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Biomaterials (v.29, #30)

Editorial board (pp. ifc).

Reduced bacterial adhesion to fibrinogen-coated substrates via nitric oxide release by Gregory W. Charville; Evan M. Hetrick; Carri B. Geer; Mark H. Schoenfisch (pp. 4039-4044).
The ability of nitric oxide (NO)-releasing xerogels to reduce fibrinogen-mediated adhesion of Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli is described. A negative correlation was observed between NO surface flux and bacterial adhesion for each species tested. For S. aureus and E. coli, reduced adhesion correlated directly with NO flux from 0 to 30pmolcm−2s−1. A similar dependence for S. epidermidis was evident from 18 to 30pmolcm−2s−1. At a NO flux of 30pmolcm−2s−1, surface coverage of S. aureus, S. epidermidis, and E. coli was reduced by 96, 48, and 88%, respectively, compared to non-NO-releasing controls. Polymeric NO release was thus demonstrated to be an effective approach for significantly reducing fibrinogen-mediated adhesion of both gram-positive and gram-negative bacteria in vitro, thereby illustrating the advantage of active NO release as a strategy for inhibiting bacterial adhesion in the presence of pre-adsorbed protein.

Keywords: Polymeric nitric oxide release; Bacterial adhesion; Fibrinogen; Antimicrobial

The biocompatibility of mesoporous silicates by Sarah P. Hudson; Robert F. Padera; Robert Langer; Daniel S. Kohane (pp. 4045-4055).
Micro- and nano-mesoporous silicate particles are considered potential drug delivery systems because of their ordered pore structures, large surface areas and the ease with which they can be chemically modified. However, few cytotoxicity or biocompatibility studies have been reported, especially when silicates are administered in the quantities necessary to deliver low-potency drugs. The biocompatibility of mesoporous silicates of particle sizes ∼150nm, ∼800nm and ∼4μm and pore sizes of 3nm, 7nm and 16nm, respectively, is examined here. In vitro, mesoporous silicates showed a significant degree of toxicity at high concentrations with mesothelial cells. Following subcutaneous injection of silicates in rats, the amount of residual material decreased progressively over 3 months, with good biocompatibility on histology at all time points. In contrast, intra-peritoneal and intra-venous injections in mice resulted in death or euthanasia. No toxicity was seen with subcutaneous injection of the same particles in mice. Microscopic analysis of the lung tissue of the mice indicated that death may be due to thrombosis. Although local tissue reaction to mesoporous silicates was benign, they caused severe systemic toxicity. This toxicity might be mitigated by modification of the materials.

Keywords: Silicate; Mesoporous; Biocompatibility; Drug delivery; Controlled drug release

Topographical control of human macrophages by a regularly microstructured polyvinylidene fluoride surface by Nora E. Paul; Claudia Skazik; Marc Harwardt; Matthias Bartneck; Bernd Denecke; Doris Klee; Jochen Salber; Gabriele Zwadlo-Klarwasser (pp. 4056-4064).
In this study we investigated the influence of surface topography on the inflammatory response of human macrophages. We generated different polyvinylidene fluoride (PVDF) surfaces including (i) a smooth surface of PVDF spherulites as a control, (ii) a randomly nanotextured surface with alumina particles, and (iii) a microstructure using laser ablation. The identical chemistry of all PVDF surfaces was demonstrated by X-ray photoelectron spectroscopy. The topography was evaluated by white light interferometry and X-profile analysis. Macrophages were cultured on the different surfaces including lipopolysaccharide (LPS) treatment as an inflammatory activator. Our results demonstrate that the microstructured surface but not the nanotexured significantly affects the activation of primary human macrophages by inducing a specific cytokine and gene expression pattern. This activation resulted in a subtype of macrophages with pro- but also anti-inflammatory properties. Interestingly, the response on the topography differed from that triggered by LPS, pointing to a different activation state of the cells. Our data clearly show that a particular topography induces an inflammatory response. This suggests that the modification of topography could influence the inflammatory potency of a biomaterial and hence could affect the biocompatibility of implants.

Keywords: Microstructure; Nanotopography; Macrophage; Inflammation; Cytokine; Gene expression

Functionally graded electrospun polycaprolactone and β-tricalcium phosphate nanocomposites for tissue engineering applications by Cevat Erisken; Dilhan M. Kalyon; Hongjun Wang (pp. 4065-4073).
Fabricating functionally graded scaffolds from biodegradable polymers to enable the mimicking of native tissue is an important challenge. Here we demonstrate the fabrication and utilization of functionally graded non-woven meshes of polycaprolactone incorporated with tricalcium phosphate nanoparticles using a new hybrid twin-screw extrusion/electrospinning (TSEE) process, which allows the time-dependent feeding of various solid and liquid ingredients and their melting, dispersion, deaeration and pressurization together with electrospinning within the confines of a single process. Using this hybrid method, the concentration of tricalcium phosphate nanoparticles could be tailored to vary in a targeted/controlled manner between the two surfaces of the scaffold mesh. The graded scaffolds were seeded and cultured with mouse preosteoblast cells (MC3T3-E1). Within 4weeks, the tissue constructs revealed the formation of continuous gradations in extracellular matrix with various markers including collagen synthesis and mineralization, akin to the type of variations observed in the typical bone-cartilage interface in terms of the distributions of concentration of Ca particles and of mechanical properties associated with this. The demonstrated hybrid method should allow much better control of the distributions of various ingredients, including the concentrations of drugs/growth factors, as well as the porosity, mechanical property, wettability, biodegradation rate distributions in tissue engineering scaffolds, aiming to mimic the elegant complex distributions found in native tissue.

Keywords: Functionally graded; Extrusion; Electrospinning; Scaffold; Tissue; Interface

The promotion of osteoblastic differentiation of rat bone marrow stromal cells by a polyvalent plant mosaic virus by Gagandeep Kaur; Mani T. Valarmathi; Jay D. Potts; Qian Wang (pp. 4074-4081).
To investigate the role that the micro/nano-environment plays on the differentiation pathway of bone marrow stromal cells (BMSCs) into osteoblasts, we employed a 2D substrate coated with turnip yellow mosaic virus (TYMV) particles. TYMV is a non-enveloped icosahedral plant virus which has an average diameter 28nm and the protein cage structure consists of 180 identical subunits. The temporal effect of TYMV coated substrate on the adhesion and differentiation capacity of the BMSCs was monitored for selected time periods of 7, 14 and 21 days. We examined the gene expression profile of BMSCs cultured in primary media (undifferentiated cells) and cells induced to osteoblast lineage by real time PCR analysis. To further corroborate our findings, we investigated the expression of osteogenic markers using immunohistochemistry and cytochemical staining. As expected, the genes involved in the process of osteogenic differentiation were activated more during the growth of cells under osteogenic media. In addition, we found that the BMSCs induced to undergo osteogenic differentiation on TYMV coated substrates formed fully mineralized nodules comprising of osteoblast-like cells around day 14. Comparing the gene expression pattern of BMSCs induced to osteogenic differentiation under standard culture conditions with the cells induced on TYMV substrates, we found significant differences in the temporal expression and level of expression of several key genes. Our findings indicate that TYMV, as a biogenic nanoparticle, can be employed as a model to modulate the nano-environment of the substrates in order to gain an insight into the role that the micro/nano-environment has in regulating adhesion, growth and differentiation of BMSCs towards osteogenic lineage, which will be vital for designing compatible biomaterials for tissue engineering purposes.

Keywords: Plant virus; Bone marrow stromal cell; Mesenchcymal stem cell; Osteoblast; Nanoparticle; Viral substrate

Multilayer nanofilms as substrates for hepatocellular applications by Corinne R. Wittmer; Jennifer A. Phelps; Christin M. Lepus; William M. Saltzman; Martha J. Harding; Paul R. Van Tassel (pp. 4082-4090).
Multilayer nanofilms, formed by the layer-by-layer (LbL) adsorption of positively and negatively charged polyelectrolytes, are promising substrates for tissue engineering. We investigate here the attachment and function of hepatic cells on multilayer films in terms of film composition, terminal layer, rigidity, charge, and presence of biofunctional species. Human hepatocellular carcinoma (HepG2) cells, adult rat hepatocytes (ARH), and human fetal hepatoblasts (HFHb) are studied on films composed of the polysaccharides chitosan (CHI) and alginate (ALG), the polypeptides poly(l-lysine) (PLL) and poly(l-glutamic acid) (PGA), and the synthetic polymers poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (PSS). The influence of chemical cross-linking following LbL assembly is also investigated. We find HepG2 to reach confluence after 7 days of culture on only 2 of 18 candidate multilayer systems: (PAH–PSS) n (i.e. nPAH–PSS bilayers) and cross-linked (PLL–ALG) n–PLL. Cross-linked PLL–ALG and PLL–PGA films support attachment and function of ARH, independently of the terminal layer, provided collagen is adsorbed to the top of the film. (PAH–PSS) n, cross-linked (PLL–ALG) n, and cross-linked (PLL–PGA) n–PLL films all support attachment, layer confluence, and function of HFHb, with the latter film promoting the greatest level of function at 8 days. Overall, film composition, terminal layer, and rigidity are key variables in promoting attachment and function of hepatic cells, while film charge and biofunctionality are somewhat less important. These studies reveal optimal candidate multilayer biomaterials for human liver tissue engineering applications.

Keywords: Layer-by-layer; Multilayer film; Nanofilm biomaterial; Hepatocyte; Liver

Microporous nanofibrous fibrin-based scaffolds for bone tissue engineering by Thanaphum Osathanon; Michael L. Linnes; Rupak M. Rajachar; Buddy D. Ratner; Martha J. Somerman; Cecilia M. Giachelli (pp. 4091-4099).
The fibrotic response of the body to synthetic polymers limits their success in tissue engineering and other applications. Though porous polymers have demonstrated improved healing, difficulty in controlling their pore sizes and pore interconnections has clouded the understanding of this phenomenon. In this study, a novel method to fabricate natural polymer/calcium phosphate composite scaffolds with tightly controllable pore size, pore interconnection, and calcium phosphate deposition was developed. Microporous, nanofibrous fibrin scaffolds were fabricated using sphere-templating methods. Composite scaffolds were created by solution deposition of calcium phosphate on fibrin surfaces or by direct incorporation of nanocrystalline hydroxyapatite (nHA). The SEM results showed that fibrin scaffolds exhibited a highly porous and interconnected structure. Osteoblast-like cells, obtained from murine calvaria, attached, spread and showed a polygonal morphology on the surface of the biomaterial. Multiple cell layers and fibrillar matrix deposition were observed. Moreover, cells seeded on mineralized fibrin scaffolds exhibited significantly higher alkaline phosphatase activity as well as osteoblast marker gene expression compared to fibrin scaffolds and nHA incorporated fibrin scaffolds (0.25 and 0.5g). All types of scaffolds were degraded both in vitro and in vivo. Furthermore, these scaffolds promoted bone formation in a mouse calvarial defect model and the bone formation was enhanced by addition of rhBMP-2.

Keywords: Fibrin; Calcium phosphate; Hydroxyapatite; Bone tissue engineering

Electrospun poly(lactic acid- co-glycolic acid) scaffolds for skin tissue engineering by Sangamesh G. Kumbar; Syam P. Nukavarapu; Roshan James; Lakshmi S. Nair; Cato T. Laurencin (pp. 4100-4107).
Electrospun fiber matrices composed of scaffolds of varying fiber diameters were investigated for potential application of severe skin loss. Few systematic studies have been performed to examine the effect of varying fiber diameter electrospun fiber matrices for skin regeneration. The present study reports the fabrication of poly[lactic acid- co-glycolic acid] (PLAGA) matrices with fiber diameters of 150–225, 200–300, 250–467, 500–900, 600–1200, 2500–3000 and 3250–6000nm via electrospinning. All fiber matrices found to have a tensile modulus from 39.23±8.15 to 79.21±13.71MPa which falls in the range for normal human skin. Further, the porous fiber matrices have porosity between 38 to 60% and average pore diameters between 10 to 14μm. We evaluated the efficacy of these biodegradable fiber matrices as skin substitutes by seeding them with human skin fibroblasts (hSF). Human skin fibroblasts acquired a well spread morphology and showed significant progressive growth on fiber matrices in the 350–1100nm diameter range. Collagen type III gene expression was significantly up-regulated in hSF seeded on matrices with fiber diameters in the range of 350–1100nm. Based on the need, the proposed fiber skin substitutes can be successfully fabricated and optimized for skin fibroblast attachment and growth.

Keywords: Electrospinning; Skin; Tissue engineering; Fiber; Human skin fibroblast; ECM proteins

Molecular epidemiology of Staphylococcus aureus from implant orthopaedic infections: Ribotypes, agr polymorphism, leukocidal toxins and antibiotic resistance by Davide Campoccia; Lucilla Baldassarri; Valter Pirini; Stefano Ravaioli; Lucio Montanaro; Carla R. Arciola (pp. 4108-4116).
Staphylococcus aureus is a leading pathogen of implant-related infections. In the field of biomaterials a variety of alternative approaches are currently proposed for prophylaxis and treatment of implant infections, but little is known on the role of the different pathogenetic mechanisms and spreading strategies that lead selected S. aureus clones to prevail and become epidemic. This study aimed at identifying and characterizing the major clones in a collection of 200 S. aureus isolates from implant orthopaedic infections. Strain typing by automated ribotyping identified 98 distinct ribogroups. Ribogroups corresponded to specific accessory gene regulatory ( agr) polymorphisms and possessed peculiar arrangements of toxins. The agr type II allele was more represented in epidemic clones, while agr type I in sporadic clones. A clear trend was observed, where epidemic clones resisted antibiotics more than sporadic ones. Conversely, the gene for lukD/lukE leukotoxin, found in 68% of the isolates, was unrelated to the level of clonal spreading.Surprisingly, the isolates of the most prevalent ribogroup were susceptible to almost all antibiotics and never possessed the lukD/ lukE gene, thus suggesting the role of factors other than antibiotic resistance and the here investigated toxins in driving the major epidemic clone to the larger success.

Keywords: Orthopaedic implant infections; Staphylococcus aureus; Automated ribotyping; Leukocidal toxins; Antibiotic resistance; agr; Polymorphism

Bacterial adhesion and growth on a polymer brush-coating by M. Reza Nejadnik; Henny C. van der Mei; Willem Norde; Henk J. Busscher (pp. 4117-4121).
Biomaterials-related infections pose serious problems in implant surgery, despite the development of non-adhesive coatings. Non-adhesive coatings, like polymer brush-coatings, have so far only been investigated with respect to preventing initial bacterial adhesion, but never with respect to effects on kinetics of bacterial growth. Here, we compare adhesion and 20h growth of three bacterial strains ( Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa) on pristine and brush-coated silicone rubber in a parallel plate flow chamber. Brush-coatings were made using a tri-block copolymer of polyethylene oxide (PEO) and polypropylene oxide (PPO). Brush-coatings prevented adhesion of staphylococci to below 5×105cm−2 after 30min, which is a 10-fold reduction compared to pristine silicone rubber. Biofilms grew on both brush-coated and pristine silicone rubber, while the viability of biofilms on brush-coatings was higher than on pristine silicone rubber. However, biofilms on brush-coatings developed more slowly and detached almost fully by high fluid shear. Brush-coating remained non-adhesive after S. epidermidis biofilm formation and subsequent removal whereas a part of its functionality was lost after removal of S. aureus biofilms. Adhesion, growth and detachment of P. aeruginosa were not significantly different on brush-coatings as compared with pristine silicone rubber, although here too the viability of biofilms on brush-coatings was higher. We conclude that polymer brush-coatings strongly reduce initial adhesion of staphylococci and delay their biofilm growth. In addition, biofilms on brush-coatings are more viable and easily removed by the application of fluid shear.

Keywords: Biofilm; Polyethylene oxide; Parallel plate flow chamber; Antifouling; Biomaterials-related infection; Pluronic F-127

Biocompatibility of silica coated NaYF4 upconversion fluorescent nanocrystals by Rufaihah Abdul Jalil; Yong Zhang (pp. 4122-4128).
Here we report the synthesis of uniform silica coated hexagonal-phase NaYF4 nanocrystals with strong NIR-to-visible upconversion fluorescence and its cytotoxicity and biodistribution in a rat model. The silica coated NaYF4 nanocrystals were incubated with rat skeletal myoblasts and bone marrow-derived mesenchymal stem cells and cytotoxicity was assessed by using MTS and LDH assay. Healthy rats were injected intravenously with the nanocrystals so as to further investigate their biocompatibility and tissue distribution. The results from this study revealed that the silica coated NaYF4 upconversion nanocrystals displayed good in vitro and in vivo biocompatibility, demonstrating their potential applications in both cellular and animal imaging systems.

Keywords: Near infrared; Upconversion nanocrystals; Imaging; Toxicity; Biodistribution

The role of daidzein-loaded sterically stabilized solid lipid nanoparticles in therapy for cardio-cerebrovascular diseases by Yu Gao; Wangwen Gu; Lingli Chen; Zhenghong Xu; Yaping Li (pp. 4129-4136).
Daidzein is a very good candidate for treating cardio-cerebrovascular diseases, but its poor oral absorption and bioavailability limit its curative efficacy. In this work, daidzein-loaded solid lipid nanoparticles (SLNs) with PEGylated phospholipid as stabilizer were successfully prepared by hot homogenization method. SLNs showed the mean particle size 126±14nm with entrapment efficiency 82.5±3.7%. In vitro release of SLNs demonstrated a sustained release manner with cumulative release over 90% within 120h in bovine serum albumin solution (4%, w/v). The pharmacokinetic behavior showed that SLNs loading daidzein could significantly increase circulation time compared with orally administrated daidzein suspension or intravenously delivered daidzein solution. SLNs showed the better effect on cardiovascular system of the anesthetic dogs by reducing the myocardial oxygen consumption (MOC) and the coronary resistance (CR) in heart compared with oral suspension or intravenous solution. The SLNs demonstrated the best effect on cerebrovascular system by increasing cerebral blood flow (CeBF) and reducing cerebrovascular resistance (CeR) in anesthetized dogs, and the protective effect on rats with ischemia-reperfusion injury model among three formulations. These results suggested that SLNs could be a potential candidate for the treatment of cardio-cerebrovascular diseases.

Keywords: Daidzein; Cardiovascular disease; Cerebrovascular diseases; Nanoparticles

The effect of magnetic targeting on the uptake of magnetic-fluid-loaded liposomes by human prostatic adenocarcinoma cells by Marie-Sophie Martina; Claire Wilhelm; Sylviane Lesieur (pp. 4137-4145).
Interactions of magnetic-fluid-loaded liposomes (MFL) with human adenocarcinoma prostatic cell line PC3 were investigated in vitro. MFL consisted of unilamellar phosphatidylcholine vesicles (mean hydrodynamic diameter close to 180nm) encapsulating 8-nm nanocrystals of maghemite (γ-Fe2O3) and sterically stabilized by introducing 5mol.% of distearylphosphatidylcholine poly(ethylene glycol)2000 (DSPE-PEG2000) in the vesicle bilayer. The association processes with living cells, including binding and effective internalization, were followed versus time at two levels. On one hand, the lipid vesicles labeled by 1mol.% of rhodamine-marked phosphatidylethanolamine were imaged by confocal fluorescence microscopy. On the other hand, the iron oxide particles associated with cells were independently quantified by magnetophoresis. This allowed modeling of MFL uptake kinetics as a two-step process involving first binding adsorption onto the outer cell membrane followed by subsequent internalization. Capture efficiency was significantly improved by guiding MFL in the near vicinity of the cells by means of a 0.29-T external magnet developing a magnetic field gradient close to 30mT/mm. Double detection of lipids by fluorescence tracking and of iron oxide by magnetophoresis showed excellent correlation. This demonstrated that MFL associate with tumor cells as intact vesicle structures which conserve their internal content.

Keywords: Magnetoliposome; Iron oxide nanoparticles; Confocal fluorescence microscopy; Magnetic cell labeling; Magnetophoresis; Uptake kinetics

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