;

This paper seeks to examine the effects of electron beam (e-beam) radiation on biodegradable polymers (PLGA and PLLA), and to understand their radiation-induced degradation mechanisms. PLGA (80:20) and PLLA polymer films were e-beam irradiated at doses from 2.5 to 50 Mrad and the degradation of these films were studied by measuring the changes in their molecular weights, FTIR spectra, thermal and morphological properties. The dominant effect of e-beam irradiation on both PLGA and PLLA is chain scission. Chain scission occurs first through scission of the polymer main chain, followed by hydrogen abstraction. Chain scission, though responsible for the reduction in the average molecular weight, T c,T g and T m of both polymers, encourages crystallization in PLGA. PLLA also undergoes chain scission upon irradiation but to a lesser degree compared to PLGA. The higher crystallinity of PLLA is the key factor in its greater stability to e-beam radiation compared to PLGA. A linear relationship is also established between the decrease in molecular weight with respect to radiation dose.
Keywords: Polyglycolic acid; Polylactic acid; Electron beam; Degradation; Cross-linking; Free radical;

During initial stages of wound healing, fibrin clots provide a three-dimensional scaffold that induces cell infiltration and regeneration. Here, L1Ig6, a ligand for αvβ3 integrin was covalently incorporated within fibrin matrices to explore it as a matrix-immobilized angiogenic factor. Incorporation at concentrations greater than 1 μg/ml reduced the fibrin crosslink density, as reflected by measurements of elastic modulus and swelling. The influence of crosslink density on endothelial cell process extension was characterized by modulating factor XIII concentrations in the coagulation mixture. At low incorporated concentrations of L1Ig6, it was possible to compensate gel elastic modulus via increased factor XIII, but not at high concentrations of L1Ig6. Similar findings were found when matrix swelling was analyzed. Fibrin crosslink density strongly influenced endothelial cell process extension, fewer and shorter processes were observed at high crosslink density. Matrix metalloproteinases (MMPs) were required for process extension and zymography and Western blots identified MMP-2 but not MMP-9. The amount of active MMP-2 increased for endothelial cells cultured in native and L1Ig6-modified matrices or when stimulated with VEGF-A165. The data indicate that distinct matrix properties can be tailored such that they become biologically stimulating and respond to cellular proteolytic activities, being a prerequisite for potential use of such matrices in biomedical applications.
Keywords: Fibrin; Hydrogels; Mechanical properties; Matrix metalloproteinases; Endothelial cell; Angiogenesis;

Photo-immobilization of a phospholipid polymer for surface modification by Tomohiro Konno; Hirokazu Hasuda; Kazuhiko Ishihara; Yoshihiro Ito (1381-1388).
A photo-reactive polymer having a phospholipid polar group was prepared, and the polymer was photo-immobilized on polymeric surfaces, where its interactions with biocomponents were investigated. By using a photo-immobilization method, the polymer was used for surface modification of polyethylene and polypropylene, polymers whose surfaces were not treated in our previous development of the phosphorylcholine-derived polymer. The photo-reactive polymer was synthesized by a coupling reaction involving copolymer consisting of 2-methacryloyloxyethyl phosphorylcholine and methacrylic acid with 4-azidoaniline. When the polymer was unpattern immobilized on the surface, X-ray photo-electron spectroscopic analysis and static contact angle measurements were performed. It was shown that the surface was covered with phospholipid polar groups. Micropattern immobilization was carried out using a micropatterned photo-mask. Measurements using atomic force microscopy showed that the swelled micropatterned polymer was five times as thick as the dried one. Protein adsorption and platelet adhesion were reduced on the polymer-immobilized regions. Mammalian cells did not adhere, and formed aggregates on the immobilized regions. In conclusion, the photo-reactive phospholipid polymer was covalently immobilized on the conventional polymer surfaces and it tended to reduce interactions with proteins and cells.
Keywords: Photo-immobilization; Phospholipid polymer; Surface modification; Protein adsorption; Cell adhesion;

The tissue response to an alkylene bis(dilactoyl)-methacrylate bone adhesive by Christian Heiss; Niels Hahn; Sabine Wenisch; Volker Alt; Peter Pokinskyj; Uwe Horas; Olaf Kilian; Reinhard Schnettler (1389-1396).
Gluing is an attractive technique to fix small bone fragments. However, to date no bone adhesive could be established successfully for all day clinical use.The purpose of this experimental study was to investigate the biocompatibility of a new bone glue based on alkylene bis(dilactoyl)-methacrylate in 36 rabbits. Monocondylar osteotomy of the distal femur was performed and bone glue was applied into the osteotomy gap in 24 rabbits. The remaining 12 animals served as controls. In all rabbits the osteotomy was subsequently stabilized by K-wire osteosynthesis. Six animals of the glue group and 3 controls were euthanized after 7, 21, 42, and 84 days, respectively. Fracture healing and degradation pattern of the glue was studied using histological, histomorphometrical, scanning electron microscopical, and radiological methods.Good resorption of the glue by mononuclear and multinucleated giant cells without prolonged inflammatory processes was observed in the glue group. Histomorphometrical analysis did not reveal any significant differences in fracture healing between the glue and control group at any time. Complete remodelling of the former osteotomy gap was found in all rabbits after 84 days.This bioresorbable bone adhesive exhibited good biocompatibility and its degradation did not interfere with physiological fracture healing.
Keywords: Bone adhesive; Polymethylmethacrylate; Biocompatibility; Degradation; Bone remodelling; Histomorphometry;

Material-specific thrombin generation following contact between metal surfaces and whole blood by Jaan Hong; Andris Azens; Kristina Nilsson Ekdahl; Claes Göran Granqvist; Bo Nilsson (1397-1403).
Little is known about the blood compatibility of metals used in various medical devices. We have previously shown that titanium and derivatives thereof are among the most thrombogenic materials which may explain its outstanding osteointegrating properties. The aim of the present study was to characterize the thrombogenic and complement-activating properties of various metals used today in medical applications.Polyester chips were coated with 50- to 100-nm thick layers of aluminium, iridium, indium, nickel, tantalum, tin, titanium, or zirconium using magnetron sputtering. The metal-coated chips were then incubated in direct contact with whole blood in an in vitro chamber model, and the blood was then analyzed for platelet counts, thrombin-antithrombin (AT), fXIIa-AT, fXIa-AT and fXIIa-C1INH complexes and the complement parameters C3a and sC5b-9.Titanium, tantalum and indium were found to exhibit pronounced thrombogenic properties, whereas aluminium, nickel and, in particular, iridium were essentially non-thrombogenic. Tin and zirconium were intermediate activators. All metals activated complement to a similar degree, with the exception of aluminium, which had more pronounced activating properties.This study clearly indicates that metals indeed have varying thrombogenic and complement activating properties. These studies have implications for the selection of metals intended for medical applications.
Keywords: Metals; Hemocompatibility; Thrombogenicity; Platelet activation; Coagulation; Complement;

Mechano-active tissue engineering of vascular smooth muscle using pulsatile perfusion bioreactors and elastic PLCL scaffolds by Sung In Jeong; Jae Hyun Kwon; Jin Ik Lim; Seung-Woo Cho; Youngmee Jung; Won Jun Sung; Soo Hyun Kim; Young Ha Kim; Young Moo Lee; Byung-Soo Kim; Cha Yong Choi; Soo-Ja Kim (1405-1411).
Blood vessels are subjected in vivo to mechanical forces in a form of radial distention, encompassing cyclic mechanical strain due to the pulsatile nature of blood flow. Vascular smooth muscle (VSM) tissues engineered in vitro with a conventional tissue engineering technique may not be functional, because vascular smooth muscle cells (VSMCs) cultured in vitro typically revert from a contractile phenotype to a synthetic phenotype. In this study, we hypothesized that pulsatile strain and shear stress stimulate VSM tissue development and induce VSMCs to retain the differentiated phenotype in VSM engineering in vitro. To test the hypothesis, rabbit aortic smooth muscle cells (SMCs) were seeded onto rubber-like elastic, three-dimensional PLCL [poly(lactide-co-caprolactone), 50:50] scaffolds and subjected to pulsatile strain and shear stress by culturing them in pulsatile perfusion bioreactors for up to 8 weeks. As control experiments, VSMCs were cultured on PLCL scaffolds statically. The pulsatile strain and shear stress enhanced the VSMCs proliferation and collagen production. In addition, a significant cell alignment in a direction radial to the distending direction was observed in VSM tissues exposed to radial distention, which is similar to that of native VSM tissues in vivo, whereas VSMs in VSM tissues engineered in the static condition randomly aligned. Importantly, the expression of SM α-actin, a differentiated phenotype of SMCs, was upregulated by 2.5-fold in VSM tissues engineered under the mechano-active condition, compared to VSM tissues engineered in the static condition. This study demonstrates that tissue engineering of VSM tissues in vitro by using pulsatile perfusion bioreactors and elastic PLCL scaffolds leads to the enhancement of tissue development and the retention of differentiated cell phenotype.
Keywords: Vascular tissue engineering; Pulsatile perfusion bioreactor; Elastic poly-(l-lactide-co-ε-caprolactone) scaffold; Smooth muscle cell;

Formation of melanocyte spheroids on the chitosan-coated surface by Sung-Jan Lin; Shiou-Hwa Jee; Wen-Chu Hsaio; Shu-Jen Lee; Tai-Horng Young (1413-1422).
The search for biocompatible materials that can maintain function of melanocytes as the cellular patch is a feasible alternative for use in the autologous melanocyte transplantation for vitiligo. In this study, we demonstrated that the surface of chitosan-coated polystyrene wells supported the growth and phenotype expression of melanocytes. Depending on the seeding density and culture time, melanocytes were monolayered or spheroidal in morphology. At seeding densities above 10×103  cells/cm2, human melanocytes started to aggregate on the surface of chitosan after 2 days in culture. These aggregates grew into compact melanocyte spheroids on day 3 and more melanocyte spheroids were observed when a higher seeding density was used. Cells remained viable in the spheroids and grew into dendritic melanocytes when they were reinoculated on polystyrene wells. Conversely, the time for the formation of melanocyte spheroids needed a longer period at lower seeding density. For example, melanocytes at as low as 1.25×103  cells/cm2 did not aggregate until the 20th day of culture. In order to interpret the phenomenon further, we proposed the formation of melanocyte spheroids on the chitosan is mediated by a balance between two competing forces: the interactions of cell–chitosan and cell–cell.
Keywords: Melanocytes; Spheroids; Chitosan; Cell density;

Microcontact printed polymeric substrates were evaluated for their ability to control Schwann cell attachment and direct proliferation, as Schwann cell guidance is a crucial factor in directing peripheral nerve regeneration. Elastomeric stamps of poly(dimethylsiloxane) were “inked” with laminin, a permissive protein for Schwann cell adhesion, and stamped onto poly(methyl methacrylate) substrates to create patterns of lines and intervals varying from 10 to 50 μm wide. Schwann cells were seeded onto the substrates in serum-free media. After 4 h, media was replaced with serum-containing growth media and changed daily thereafter. The addition of growth media to stimulate proliferation initially caused some loss in cell orientation relative to the laminin pattern, but when monolayer formation was complete, a high degree of cell orientation was observed. As both cell–cell contacts and surface coverage were maximized, the Schwann cells achieved an even higher order of orientation than observed during the early stages of proliferation. Significantly, smaller pattern widths increased the degree of orientation, regardless of interval width. Our results indicate that patterned polymeric substrates may enhance peripheral nerve regeneration by creating a highly ordered Schwann cell matrix for guidance of neurons.
Keywords: Micropatterning; Schwann cells; Cell proliferation; Protein adsorption;

Production and characterization of ECM powder: implications for tissue engineering applications by Thomas W. Gilbert; Donna Beer Stolz; Frank Biancaniello; Abby Simmons-Byrd; Stephen F. Badylak (1431-1435).
Two methods to produce a particulate form of extracellular matrix (ECM) from porcine urinary bladder were investigated. One method to produce the powder involved snap freezing a lyophilized form of the material and then pulverizing it in a grinding mill. The second method was similar except that the ECM was saturated in a solution of NaCl prior to snap freezing to precipitate salt crystals within the matrix before grinding. Several methods were utilized to analyze the particle size distribution and ultrastructure including sonic sifting, laser diffraction, and scanning electron microscopy (SEM). The salt precipitation method yielded a more uniform distribution of particles with a smaller mean diameter (158 vs. 191 μm). SEM showed that the particles produced by grinding without salt precipitation were irregularly shaped, sheet-like particles. ECM particles produced by the salt precipitation method were round and porous in appearance with many particles in the range of 1 μm which tended to agglomerate with the larger particles and with each other. We conclude that the production of a comminuted form of ECM is possible and that the uniformity of particle size and shape are dependent upon the manufacturing methodology.
Keywords: Extracellular matrix; Urinary bladder matrix; Powder; Particle size analysis; Scanning electron microscopy; Tissue engineering;

Hemocompatibility and anaphylatoxin formation of protein-immobilizing polyacrylonitrile hemodialysis membrane by Ting-Yu Liu; Wen-Ching Lin; Li-Ying Huang; San-Yuan Chen; Ming-Chien Yang (1437-1444).
Plasma proteins were covalently immobilized onto polyacrylonitrile (PAN) membrane to evaluate the hemocompatibility and anaphylatoxin formation. This is used as a model to study the effect of protein-adsorption on the blood-contacting response of hemodializing membranes. The proteins used were either platelet-adhesion-promoting collagen (COL) or platelet-adhesion-inhibiting human serum albumin (HSA). The microstructure and characterization of the protein-immobilizing PAN membranes were evaluated by Coomassie dye assay, atomic force microscopy, X-ray photoelectron spectroscopy and water contact angle measurement. PAN–HSA membrane improved not only hemocompatibility including less platelet adhesion, longer blood coagulation times, and higher thrombin inactivity level, but also induced lower complement activation. On the other hand, PAN–COL membrane exhibited blood incompatibility, although induced less increase of C3, C4 antigens of serum. Overall results of this study demonstrated that the immobilization of HSA onto the surface of PAN membrane would be beneficial to improve the hemocompatibility and to reduce the anaphylatoxin formation during hemodialysis.
Keywords: Polyacrylonitrile; Hemocompatibility; Platelet adhesion; Anaphylatoxin formation; Leukopenia;

Localisation of osteogenic and osteoclastic cells in porous β-tricalcium phosphate particles used for human maxillary sinus floor elevation by Ilara R. Zerbo; Antonius L.J.J. Bronckers; Gert de Lange; Elisabeth H. Burger (1445-1451).
We and others have shown earlier that porous β-tricalcium phosphate (TCP) (Cerasorb®) can be used in patients to augment the maxillary sinus floor prior to placement of oral dental implants. To better understand the transformation of TCP particles into bone tissue, we analyse here the appearance of cells with osteogenic or osteoclastic potential in relation to these particles. In biopsies taken at 6 months after sinus floor augmentation we observed bone growth into the TCP particles but also replacement by soft connective tissue. To identify possible osteoprogenitor cells in this tissue, histological sections were immunostained with an antibody to Runx2/Cbfa1, an essential and early transcription factor for osteoblast differentiation. The osteogenic potential of cells was further confirmed by immunostaining for bone sialoprotein (BSP) and osteopontin (OPN). Other sections were stained for Tartrate Resistant Acid Phosphatase (TRAP) activity to identify cells with osteoclastic capacity.Runx2/Cbfa1 positive connective tissue cells were found in abundance throughout and around the TCP particles, even at a distance of several millimetres from the maxillary bone surface. About 95% of the cells found within TCP particles stained positive for Runx2/Cbfa1. Fewer cells stained positive for BSP and OPN, suggesting more mature osteoblastic properties. Mono- and binucleate TRAP-positive cells, but no multinucleate TRAP-positive osteoclasts, were found in the soft tissue infiltrating the TCP and at the surface of the TCP particles. Both the Runx2/Cbfa1 positive and the TRAP-positive cells decreased apically with increasing vertical distance from the maxillary bone surface.This data suggests that the TCP particles attract osteoprogenitor cells that migrate into the interconnecting micropores of the bone substitute material by 6 months. The lack of large multinucleate TRAP positive cells suggests that resorption of the TCP material by osteoclasts plays only a minor role in its replacement by bone. Chemical dissolution, possibly favoured by a high cell metabolism in the particles, seems the predominant cause of TCP degradation. The abundance of Runx2/Cbfa1 positive cells would indicate that with a greater time of healing there will be further bone deposition into these particles.
Keywords: Immunochemistry; Bone regeneration; Bone ingrowth;