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Biomaterials (v.27, #33)

Editorial board (pp. co2).
Calendar (pp. i).

The effect of water uptake on the behaviour of hydrophilic cements in confined environments by L.F. Luciano F. Boesel; Rui L. Reis (pp. 5627-5633).
Physiological fluids will be in contact with the implant components from the first moments after a surgery. Therefore, the study of the effect of water on the properties of the bone cements that are part of the arthroplasty procedure is of critical importance to predict the long-term performance of the whole system. In our research group, we have developed a novel concept, the hydrophilic, partially degradable and bioactive cements which uptake considerably more water than standard bone cements. In this paper, we aimed to study the effect of water uptake (WU) by these cements on their behaviour. The tests were carried out in confined cavities, which represent more accurately the in vivo situation the cement will face (constrained by the bone and prosthesis surfaces). We observed that the equilibrium WU decreased up to 60% (as compared to non-confined situations), depending of the formulation. This decrease resulted in a latent tendency of the cements to swell, and the hindering of such swelling generated a swelling pressure against the constraining walls. The pressure, and consequent press-fitting effect, could be controlled by a number of mechanisms, and resulted in higher stability of the hydrophilic cements, expressed as an increase in the push-out force, required to extract the specimens from such constrained cavities. This effect was only observed in hydrophilic cements, not in commercial, hydrophobic ones used as controls. We conclude that such cements will provide an additional and very useful source of immediate adhesion in the short-term after surgery: water induced press fitting.

Keywords: Press-fitting; Swelling; Acrylic cements; Starch; Interfaces

Granulocyte-based immune response against decellularized or glutaraldehyde cross-linked vascular tissue by Erwin Rieder; Anneliese Nigisch; Barbara Dekan; M.-T. Marie-Theres Kasimir; Muhlbacher Ferdinand Mühlbacher; Ernst Wolner; Paul Simon; G. Günter Weigel (pp. 5634-5642).
Supporting structures derived from biological tissue have been used in numerous tissue-engineering applications. This study focuses on the immune response of human leukocytes toward decellularized or glutaraldehyde (GA) cross-linked vascular tissue in vitro.Porcine and human pulmonary roots were sterilized with antibiotics, decellularized or cross-linked with GA. Proteins of the vascular tissue were extracted and the migratory response of human leukocytes toward protein extracts was examined using an in vitro migration chamber. Transmigrated leukocytes were counted and subsets (lymphocytes, monocytes, granulocytes) analyzed by flow cytometry.Decellularization significantly reduced the migration of monocytes compared to native porcine tissue. Although the proportion of transmigrating lymphocytes was much lower, decellularization again reduced the migratory response. Surprisingly, after decellularization granulocyte migration was still significantly higher than the negative control. Results comparable to those obtained with porcine material were found when human tissue was used for the experiments. Interestingly, migratory behavior toward extracts of GA-fixed porcine tissue was similar to that of decellularized specimens.We have shown that decellularization of vascular tissue reduces lymphocyte and monocyte recruitment comparable to cross-linking treatment. However, the migration of granulocytes, which are also known to be strongly involved in early inflammatory reactions, could be abolished neither by decellularization nor by fixation with GA.

Keywords: Tissue engineering; Immune response; ECM; Leukocytes

Procoagulant activity of surface-immobilized Hageman factor by Kaushik Chatterjee; Erwin A. Vogler; Christopher A. Siedlecki (pp. 5643-5650).
Procoagulant activity of surface-immobilized coagulation factor XIIa (activated Hageman factor) is reported. Activity of FXIIa immobilized onto the surfaces of three silanized-glass procoagulants spanning a wide range of wettability was assayed in normal and FXII-deficient plasmas. Previously published mathematical models were used to characterize the procoagulant activity of protein-immobilized materials and soluble enzymes. Results show that FXIIa activity is unrelated to underlying procoagulant surface chemistry and is similar to soluble FXIIa activity. The uninfluential role of the surface on FXIIa suggests that the solid surface activates FXII in biomaterial-induced blood coagulation but is not otherwise involved in FXIIa activity as described by the classical mechanism.

Keywords: Coagulation; Blood compatibility; Plasma proteins

Proliferation and osteoblastic differentiation of human bone marrow-derived stromal cells on akermanite-bioactive ceramics by Hongli Sun; Chengtie Wu; Kerong Dai; Jiang Chang; Tingting Tang (pp. 5651-5657).
In the present study, the effects of a calcium magnesium silicate bioactive ceramic (akermanite) on proliferation and osteoblastic differentiation of human bone marrow stromal cells (hBMSC) have been investigated and compared with the classical ceramic ( β-tricalcium phosphate, β-TCP). Akermanite and β-TCP disks were seeded with hBMSC and kept in growth medium or osteogenic medium for 10 days. Proliferation and osteoblastic differentiation were evaluated on day 1, 4, 7 and 10. The data from the Alamar Blue assay and lactic acid production assay showed that hBMSC proliferated more significantly on akermanite than on β-TCP. The analysis of osteoblast-related genes, including alkaline phosphatase (ALP), osteopontin (OPN), bone sialoprotein (BSP) and osteocalcin (OC), indicated that akermanite ceramics enhanced the expression of osteoblast-related genes, but type I collagen (COL I) showed no noticeable difference among akermanite and β-TCP ceramics. Furthermore, this stimulatory effect was observed not only in osteogenic medium, but also in normal growth medium without osteogenic reagents such asl-ascorbic acid, glycerophosphate and dexamethasone. This result suggests that akermanite can promote osteoblastic differentiation of hBMSC in vitro even without osteogenic reagents, and may be used as a bioactive material for bone regeneration and tissue engineering applications.

Keywords: Akermanite; β; -tricalcium phosphate; Osteoblastic differentiation

The performance of human dental pulp stem cells on different three-dimensional scaffold materials by Weibo Zhang; X. Frank Walboomers; Toin H. van Kuppevelt; Willeke F. Daamen; Zhuan Bian; John A. Jansen (pp. 5658-5668).
The aim of this study was to investigate the in vitro and in vivo behavior of human dental pulp stem cells (DPSCs) isolated from impacted third molars, when seeded onto different 3-dimensional (3-D) scaffold materials: i.e. a spongeous collagen, a porous ceramic, and a fibrous titanium mesh. Scaffolds were loaded with DPSC, and subsequently divided into two groups. The first group was cultured in osteogenic differentiation medium in vitro for 4 weeks. The second group of samples was implanted subcutaneously in nude mice for 6 or 12 weeks. Samples cultured in vitro were analyzed by scanning electron microscopy and RT-PCR for dentin sialophosphoprotein (DSPP) expression. In vivo samples were evaluated by histology, RT-PCR and immunohistochemistry. The results indicated that in vitro, cells developed abundant deposition of mineralized extracellular matrix (ECM) with expression of DSPP in all 3-D materials. The simultaneous implantation experiment showed formation of tissue that was DSPP positive in all three scaffolds materials. However, the aspect of the formed tissues in all scaffolds resembled more connective tissue than a dentin-like tissue. Limited calcification of the ECM was only seen in the ceramic scaffold. In both experiments, no other differences could be attributed to the different materials used. In conclusion, the in vivo behavior of DPSC and their relations with 3-D scaffold materials should be further studied before clinical use can be considered.

Keywords: Dentine; Stem cell; Titanium; Collagen; Calcium phosphate

3D hepatocyte monolayer on hybrid RGD/galactose substratum by Yanan Du; Ser-mien Chia; Rongbin Han; Shi Chang; Huihuan Tang; Hanry Yu (pp. 5669-5680).
Hepatocyte-based applications such as xenobiotics metabolism and toxicity studies usually require hepatocytes anchoring onto flat substrata that support their functional maintenance. Conventional cell culture plates coated with natural matrices or synthetic ligands allow hepatocytes to adhere tightly as two-dimensional (2D) monolayer but these tightly anchored hepatocytes rapidly lose their differentiated functions. On galactosylated substrata, hepatocytes adhere loosely; and readily form three-dimensional (3D) spheroids that can maintain high levels of cellular functions. These spheroids detach easily from the substrata and exhibit poor mass transport properties unsuitable for many applications. Here, we have developed a hybrid RGD/galactose substratum based on polyethylene terephthalate film conjugated with both RGD peptide and galactose ligand to enhance cell adhesion and functions synergistically. Primary hepatocytes adhere effectively onto the transparent hybrid substratum in 96-well plates as monolayer while exhibiting high levels of liver-specific functions, morphology and cell–cell interactions typically seen in the 3D hepatocyte spheroids. The hepatocytes cultured onto the hybrid substratum also exhibit high levels of sensitivity to a model drug acetaminophen similar to the 3D hepatocyte spheroids. The monolayer of hepatocytes exhibiting the 3D cell behaviors on this flat hybrid substratum can be useful for various applications requiring both effective mass transfer and cellular support.

Keywords: Surface modification; RGD peptide; Galactosylation; Hybrid biomaterial; Hepatocyte; Hepatotoxicity

Effect of fiber diameter and orientation on fibroblast morphology and proliferation on electrospun poly(d,l-lactic- co-glycolic acid) meshes by Chris A. Bashur; Linda A. Dahlgren; Aaron S. Goldstein (pp. 5681-5688).
Engineered ligament tissues are promising materials for the repair of tears and ruptures, but require the development of biomaterial scaffolds that not only support physiologically relevant loads, but also possess architectures capable of orienting cell adhesion and extracellular matrix deposition. Based on evidence that micron-scale topographic features induce cell orientation through a contact guidance phenomenon, we postulate that oriented micron-scale fiber meshes—formed by the electrospinning process—can regulate cell morphology. To test this, fused fiber meshes of poly(d,l-lactic- co-glycolic acid) (PLGA) were electrospun onto rigid supports under conditions that produced mean fiber diameters of 0.14–3.6μm, and angular standard deviations of 31–60°. Analysis of the morphology of adherent NIH 3T3 fibroblasts indicated that projected cell area and aspect ratio increased systematically with both increasing fiber diameter and degree of fiber orientation. Importantly, cell morphology on 3.6μm fibers was similar to that on spincoated PLGA films. Finally, cell densities on electrospun meshes were not significantly different from spincoated PLGA, indicating that cell proliferation is not sensitive to fiber diameter or orientation.

Keywords: Ligament; Fibroblast; Electrospin; Cell adhesion; Cell proliferation; Cell morphology

The effect of gelatin–chondroitin sulfate–hyaluronic acid skin substitute on wound healing in SCID mice by Tzu-Wei Wang; Jui-Sheng Sun; Hsi-Chin Wu; Yang-Hwei Tsuang; Wen-Hsi Wang; Feng-Huei Lin (pp. 5689-5697).
Tissue-engineered skin substitutes provided a feasibility to overcome the shortage of skin autograft by culturing keratinocytes and dermal fibroblasts in vitro. In this study, we applied bi-layer gelatin–chondrointin-6-sulfate–hyaluronic acid (gelatin–C6S–HA) biomatrices onto the severe combined immunodeficiency (SCID) mice to evaluate its effect on promoting wound healing. Human foreskin keratinocytes and dermal fibroblasts were cultured with reconstructed skin equivalent (rSE) for 7 days. The rSE was then grafted to the dorsum of SCID mice to evaluate its biocompatibility by histologic and immunohistochemistry analysis. The results showed that human epidermis were well-developed with the expression of differentiated markers and basement membrane-specific proteins at 4 weeks. After implantation, the percentages of skin graft take were satisfactory, while cell-seeded group was better than non-cell-seeded one. The basement membrane proteins including laminin, type IV collagen, type VII collagen, integrin α6, and integrin β4 were all detected at the dermal–epidermal junction, which showed a continuous structure in the 4 weeks after grafting. This bi-layer gelatin–C6S–HA skin substitute not only has positive effect on promoting wound healing, but also has high rate of graft take. This rSE would have the potential to be applied on the extensively and deeply burned patients who suffer from severe skin defect in the near future.

Keywords: Wound healing; Transplantation; Biomimetic material

Elastogenic effects of exogenous hyaluronan oligosaccharides on vascular smooth muscle cells by Binata Joddar; Anand Ramamurthi (pp. 5698-5707).
Prior studies suggest that hyaluronan (HA), a glycosaminoglycan, may upregulate innately poor elastin matrix synthesis by adult vascular smooth muscle cells (SMCs). HA scaffolds could thus be useful to regenerate damaged vascular elastin. In an earlier study, we established that the elastogenic effects of non-oligomeric HA are fragment size- and/or dose-specific. We currently investigate the pro-elastogenic effects of exogenous HA oligomers on rat aortic smooth muscle cells (RASMCs). RASMCs were cultured with pure HA oligomers (4-mers) and mixtures (4–8mers) obtained by enzymatic digestion of long-chain HA (MW ∼2000kDa). Polyacrylamide gel electrophoresis (PAGE)/ Matrix Assisted Laser Desorption/ Ionization Spectroscopy Time- Of- Flight Analysis (MALDI-TOF) showed HA digestates to contain a mixture of 4–8mers with a predominance of 4-mers (75±0.4% w/w). Cell layers supplemented with both pure HA 4-mers or oligomer mixtures showed proliferation levels similar to non-HA controls over 21 days of culture. Pure 4-mers and oligomer mixtures enhanced DNA-normalized output of tropoelastin by 1.6 and 1.8 times, respectively, and that of matrix elastin by ∼2.7 times relative to controls. Sodium dodecyl sulfate (SDS)-PAGE/Western Blot and a desmosine assay semi-quantitatively confirmed the observed biochemical trends for tropoelastin and matrix elastin, respectively. HA oligomers induced enhanced synthesis of the elastin crosslinker, desmosine, and appeared to stabilize the elastin matrix by suppression of elastin–laminin receptor (ELR) activity relative to controls. Transmission electron micrographs (TEMs) showed elastin deposits within oligomer-supplemented cultures to be distinct, longitudinally oriented, aggregating fibrils, and clumps, and to be less abundant and mostly amorphous in controls. HA oligomers preserved normal fibrillin-mediated elastin matrix deposition. Results suggest that HA oligos are highly pro-elastogenic, promote elastin fibril formation, and stabilize elastin matrix and may thus be usefully incorporated into scaffolds for guided elastin regeneration.

Keywords: Glycosaminoglycans; Hyaluronan; Oligomers; Regeneration; Extracellular Matrix

The effect of collagen-targeting platelet-derived growth factor on cellularization and vascularization of collagen scaffolds by Hang Lin; Bing Chen; Wenjie Sun; Wenxue Zhao; Yannan Zhao; Jianwu Dai (pp. 5708-5714).
Platelet-derived growth factor BB (PDGF-BB) was an important inductive factor during wound healing, but the lack of efficient delivery system limited its clinical application. Here, a peptide of seven amino acids was firstly utilized to engineer PDGF-BB to construct a collagen-targeting system. This peptide functioned as collagen-binding domain (CBD) to specially target the PDGF-BB to the collagen and restrict its diffusion. In our study, in vitro activity assay showed that the CBD-fused PDGF (CBD-PDGF) and native PDGF (NAT-PDGF) possessed similar activity to stimulate the human fibroblast proliferation. As expected, this peptide promoted the binding of PDGF to collagen scaffolds, and CBD-PDGF caused more cells to proliferate on the collagen gel than NAT-PDGF when the same amounts of PDGF were added. In the in vivo experiment, CBD-PDGF-loaded collagen scaffolds were uniformly cellularized and vascularized compared to that of NAT-PDGF-loaded scaffolds. Our study demonstrated that the CBD targeted PDGF to the collagen scaffold to exert its functions, and it suggested this could be an effective system for targeting tissue regeneration and wound repair.

Keywords: Platelet-derived growth factor BB (PDGF-BB); Collagen; Collagen-targeting system; Collagen-binding domain (CBD)

The generation of germline transgenic silkworms for the production of biologically active recombinant fusion proteins of fibroin and human basic fibroblast growth factor by Rika Hino; Masahiro Tomita; Katsutoshi Yoshizato (pp. 5715-5724).
We generated germline transgenic silkworms bearing a fibroin light chain (FL) promoter-driven FL gene whose 3′-end was flanked with human basic fibroblast growth factor (bFGF) gene, FL/bFGF gene. The cocoons from transgenic worms were trypsinized to remove sericin layers, and treated with solution containing CaCl2, ethanol, and water at a molar ratio of 1:2:8 (CaCl2/ethanol/water) to solubilize fibroin layers. Western blot analysis showed that the recombinant protein, r(FL/bFGF), was solubilized with CaCl2/ethanol/water, but not with trypsin, indicating that r(FL/bFGF) was in fibroin layers. Thus, it was concluded that the worms spun cocoons whose fibroin layers were composed of the inherent gene-derived natural fibroin (nF) and r(FL/bFGF). The mixture of nF and r(FL/bFGF) was dubbed r(FL/bFGF)nF. The solubilized r(FL/bFGF)nF was refolded using the glutathione redox system. Human umbilical vein endothelial cells (HUVECs) grew in the refolded r(FL/bFGF)nF-containing culture media, showing that bFGF in r(FL/bFGF) was biologically active. r(FL/bFGF)nF immobilized on a culture dish also supported the growth of HUVECs in bFGF-free media, suggesting the usefulness of r(FL/bFGF)nF as a new biomaterial for tissue engineering. The currently developed transgenic silkworms will be suitable for mass production of fibroins bearing a variety of biological activities.

Keywords: Silk; Transgenic silkworms; Human fibroblast growth factor; Bioactive fibroin; Scaffold; Human umbilical vein endothelial cells

Synthesis and in vitro anti-cancer evaluation of tamoxifen-loaded magnetite/PLLA composite nanoparticles by F.X. Hu; K.G. Neoh; E.T. Kang (pp. 5725-5733).
The present study deals with the synthesis and characterization of tamoxifen-loaded magnetite/poly(l-lactic acid) composite nanoparticles (TMCN), and their in vitro anti-cancer activity against MCF-7 breast cancer cells. The composite nanoparticles with an average size of ∼200nm, were synthesized via a solvent evaporation/extraction technique in an oil/water emulsion. The superparamagnetic property (saturation magnetization value of ∼7emu/g) of the TMCN is provided by Fe3O4 nanoparticles of ∼6nm encapsulated in the poly(l-lactic acid) matrix. The encapsulation efficiency of the Fe3O4 and tamoxifen as a function of the concentration in the organic phase was investigated. The uptake of TMCN and tamoxifen by MCF-7 was estimated from the intracellular iron concentration. After 4h incubation of MCF-7 with TMCN, significant changes in the cell morphology were discernible from phase contrast microscopy. Cytotoxicity assay shows that while the Fe3O4-loaded poly(l-lactic acid) composite nanoparticles exhibit no significant cytotoxicity against MCF-7, ∼80% of the these cells were killed after incubation for 4 days with TMCN.

Keywords: Composite nanoparticles; Magnetic drug targeting; Controlled release; Tamoxifen; MRI

Combined topographical and chemical micropatterns for templating neuronal networks by Jiayi Zhang; Sowmya Venkataramani; Heng Xu; Y.-K Yoon-Kyu Song; H.-K. Hyun-Kon Song; G. Tayhas R. Palmore; Justin Fallon; Arto V. Nurmikko (pp. 5734-5739).
In vitro neuronal networks with geometrically defined features are desirable for studying long-term electrical activity within the neuron assembly and for interfacing with external microelectronic circuits. In standard cultures, the random spatial distribution and overlap of neurites makes this aim difficult; hence, many recent efforts have been made on creating patterned cellular circuits. Here, we present a novel method for creating a planar neural network that is compatible with optical devices. This method combines both topographical and chemical micropatterns onto which neurons can be cultured. Compared to other reported patterning techniques, our approach and choice of template appears to show both geometrical control over the formation of specific neurite connections at low plating density and compatibility with microelectronic circuits that stimulate and record neural activity.

Keywords: Neural network; Micropatterning; Cell culture; Photolithography; Bioelectronic interface

Hydroxyl-containing antimony oxide bromide nanorods combined with chitosan for biosensors by Xianbo Lu; Zhenhai Wen; Jinghong Li (pp. 5740-5747).
A hydroxyl-containing antimony oxide bromide (AOB) nanorods was synthesized by a hydrothermal method. TEM and SEM images showed that the as-prepared AOB nanorods were very copious with diameters of about 50nm. The AOB nanorods could be easily combined with biopolymer chitosan (Chi) to form an organic–inorganic hybrid material, and a biocompatible, crack-free and porous Chi–AOB composite film could be readily obtained. Horseradish peroxidase (HRP) was chosen as a model protein to construct a reagentless mediator-free third-generation HRP biosensor. UV–visible and FTIR spectroscopy revealed that HRP entrapped in the composite film could retain its native secondary structure. A pair of stable and well-defined redox peaks of HRP with a formal potential of about −0.24V (vs. Ag/AgCl) in a pH 7.0 phosphate-buffered solution (PBS) were obtained at the HRP–Chi–AOB composite film modified glassy carbon (GC) electrode. With advantages of organic–inorganic hybrid materials, dramatically facilitated direct electron transfer of HRP and excellent bioelectrocatalytic activity towards H2O2 were demonstrated. The apparent Michaelis–Menten constantKMapp was calculated to be 7.5μm, indicating that HRP entrapped in the composite film possessed high affinity to H2O2 and exhibited high enzymatic activity. The prepared biosensor displayed good sensitivity and reproducibility, wide linear range, low detection limit, fast response and excellent long-term stability. The Chi–AOB composite film could be used efficiently for the entrapment of other redox-active proteins and may find wide potential applications in biosensors, biocatalysis, biomedical devices and bioelectronics.

Keywords: Biosensor; Direct electron transfer; Antimony oxide bromide; Chitosan; Nanorods; Horseradish peroxidase

Fabrication of hydroxyapatite ultra-thin layer on gold surface and its application for quartz crystal microbalance technique by Akira Monkawa; Toshiyuki Ikoma; Shunji Yunoki; Tomohiko Yoshioka; Junzo Tanaka; Dinko Chakarov; Bengt Kasemo (pp. 5748-5754).
We present a method for coating gold quartz crystal microbalance with dissipation (QCM-D) sensor with ultra-thin layer of hydroxyapatite nanocrystals evenly covering and tightly bound to the surface. The hydroxyapatite layer shows a plate-like morphology and less than 20nm in thickness. The hydroxyapatite sensor operated in liquid with high stability and sensitivity. The in-situ adsorption mechanism and conformational change of fibrinogen on gold, titanium and hydroxyapatite surfaces were investigated by QCM-D technique and Fourier-transform infrared spectroscopy. The change of secondary structures of fibrinogen adsorbed on the surfaces depended on the adsorbed amounts of protein. The secondary structure of fibrinogen adsorbed on the surfaces changes with increasing coverage. This is explained by repulsion among fibrinogens, affecting water structure and thus the strength of fibrinogen interactions on the surface. The study indicates that the hydroxyapatite sensor is applicable for qualitative and conformational analysis of protein adsorption.

Keywords: Hydroxyapatite; QCM-D; FT-IR; Fibrinogen; Adsorption

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