Biomaterials (v.28, #9)

Vitrification as a prospect for cryopreservation of tissue-engineered constructs by L.L. Kuleshova; S.S. Gouk; D.W. Hutmacher (1585-1596).
Cryopreservation plays a significant function in tissue banking and will presume yet larger value when more and more tissue-engineered products will routinely enter the clinical arena. The most common concept underlying tissue engineering is to combine a scaffold (cellular solids) or matrix (hydrogels) with living cells to form a tissue-engineered construct (TEC) to promote the repair and regeneration of tissues. The scaffold and matrix are expected to support cell colonization, migration, growth and differentiation, and to guide the development of the required tissue. The promises of tissue engineering, however, depend on the ability to physically distribute the products to patients in need. For this reason, the ability to cryogenically preserve not only cells, but also TECs, and one day even whole laboratory-produced organs, may be indispensable. Cryopreservation can be achieved by conventional freezing and vitrification (ice-free cryopreservation). In this publication we try to define the needs versus the desires of vitrifying TECs, with particular emphasis on the cryoprotectant properties, suitable materials and morphology. It is concluded that the formation of ice, through both direct and indirect effects, is probably fundamental to these difficulties, and this is why vitrification seems to be the most promising modality of cryopreservation.
Keywords: Tissue-engineered constructs; Vitrification; Freezing; Cryopreservation;

In this study, 2-amino-4-chloro-6-hydroxy-s-triazine (ACHT) was synthesized through controlled hydrolysis of 2-amino-4,6-dichloro-s-triazine (ADCT). A simple pad-dry-cure approach was employed to immobilize ACHT onto cellulosic fibrous materials. After treatment with diluted chlorine bleach, the covalently bound ACHT moieties were transformed into chloromelamines. The structures of the samples were fully characterized with NMR, UV/VIS, DSC, TG, iodometric titration and elemental analyses. The chloromelamine-based fibrous materials provided potent, durable, and rechargeable biocidal functions against bacteria (including multi-drug resistant species), yeasts, viruses, and bacterial spores. SEM studies demonstrated that the new fibrous materials could effectively prevent the formation of biofilms, and controlled release investigations in vitro suggested that the biocidal activities were bioresponsive. Biocidal mechanisms of the chloromelamine-based fibrous materials were further discussed.
Keywords: Biocidal polymers; Cotton cellulose; Biofilm; Bioresponsive; Controlled release;

Branched polyesters of the general structure poly[vinyl-3-(dialkylamino)alkylcarbamate-co-vinyl acetate-co-vinyl alcohol]-graft-poly(d,l-lactide-co-glycolide) have shown potential for nano- and micro-scale drug delivery systems. For further optimization of this polymer class their cytotoxicity needs to be characterized establishing structure-toxicity relationships. Effects of type and degree of amine substitution as well as molecular weight on cytotoxicity were evaluated in L929 mouse fibroblasts using a MTT assay whereas interactions with cell membranes were quantified by LDH release and caspase (3/7)-activation. Finally, direct cell-polymer contact assays were conducted. Ungrafted amine-modified polymer backbone yielded IC50 values in the range of 0.05–10 mg/ml. Generally higher toxicities were observed with an increasing degree of amine substitution. Amine substituents could be ranked as diethylaminopropylamine (DEAPA)<diethylaminoethylamine (DEAEA)<dimethylaminopropylamine (DMAPA) but the degree of amine substitution was more dominant than the type of amine function. Membrane interactions seem to cause necrotic cell reactions in a dose-dependent manner for highly charged amine-poly(vinyl alcohol) (PVA) backbones. To attenuate cytotoxic effects DEAPA-PVA backbones were grafted with biodegradable PLGA side chains at molecular ratios of 1:10 and 1:20. Cytotoxicity of extracts of these polymers was significantly lower compared to ungrafted polymers possibly caused by shielding of polycationic backbone with hydrophobic PLGA side chains. P(33)-20, a polymer containing a sufficiently high degree of amine substitution could serve as a lead candidate for further investigations. In conclusion, structure–toxicity relationships could be established and shielding the polycationic backbone using PLGA side chains seems to be a promising strategy meriting further investigations.
Keywords: Cytotoxicity; Copolymer; Poly(vinyl alcohol); Polyglycolic acid; Polylactic acid; Degradation;

DLC coatings: Effects of physical and chemical properties on biological response by Wen J. Ma; Andrew J. Ruys; Rebecca S. Mason; Phil J. Martin; Avi Bendavid; Zongwen Liu; Mihail Ionescu; Hala Zreiqat (1620-1628).
Recent trials on diamond-like carbon (DLC) coated medical devices have indicated promise for blood interfacing applications. The literature is sparse regarding structural and compositional effects of DLC on cellular response. An important goal in optimizing blood-interfacing implants is minimal macrophage attachment, and maximal albumin:fibrinogen adsorption ratio. DLC coatings deposited by PACVD and FAD, were analysed with respect to sp3 content (EELS), hydrogen content (ERDA), surface composition (XPS), surface roughness (AFM), surface energy, albumin:fibrinogen adsorption ratio, and macrophage viability and attachment. We found that increasing surface roughness and surface energy enhanced the macrophage viability and the albumin:fibrinogen adsorption ratio. We also found that the higher the hydrogen content for a-C:Hs deposited by PACVD, the lower the albumin:fibrinogen adsorption ratio, and macrophage attachment. This suggests that hydrogen content may be an important factor for influencing the biological response of DLC surfaces. Macrophage cells spread well on all DLC surfaces, and the surface results indicated the non-toxic nature of the surfaces on the cells at the time points tested.
Keywords: Biocompatibility; Amorphous carbon; Macrophage; Protein; Surface energy; Hydrogen content;

Effect of ultrasmall superparamagnetic iron oxide nanoparticles (Ferumoxtran-10) on human monocyte-macrophages in vitro by Karin Müller; Jeremy N. Skepper; Mihaly Posfai; Rikin Trivedi; Simon Howarth; Claire Corot; Eric Lancelot; Paul W. Thompson; Andrew P. Brown; Jonathan H. Gillard (1629-1642).
Ferumoxtran-10, a dextran-coated ultrasmall superparamagnetic iron oxide particle, has the potential to reveal macrophages in vivo using magnetic resonance imaging potentially acting as a marker of inflammatory status. Pending clinical trials, we examined the interactions of Ferumoxtran-10 with human monocyte-macrophages (HMMs) in vitro to assess its safety and lack of pro-inflammatory activity. After 72 h, Ferumoxtran-10 was not toxic at 1 mg/ml and may be only mildly toxic at 10 mg/ml. Viability in cells with a high intracellular Ferumoxtran-10 load was not affected over 14 days. Ferumoxtran-10 did not interfere with baseline or stimulated cytokine (interleukin-12, interleukin-6, tumour necrosis factor-α or interleukin-1β) or superoxide anion production or with Fc-receptor-mediated phagocytosis. Similarly, Ferumoxtran-10 did not induce cytokine production and was not chemotactic. High-resolution electron microscopy and selected-area electron diffraction confirmed the core of Ferumoxtran-10 is composed of crystalline magnetite. Bright field transmission electron microscopy of thin sections demonstrated that Ferumoxtran-10 was retained in lysosomes of HMM for several days. Ferumoxtran-10 is not toxic to HMMs in vitro, does not activate them to produce pro-inflammatory cytokines or superoxide anions, is not chemotactic and does not interfere with Fc-receptor-mediated phagocytosis. Furthermore, extremely high intracellular Ferumoxtran-10 concentrations had only slight or no effects on these key activities.
Keywords: Nanoparticle; Macrophage; Cytotoxicity; Inflammation; Cell activation; Electron microscopy;

Biocompatibility evaluation of silk fibroin with peripheral nerve tissues and cells in vitro by Yumin Yang; Xuemei Chen; Fei Ding; Peiyun Zhang; Jie Liu; Xiaosong Gu (1643-1652).
Silk-based materials have been used in the field of bone or ligament tissue engineering. In order to explore the feasibility of using purified silk fibroin to construct artificial nerve grafts, it is necessary to evaluate the biocompatibility of silk fibroin material with peripheral nerve tissues and cells. We cultured rat dorsal root ganglia (DRG) on the substrate made up of silk fibroin fibers and observed the cell outgrowth from DRG during culture by using light and electron microscopy coupled with immunocytochemistry. On the other hand, we cultured Schwann cells from rat sciatic nerves in the silk fibroin extract fluid and examined the changes of Schwann cells after different times of culture. The results of light microscopy, MTT test and cell cycle analysis showed that Schwann cells cultured in the silk fibroin extract fluid showed no significant difference in their morphology, cell viability and proliferation as compared to that in plain L15 medium. Furthermore, no significant difference was found in expression of the factors secreted by Schwann cells, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and S-100, between Schwann cells cultured in the silk fibroin extraction fluid and in plain L15 medium by the aid of immunocytochemistry, RT-PCR and Western analysis. Collectively, these data indicate that silk fibroin has good biocompatibility with DRG and is also beneficial to the survival of Schwann cells without exerting any significant cytotoxic effects on their phenotype or functions, thus providing an experimental foundation for the development of silk fibroin as a candidate material for nerve tissue engineering applications.
Keywords: Silk fibroin; Dorsal root ganglia; Schwann cell; Biocompatibility; Cytotoxicity;

Extracellular matrix formation and mineralization on a phosphate-free porous bioactive glass scaffold using primary human osteoblast (HOB) cells by Julian R. Jones; Olga Tsigkou; Emily E. Coates; Molly M. Stevens; Julia M. Polak; Larry L. Hench (1653-1663).
Sol–gel derived bioactive glasses of the 70S30C (70 mol% SiO2, 30 mol% CaO) composition have been foamed to produce 3D bioactive scaffolds with hierarchical interconnected pore morphologies similar to trabecular bone. The aim of this study was to investigate primary human osteoblast response to porous bioactive glass scaffolds. The scaffolds supported osteoblast growth and induced differentiation, within the 3-week culture period, as depicted by enhanced ALPase enzymatic activity, without the addition of supplementary factors such as ascorbic acid, β-glycerophosphate and dexamethasone. This is the first time this has been observed on a bioactive glass that does not contain phosphate. Deposition of extracellular matrix was also confirmed by enhanced production of the extracellular matrix protein collagen type I. SEM showed indications of mineralized bone nodule formation without the addition of growth factors. The 70S30C bioactive glass scaffolds therefore fulfil many of the criteria for an ideal scaffold for bone tissue engineering applications.
Keywords: Osteoblast; Bioactive glass; Porous scaffold; Ion release; Mineralization;

Polycaprolactone (PCL) cylindrical scaffolds with gradually increasing pore size along the longitudinal direction were fabricated by a novel centrifugation method to investigate pore size effect on cell and tissue interactions. The scaffold was fabricated by the centrifugation of a cylindrical mold containing fibril-like PCL and the following fibril bonding by heat treatment. The scaffold showed gradually increasing pore size (from ∼88 to ∼405 μm) and porosity (from ∼80% to ∼94%) along the cylindrical axis by applying the centrifugal speed, 3000 rpm. The scaffold sections were examined for their in vitro cell interactions using different kinds of cells (chondrocytes, osteoblasts, and fibroblasts) and in vivo tissue interactions using a rabbit model (skull bone defects) in terms of scaffold pore sizes. It was observed that different kinds of cells and bone tissue were shown to have different pore size ranges in the scaffold for effective cell growth and tissue regeneration. The scaffold section with 380–405 μm pore size showed better cell growth for chondrocytes and osteoblasts, while the scaffold section with 186–200 μm pore size was better for fibroblasts growth. Also the scaffold section with 290–310 μm pore size showed faster new bone formation than those of other pore sizes. The pore size gradient scaffolds fabricated by the centrifugation method can be a good tool for the systematic studies of the interactions between cells or tissues and scaffolds with different pore size.
Keywords: Pore size; Scaffold; Porosity; Polycaprolactone (PCL); Cell adhesion; Bone regeneration;

Cell interaction with three-dimensional sharp-tip nanotopography by Chang-Hwan Choi; Sepideh H. Hagvall; Benjamin M. Wu; James C.Y. Dunn; Ramin E. Beygui; Chang-Jin “CJ” Kim (1672-1679).
Cells in their native microenvironment interact with three-dimensional (3D) nanofeatures. Despite many reports on the effects of substrate nanotopography on cells, the independent effect of 3D parameters has not been investigated. Recent advances in nanofabrication for precise control of nanostructure pattern, periodicity, shape, and height enabled this systematic study of cell interactions with 3D nanotopographies. Two distinct nanopatterns (posts and grates) with varying three-dimensionalities (50–600 nm in nanostructure height) were created, while maintaining the pattern periodicity (230 nm in pitch) and tip shape (needle- or blade-like sharp tips). Human foreskin fibroblasts exhibited significantly smaller cell size and lower proliferation on needle-like nanoposts, and enhanced elongation with alignment on blade-like nanogrates. These phenomena became more pronounced as the nanotopographical three-dimensionality (structural height) increased. The nanopost and nanograte architectures provided the distinct contact guidance for both filopodia extension and the formation of adhesion molecules complex, which was believed to lead to the unique cell behaviors observed.
Keywords: Nanotopography; Fibroblast; Cell proliferation; Cell morphology; Cell spreading; Cell adhesion;

Morphological regulation of rabbit chondrocytes on glucose-displayed surface by Masahiro Kino-oka; Yoshiki Morinaga; Mee-Hae Kim; Yasunori Takezawa; Masaya Kawase; Kiyohito Yagi; Masahito Taya (1680-1688).
A culture surface was designed to regulate morphology of rabbit chondrocytes by changing the ratio of d- and l-glucose isomers displayed on a glass plate. With increasing ratio of d-glucose displayed on the surfaces, the efficiency of cell attachment improved, meaning that the attachment exclusively occurred via mediation of an affinity between d-glucose displayed and glucose transporter on cell membrane. At 0% and 100% d-glucose display, the round-shaped cells appeared dominantly, and most of cells became stretched in shape at 50% d-glucose display, indicating that the frequency of round-shaped cells depicted a concave profile against the ratio of d-glucose displayed. From the cytoskeletal staining of F-actin and vinculin, the immature stress fibers with fewer focal contacts were recognized in both the round shaped cells and those stretched in shape on 100% d-glucose-displayed surface. The time-lapse observation revealed that the cells on 100% d-glucose-displayed surface conducted active migration and aggregation with formation of collagen type II. These results suggest that 100% d-glucose-displayed surface can offer culture environment to maintain the chondrocytic phenotype of cells, similarly to the conditions achieved in three-dimensional (3-D) culture.
Keywords: Chondrocyte; Morphology; Glucose-displayed surface; ECM formation; Cytoskeletal formation;

Coronary stents: A materials perspective by Gopinath Mani; Marc D. Feldman; Devang Patel; C. Mauli Agrawal (1689-1710).
The objective of this review is to describe the suitability of different biomaterials as coronary stents. This review focuses on the following topics: (1) different materials used for stents, (2) surface characteristics that influence stent–biology interactions, (3) the use of polymers in stents, and (4) drug-eluting stents, especially those that are commercially available.
Keywords: Stent; Surface treatment; Surface modification; Drug delivery;

Polysaccharide intercellular adhesin or protein factors in biofilm accumulation of Staphylococcus epidermidis and Staphylococcus aureus isolated from prosthetic hip and knee joint infections by Holger Rohde; Eike C. Burandt; Nicolaus Siemssen; Lars Frommelt; Christoph Burdelski; Sabine Wurster; Stefanie Scherpe; Angharad P. Davies; Llinos G. Harris; Matthias A. Horstkotte; Johannes K.-M. Knobloch; Chandran Ragunath; Jeffrey B. Kaplan; Dietrich Mack (1711-1720).
Nosocomial staphylococcal foreign-body infections related to biofilm formation are a serious threat, demanding new therapeutic and preventive strategies. As the use of biofilm-associated factors as vaccines is critically restricted by their prevalence in natural staphylococcal populations we studied the distribution of genes involved in biofilm formation, the biofilm phenotype and production of polysaccharide intercellular adhesin (PIA) in clonally independent Staphylococcus aureus and Staphylococcus epidermidis strains isolated from prosthetic joint infections after total hip or total knee arthroplasty. Biofilm formation was detected in all S. aureus and 69.2% of S. epidermidis strains. Importantly, 27% of biofilm-positive S. epidermidis produced PIA-independent biofilms, in part mediated by the accumulation associated protein (Aap). Protein-dependent biofilms were exclusively found in S. epidermidis strains from total hip arthroplasty (THA). In S. aureus PIA and proteins act cooperatively in biofilm formation regardless of the infection site. PIA and protein factors like Aap are of differential importance for the pathogenesis of S. epidermidis in prosthetic joint infections (PJI) after THA and total knee arthroplasty (TKA), implicating that icaADBC cannot serve as a general virulence marker in this species. In S. aureus biofilm formation proteins are of overall importance and future work should focus on the identification of functionally active molecules.
Keywords: Staphylococcus epidermidis; Staphylococcus aureus; Biofilm formation; Polysaccharide intercellular adhesin; MSCRAMM; icaADBC;

Risk of infection is considerable in open fractures and its management is challenging, especially when fracture fixation material is used. Thus, it may be desirable to use a device from which antibiotics can be released in a controlled way. Room temperature processed silica sol–gels are novel, resorbable and biocompatible, controlled release materials. Vancomycin, a potent antibiotic used in treating osteomyelitis, can be released from silica sol–gels. Herein, we describe the synthesis of thin, resorbable, controlled release bactericidal sol–gel films on a Ti-alloy substrate and determine the effect of processing parameters on its degradation and vancomycin release. A close correlation between release and degradation rates suggests that film degradation is the main mechanism underlying the control of release. Using a multi-layer process and various concentrations of vancomycin, released concentrations exceed the minimal inhibitory concentration (MIC) of vancomycin against Staphylococcus aureus. The findings enable the tailoring of release and degradation properties of the films to therapeutic needs by controlling sol–gel processing parameters. Given the bactericidal properties of released vancomycin, and the biocompatibility of the sol–gel films, the present data suggest great promise to prevent and treat bone infections in a clinical setting.
Keywords: Sol–gel techniques; Controlled drug release; Antibacterial; In vitro test;

Natural paclitaxel (Taxol®) is an effective anti-cancer drug, although a critical disadvantage is its non-targeting nature. To address this issue, cholesterol-grafted poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide-co-undecenoic acid) was synthesized with different starting monomer ratios via a free radical copolymerization route. Folate was subsequently attached to the hydrophilic segment of the polymer in order to target folate receptors-overexpressing cancer cells. The success of synthesis was confirmed with 1H-NMR carried out in CDCl3/D2O. Using a membrane dialysis method, the polymer was then self-assembled into micelles whose hydrophobic cores could be utilized to encapsulate paclitaxel, an extremely hydrophobic compound. The polymer had a low CMC of ∼20 mg/L in water. Dynamic light scattering further showed that the sizes of blank micelles formed from the polymer were below 180 nm at different pH values tested and ∼220 nm upon drug incorporation. More importantly, it was demonstrated that the micelles exhibited a useful pH-induced thermo-sensitivity, such that drug was released more rapidly at pH 5.0 (acidic endosomal/lysosomal environment) than at pH 7.4 (normal extracellular pH). In vitro cytotoxicity assays performed against KB cells then provided concluding evidences that the cellular uptake of micelles surface-functionalised with folate was indeed enhanced due to a receptor-assisted endocytosis process. This novel polymeric design thus has the potential to be a useful paclitaxel vehicle for the treatment of folate-receptor positive cancers.
Keywords: Paclitaxel; Targeted and intracellular delivery; Micelles; pH- and temperature-sensitive; Folate;