Biomaterials (v.27, #16)

Calendar (I).

UHMWPE and vitamin E bioactivity: An emerging perspective by Filippo Renò; Mario Cannas (3039-3043).
Ultra-high molecular weight polyethylene (UHMWPE) is a semicrystalline biomaterial widely used in the components for articular prosthesis for its excellent mechanical qualities.Two major problems limit the UHMWPE prosthesis life—wearing and delamination, both phenomena being mainly due to chemical oxidation of polymer. Wearing causes the release of generated particulate that triggers a macrophage reaction leading to chronic inflammation and osteolysis, while delamination, due to the mechanical stress, macroscopically alters the surfaces. The most diffused method to reduce wearing is UHMWPE molecular cross-linking by high-energy irradiation followed by melting that also reduces polymer fatigue strength. For this reason, the use of vitamin E (α-tocopherol), as an anti-oxidative and biocompatible additive for normal and cross-linked UHMWPE, has been suggested as an alternative method to improve polymer wearing resistance without altering its mechanical strength. This paper describes briefly the rationale of vitamin E as UHMWPE additive and its possible use as an emerging perspective in the orthopaedic field.
Keywords: UHMWPE; Vitamin E; Oxidation; Cross-linking;

Surface engineering approaches to micropattern surfaces for cell-based assays by Didier Falconnet; Gabor Csucs; H. Michelle Grandin; Marcus Textor (3044-3063).
The ability to produce patterns of single or multiple cells through precise surface engineering of cell culture substrates has promoted the development of cellular bioassays that provide entirely new insights into the factors that control cell adhesion to material surfaces, cell proliferation, differentiation and molecular signaling pathways. The ability to control shape and spreading of attached cells and cell–cell contacts through the form and dimension of the cell-adhesive patches with high precision is important. Commitment of stem cells to different specific lineages depends strongly on cell shape, implying that controlled microenvironments through engineered surfaces may not only be a valuable approach towards fundamental cell-biological studies, but also of great importance for the design of cell culture substrates for tissue engineering. Furthermore, cell patterning is an important tool for organizing cells on transducers for cell-based sensing and cell-based drug discovery concepts. From a material engineering standpoint, patterning approaches have greatly profited by combining microfabrication technologies, such as photolithography, with biochemical functionalization to present to the cells biological cues in spatially controlled regions where the background is rendered non-adhesive (“non-fouling”) by suitable chemical modification. The focus of this review is on the surface engineering aspects of biologically motivated micropatterning of two-dimensional (flat) surfaces with the aim to provide an introductory overview and critical assessment of the many techniques described in the literature. In particular, the importance of non-fouling surface chemistries, the combination of hard and soft lithography with molecular assembly techniques as well as a number of less well known, but useful patterning approaches, including direct cell writing, are discussed.
Keywords: Microfabrication; Lithography; Surface modification; Cell patterns; Protein adsorption;

To date, long-term functions of osteoblasts leading to calcium and phosphorus mineral deposition on nanometals have not been determined. Nanometals are metals with constituent metal particles and/or surface features less than 100 nm in at least one dimension. For this reason, the objective of this in vitro study was to determine the amount of calcium and phosphorus mineral formation on microphase compared to nanophase Ti, Ti6Al4V, and CoCrMo cultured with and without osteoblasts (bone-forming cells). The results of this study provided the first evidence of significantly greater calcium and phosphorus deposition by osteoblasts and precipitation from culture media without osteoblasts on nanophase compared to respective microphase Ti6Al4V and CoCrMo after 21 days; the greatest calcium and phosphorus mineral deposition occurred on nanophase CoCrMo while the greatest calcium and phosphorus mineral precipitation without osteoblasts occurred on nanophase Ti6Al4V. No differences were found for any type of Ti: wrought, microphase, or nanophase. Moreover, increased calcium and phosphorus mineral content correlated to greater amounts of underlying aluminum content on Ti6Al4V surfaces. Since, compared to microphase Ti6Al4V, nanophase Ti6Al4V contained a higher amount of aluminum at the surface (due to greater surface area), this may provide a reason for enhanced calcium and phosphorus mineral content on nanophase Ti6Al4V. Regardless of the mechanism, this study continues to support the further investigation of nanometals for improved orthopedic applications.
Keywords: Nanotechnology; Calcium; Titanium; Ti6Al4V; Cobalt chromium alloy; Osteoblast;

Evaluation of silicon nanoporous membranes and ECM-based microenvironments on neurosecretory cells by Carlos A. Lopez; Aaron J. Fleischman; Shuvo Roy; Tejal A. Desai (3075-3083).
Understanding the interactions between microfabricated synthetic interfaces and cultured cells expressing a neuronal phenotype are critical for advancing research in the field of neural engineering such as neural recording and stimulation and neural microdevice interactions with the human brain. Here we explore the integration of these two components for therapeutic applications of neural prostheses. Microfabricated silicon nanoporous membranes were investigated for their effects on survival, proliferation, and differentiation of the well-known PC12 clonal line. Specifically, cell morphology, examined through fluorescence staining, were comparable in many respects on both silicon membrane and widely-used polystyrene culture surfaces. The attachment and differentiation of PC12 cells cultured on collagen and laminin-modified membranes and standard tissue culture surfaces were similar. Lastly, the differentiation response and tyrosine hydroxylase activity of PC12 cells embedded in a type I collagen matrix on experimental membrane substrates while exposed to NGF were significant and indistinguishable from tissue-culture polystyrene (TC-PS) surfaces. Results from this research suggest that microfabricated silicon nanoporous membranes may be useful, biocompatible permselective structures for neuroprosthetic applications and that collagen may be a useful immobilizing matrix for PC12 cells loaded in implantable macroencapsulation devices designed for the treatment of neurodegenerative disorders.
Keywords: Biocompatibility; Cell encapsulation; Nanoporous membranes; Neural prosthesis; PC12; Silicon;

Glycocalyx-mimetic dextran-modified poly(vinyl amine) surfactant coating reduces platelet adhesion on medical-grade polycarbonate surface by A. Sen Gupta; S. Wang; E. Link; E.H. Anderson; C. Hofmann; J. Lewandowski; K. Kottke-Marchant; R.E. Marchant (3084-3095).
A dextran-modified poly(vinyl amine) comb-like surfactant polymer, poly(N-vinyl dextran aldonamide-co-N-vinyl hexanamide), that can surface-adsorb on hydrophobic polymeric substrates, was designed to improve the interfacial blood-compatibility of polymeric biomaterials. Medical-grade polycarbonate was selected as a model substrate because of its extensive use in blood-contacting biomedical devices like hemodialyzers, blood pumps and oxygenators. The surfactant polymer was physisorbed from aqueous solution onto the polycarbonate substrate. The surfactant coating was stable under dynamic shear conditions in whole blood, as confirmed by fluorescence microscopy and total internal reflection fluorescence (TIRF) experiments with fluorescein-labeled surfactant polymer. The coated disks and uncoated control disks were exposed to platelet-rich plasma (PRP) and whole human blood in a rotating disk system (RDS) to study platelet-adhesion under dynamic shear stress environments. Adhered platelets were stained with fluorescein isothiocyante (FITC)-tagged anti-CD41a monoclonal antibody and imaged by epifluorescence microscopy. Complimentary images were obtained by phase-contrast microscopy. Platelet adhesion on the surfactant-coated disks was reduced by ∼90%, compared with uncoated disks. The images also showed a concomitant reduction in platelet-derived microparticles on surfactant-coated disks, compared with uncoated disks. The results suggest potential application of carbohydrate-modified surfactant polymers as a glycocalyx-mimetic non-thrombogenic interfacial coating for blood-contacting biomaterials.
Keywords: Glycocalyx-mimetic; Interface; Blood-compatibility; Coating; Platelet adhesion;

Surface-induced changes in protein adsorption and implications for cellular phenotypic responses to surface interaction by Lorcan T. Allen; Miriam Tosetto; Ian S. Miller; Darran P. O’Connor; Stephen C. Penney; Iseult Lynch; Alan K. Keenan; Stephen R. Pennington; Kenneth A. Dawson; William M. Gallagher (3096-3108).
Understanding external factors that determine cellular phenotypic responses is of key interest in the field of biomaterials. Currently, material surface characteristics, protein adsorption and cellular phenotypic responses are all considered to be interrelated and ultimately determine the biocompatibility of materials. The exact nature of the relationship between these distinct, yet related, phenomena still remains to be elucidated. Through the use of a series of thermoresponsive N-isopropylacrylamide-based co-polymer films, we aimed to shed light on the relationship between surface hydrophobicity, protein adsorption and subsequent cellular response. Despite changes in co-polymer hydrophobicity mediated by altered ratios of constituent monomers, differential cellular response was only apparent in the presence of serum. Co-polymer films displayed alterations with respect to the amount of protein adsorbed on the surface, with individual serum proteins (albumin and fibronectin) displaying contrasting adsorption characteristics. Changes in protein adsorption corresponded to changes in cell adhesion, cytoskeletal organisation and cell morphology, as well as to changes in cell movement and intracellular signalling events. Examination of focal adhesion kinase (FAK), and extracellular signal-regulated kinase (ERK 1/2), important mediators of adhesion and growth factor-related signalling events, revealed a comparative reduction in phosphorylation of these signalling proteins in cells grown on co-polymers in comparison to those cultured on tissue culture polystyrene (TCP; used as a control surface). We also associated surface-mediated phenotypic alterations of cells grown on TCP and co-polymer films with particular changes in gene expression. These results indicate that cellular response to interaction with our series of co-polymer films is determined by the surface-adsorbed protein layer, which in turn is determined by the changing surface chemistry as the ratio of the co-monomers is altered.
Keywords: Surface hydrophobicity; Protein adsorption; Fibronectin; Focal adhesion; Cell signalling; Cell migration;

Bone-like tissue formation using an equine COLLOSS® E-filled titanium scaffolding material by Marloes E.L. Nienhuijs; X. Frank Walboomers; Matthias A.W. Merkx; Paul J.W. Stoelinga; John A. Jansen (3109-3114).
COLLOSS®, a bovine extracellular matrix product containing native BMPs has already shown osteoinductive properties. To overcome problems with risk of transmissable spongiform encephalopathy (TSE) infection, an equine derived version was investigated in this study, named COLLOSS® E. Disc- and tube-shaped implants were made from titanium fibre mesh. The central space of tubes was filled and the discs were impregnated with the COLLOSS® E material to assess osteo-induction. These implants and non-loaded controls were implanted subcutaneously into the back of Wistar rats. After implantation periods of 2, 8, and 12 weeks, the implants were retrieved and sections were made. Histology showed a thin fibrous capsule surrounding the titanium mesh and a very mild tissue reaction. The disc implants, loaded or non-loaded, showed no bone formation at all. After 2 weeks of implantation 3 out of 5 of the loaded tubes showed bone formation with a mean of 0.3 mm2 areas of new formed bone, after 8 weeks 3 out of 6 and 0.7 mm2, and after 12 weeks this increased to 6 out of 6 and 1.0 mm2. In the non-loaded tubes only connective tissue in growth was seen. In conclusion, it was demonstrated that COLLOSS® E material, loaded in a titanium fibre mesh tube shows osteoinductive properties. The effect of COLLOSS® E has to be investigated further in orthotopic sites, which resemble more the final clinical application for bone reconstruction.
Keywords: Bone tissue engineering; BMP (bone morphogenetic protein); Bone graft; Growth factors; In vivo test;

Electrospun silk-BMP-2 scaffolds for bone tissue engineering by Chunmei Li; Charu Vepari; Hyoung-Joon Jin; Hyeon Joo Kim; David L. Kaplan (3115-3124).
Silk fibroin fiber scaffolds containing bone morphogenetic protein 2 (BMP-2) and/or nanoparticles of hydroxyapatite (nHAP) prepared via electrospinning were used for in vitro bone formation from human bone marrow-derived mesenchymal stem cells (hMSCs). BMP-2 survived the aqueous-based electrospinnig process in bioactive form. hMSCs were cultured for up to 31 days under static conditions in osteogenic media on the scaffolds (silk/PEO/BMP-2, silk/PEO/nHAP, silk/PEO/nHAP/BMP-2) and controls (silk/PEO, silk/PEO extracted). Electrospun silk fibroin-based scaffolds supported hMSC growth and differentiation toward osteogenic outcomes. The scaffolds with the co-processed BMP-2 supported higher calcium deposition and enhanced transcript levels of bone-specific markers than in the controls, indicating that these nanofibrous electrospun silk scaffolds were an efficient delivery system for BMP-2. X-ray diffraction (XRD) analysis revealed that the apatite formed on the silk fibroin/BMP-2 scaffolds had higher crystallinity than on the silk fibroin scaffold controls. In addition, nHAP particles were incorporated into the electrospun fibrous scaffolds during processing and improved bone formation. The coexistence of BMP-2 and nHAP in the electrospun silk fibroin fibers resulted in the highest calcium deposition and upregulation of BMP-2 transcript levels when compared with the other systems. The results suggest that electrospun silk-fibroin-based scaffolds are potential candidates for bone tissue engineering. Furthermore, the mild aqueous process required to spin the fibers offers an important option for delivery of labile cytokines and other components into the system.
Keywords: Silk; Electrospinning; Bone; Tissue engineering; BMP; Hydroxyapatite;

Characterizing the modification of surface proteins with poly(ethylene glycol) to interrupt platelet adhesion by Haiyan Xu; Joel L. Kaar; Alan J. Russell; William R. Wagner (3125-3135).
Surface protein modification with poly(ethylene glycol) (PEG) can inhibit acute thrombosis on damaged vascular and biomaterial surfaces by blocking surface protein–platelet interactions. However, the feasibility of employing protein reactive PEGs to limit intravascular and biomaterial thrombosis in vivo is contingent upon rapid and extensive surface protein modification. To characterize the factors controlling this potential therapeutic approach, the model protein bovine serum albumin was adsorbed onto polyurethane surfaces and modified with PEG-carboxymethyl succinimidyl ester (PEG-NHS), PEG-isocyanate (PEG-ISO), or PEG-diisocyanate (PEG-DISO) in aqueous buffer at varying concentrations and contact times. It was found that up to 5 PEGs could be attached per albumin molecule within one min and that adsorbed albumin PEGylation approached maximal levels by 6 min. The lability of reactive PEGs in aqueous buffer reduced total protein modification by 50% when the PEG solution was incubated for 7 min prior to application. For fibrinogen PEGylation (performed in the solution phase), PEG-NHS was more reactive than PEG-ISO or PEG-DISO. The γ peptide of fibrinogen, which contains several key platelet-binding motifs, was highly modified. A marked reduction in platelet adhesion was observed on fibrinogen-adsorbed polyurethane treated with PEG-NHS or PEG-DISO. Relative differences in platelet adhesion on PEG-NHS and PEG-DISO modified surfaces could be attributed to differences in reactivity towards fibrinogen and the size of the polymer backbone. Taken together, these findings provide insight and guidance for applying protein reactive PEGs for the interruption of acute thrombotic deposition.
Keywords: Platelet adhesion; Thrombosis; Protein adsorption; Fibrinogen; Poly(ethylene glycol); Protein modification;

Characterisation of electrospun polystyrene scaffolds for three-dimensional in vitro biological studies by Simon C. Baker; Neil Atkin; Paul A. Gunning; Nick Granville; Karen Wilson; Darren Wilson; Jennifer Southgate (3136-3146).
The purpose of this study was to produce a well-characterised electrospun polystyrene scaffold which could be used routinely for three-dimensional (3D) cell culture experimentation. A linear relationship ( p < 0.01 ) between three principal process variables (applied voltage, working distance and polymer concentration) and fibre diameter was reliably established enabling a mathematical model to be developed to standardise the electrospinning process. Surface chemistry and bulk architecture were manipulated to increase wetting and handling characteristics, respectively. X-ray photoelectron spectroscopy (XPS) confirmed the presence of oxygen-containing groups after argon plasma treatment, resulting in a similar surface chemistry to treated tissue culture plastic. The bulk architecture of the scaffolds was characterised by scanning electron microscopy (SEM) to assess the alignment of both random and aligned electrospun fibres, which were calculated to be 0.15 and 0.66, respectively. This compared to 0.51 for collagen fibres associated with native tissue. Tensile strength and strain of approximately of 0.15 MPa and 2.5%, respectively, allowed the scaffolds to be routinely handled for tissue culture purposes.The efficiency of attachment of smooth muscle cells to electrospun scaffolds was assessed using a modified 3-[4,5-dimethyl(thiazol-2yl)-3,5-diphery] tetrazolium bromide assay and cell morphology was assessed by phalloidin-FITC staining of F-actin. Argon plasma treatment of electrospun polystyrene scaffold resulted in significantly increased cell attachment ( p < 0.05 ). The alignment factors of the actin filaments were 0.19 and 0.74 for the random and aligned scaffold respectively, compared to 0.51 for the native tissue. The data suggests that electrospinning of polystyrene generates 3D scaffolds which complement polystyrene used in 2D cell culture systems.
Keywords: Non-woven fabric; Biomaterial; Scaffold; SEM; Smooth muscle cell; Tissue culture;

Effective transfection of cells with multi-shell calcium phosphate-DNA nanoparticles by Viktoriya V. Sokolova; Ina Radtke; Rolf Heumann; Matthias Epple (3147-3153).
Coated calcium phosphate nanoparticles were prepared for cell transfection. A calcium phosphate nanoparticle served as core which was then coated with DNA for colloidal stabilisation. The efficiency of transfection could be considerably increased by adding another layer of calcium phosphate on the surface, thereby incorporating DNA into the particle and preventing its degradation within the cell by lysosomes. A subsequent outermost layer of DNA on the calcium phosphate gave a colloidal stabilisation. The efficiency of such multi-shell particles was significantly higher than that of simple DNA-coated calcium phosphate nanoparticles. The transfection efficiency of EGFP-encoding DNA was tested with different cell lines (T-HUVEC, HeLa, and LTK). The dispersions were stable and could be used for transfection after 2 weeks of storage at 4 °C without loss of efficiency.
Keywords: Calcium phosphate; DNA; Transfection; Drug delivery; Gene therapy;

Combination of baculovirus-mediated gene transfer and rotating-shaft bioreactor for cartilage tissue engineering by Huang-Chi Chen; Hsiao-Ping Lee; Yi-Chen Ho; Ming-Lun Sung; Yu-Chen Hu (3154-3162).
We have previously demonstrated efficient baculovirus transduction of rat chondrocytes in 6-well plates. To further explore the potential of baculovirus in cartilage tissue engineering, the baculovirus-transduced chondrocytes were seeded into porous scaffolds and cultivated in a rotating-shaft bioreactor (RSB) which was developed for two-phase cultivation of tissue engineered cartilage. The baculovirus transduction resulted in efficiencies up to 90%, and affected neither cell adhesion to the scaffolds nor cell survival in the RSB. After 4-week RSB cultivation, the transduced cells remained highly differentiated and grew into constructs that resembled the untransduced constructs with regard to gross appearance, construct size, cell morphology, cell spatial distribution, glycosaminoglycan and collagen production and deposition. Importantly, baculovirus transduction did not alter the expression of chondrocytic genes. These data confirmed that baculovirus transduction neither harms chondrocytes nor retards the formation of cartilage-like tissues in the RSB, thus implicating the potentials of combining baculovirus-mediated gene transfer with RSB cultivation in in vitro cartilage tissue engineering.
Keywords: Baculovirus; Bioreactor; Cartilage; Chondrocyte; Gene therapy; Tissue engineering;