Biomaterials (v.28, #5)

This contribution demonstrates a simple and reproducible method for fabricating surface-tethered polymer brushes that vary in grafting density and layer thickness for peptide adsorption and cell-adhesion studies. Surface-initiated atom transfer radical polymerization was used together with thiol self-assembly to generate these nanothin polymer brush layers of poly((polyethylene glycol) methacrylate). A kinetic study was done to measure the layer thickness growth rate at room temperature from flat gold substrates presenting different polymerization initiator molecule surface densities. The polymer brush layers transition from mushroom to brush regimes with increasing grafting density. A crossover density of 0.038±0.005 chains/nm2 was determined for the PPEGMA polymer brushes. The results described in this paper show that layer properties such as wettability and dry layer thickness depend strongly on initiator surface density. Ultimately, the adsorbed concentration of an RGD-containing synthetic peptide Gly–Arg–Gly–Asp–Ser and the adhesion and spreading of cells were correlated with surface properties, which continues to be a major research theme in biomedical and biomaterials research.
Keywords: Cell adhesion; Protein adsorption; RGD peptide; Surface analysis; Surface grafting;

Effects of solvent dehydration on creep resistance of poly(vinyl alcohol) hydrogel by Jeeyoung Choi; Hatice Bodugoz-Senturk; Hsiang J. Kung; Arnaz S. Malhi; Orhun K. Muratoglu (772-780).
As a synthetic replacement material for osteochondral defect repair, poly(vinyl alcohol) (PVA) hydrogels offer a great potential due to their high water content and strong mechanical integrity. To survive the high stress environment in the joint space, high creep resistance becomes one of the key requirements for hydrogel implants. We hypothesized that reducing the equilibrium water content (EWC) of hydrogels would improve their creep resistance. We investigated the effect of dehydration of PVA theta-gels in various solvent/solution media followed by rehydration in saline solution. Decreasing EWC increased the creep resistance of PVA theta-gels. The most effective medium was isopropyl alcohol for reducing the EWC and increasing the creep resistance of PVA theta-gels.
Keywords: Poly(vinyl alcohol); Poly(ethylene glycol); Hydrogel; Dehydration; Osteochondral defects;

Modulation of nano-hydroxyapatite size via formation on chitosan–gelatin network film in situ by Junjie Li; YiPing Chen; Yuji Yin; Fanglian Yao; Kangde Yao (781-790).
Natural bone is actually an inorganic/organic composite mainly make up of nano-hydroxyapatite (Ca10(PO4)6(OH)2, nHA) and collagen fibers. It is most important to form nHA/polymer composites in order to provide good biocompatibility and integration with bone tissue. In this work, nHA was formed in-situ on the surface of chitosan–gelatin (CG) network films in tris-buffer solution containing Ca(NO3)2-Na3PO4. The interaction between CG network film and nHA crystalline were studided using the diffuse reflectance FT-IR (ATR-FTIR), thermal analysis (TGA) and X-ray diffraction analysis (XRD), and the influence the nHA size factors, e.g. the ratio of chitosan (CS) and gelatin (Gel), concentration of calcium ions and reaction temperature, were elucidated by XRD and transmission electron microscope (TEM). Results suggested that carboxyl groups, C=O and amino groups play crucial roles for HA formatting on the surface of CG network films and the average size of nHA crystalline decreasing with enhancing Gel content and increase with the increasing calcium and phosphate concentration, and when the reaction temperature below 50 °C the nHA crystalline size is almost fixedness (range from 17.2 to 19.2 nm) but when the temperature arrived at 70 °C it increase to 52.3 nm.
Keywords: Nano-hydroxyapatite; Chitosan; Gelatin; Formation in-situ; Bone tissue engineering;

Biomolecular immobilization on conducting polymers for biosensing applications by Tarushee Ahuja; Irfan Ahmad Mir; Devendra Kumar; Rajesh (791-805).
A detail study on different aspects of biomolecule immobilization techniques on conducting polymers (CP) for applications in biosensors is described. Comparative studies are conducted in between the different mode of biomolecule immobilization techniques, viz. physical, covalent and electrochemical immobilization onto the conducting polymer films for the fabrication of electrochemical biosensors for clinical, food and environmental monitoring applications. This review focuses on the current status of biomolecule immobilization techniques on CP and their applications in the development of amperometric biosensors.
Keywords: Conducting polymers; Biosensors; Electrochemical polymerization; Physical adsorption; Covalent immobilization; Electrochemical immobilization;

Response of human endothelial cells to oxidative stress on Ti6Al4V alloy by Roman Tsaryk; Marie Kalbacova; Ute Hempel; Dieter Scharnweber; Ronald E. Unger; Peter Dieter; C.James Kirkpatrick; Kirsten Peters (806-813).
Titanium and its alloys are amongst the most frequently used materials in bone and dental implantology. The good biocompatibility of titanium(-alloys) is attributed to the formation of a titanium oxide layer on the implant surface. However, implant failures do occur and this appears to be due to titanium corrosion. Thus, cells participating in the wound healing processes around an implanted material, among them endothelial cells, might be subjected to reactive oxygen species (ROS) formed by electrochemical processes during titanium corrosion. Therefore, we studied the response of endothelial cells grown on Ti6Al4V alloy to H2O2 and compared this with the response of endothelial cells grown on cell culture polystyrene (PS). We could show that although the cell number was the same on both surfaces, metabolic activity of endothelial cells grown on Ti6Al4V alloy was reduced compared to the cells on PS and further decreased following prototypic oxidative stress (H2O2-treatment). The analysis of H2O2-induced oxidative stress showed a higher ROS formation in endothelial cells on Ti6Al4V than on PS. This correlated with the depletion of reduced glutathione (GSH) in endothelial cells grown on Ti6Al4V surfaces and indicated permanent oxidative stress. Thus, endothelial cells in direct contact with Ti6Al4V showed signs of oxidative stress and higher impairment of cell vitality after an additional oxidative stress. However, the exact nature of the agent of oxidative stress generated from Ti6Al4V remains unclear and requires further investigation.
Keywords: Titanium alloy; Endothelial cells; Free radicals; Corrosion; In vitro; Oxidative stress;

Combined marrow stromal cell-sheet techniques and high-strength biodegradable composite scaffolds for engineered functional bone grafts by Yefang Zhou; Fulin Chen; Saey Tuan Ho; Maria Ann Woodruff; Tit Meng Lim; Dietmar W. Hutmacher (814-824).
In this study, cell sheets comprising multilayered porcine bone marrow stromal cells (BMSC) were assembled with fully interconnected scaffolds made from medical-grade polycaprolactone–calcium phosphate (mPCL–CaP), for the engineering of structural and functional bone grafts. The BMSC sheets were harvested from culture flasks and wrapped around pre-seeded composite scaffolds. The layered cell sheets integrated well with the scaffold/cell construct and remained viable, with mineralized nodules visible both inside and outside the scaffold for up to 8 weeks culture. Cells within the constructs underwent classical in vitro osteogenic differentiation with the associated elevation of alkaline phosphatase activity and bone-related protein expression. In vivo, two sets of cell-sheet-scaffold/cell constructs were transplanted under the skin of nude rats. The first set of constructs (5×5×4 mm3) were assembled with BMSC sheets and cultured for 8 weeks before implantation. The second set of constructs (10×10×4 mm3) was implanted immediately after assembly with BMSC sheets, with no further in vitro culture. For both groups, neo cortical and well-vascularised cancellous bone were formed within the constructs with up to 40% bone volume. Histological and immunohistochemical examination revealed that neo bone tissue formed from the pool of seeded BMSC and the bone formation followed predominantly an endochondral pathway, with woven bone matrix subsequently maturing into fully mineralized compact bone; exhibiting the histological markers of native bone. These findings demonstrate that large bone tissues similar to native bone can be regenerated utilizing BMSC sheet techniques in conjunction with composite scaffolds whose structures are optimized from a mechanical, nutrient transport and vascularization perspective.
Keywords: Bone engineering; Bone marrow stromal cells; Mesenchymal stem cell; Composites; Biodegradable polymers; Functional tissue engineering;

A surface-tethered model to assess size-specific effects of hyaluronan (HA) on endothelial cells by Samir Ibrahim; Binata Joddar; Matthew Craps; Anand Ramamurthi (825-835).
Crosslinked gels (hylans) containing long-chain (MW>1×106  Da) hyaluronan (HA), a connective tissue GAG, show exceptional biocompatibility for vascular implantation but poorly interact with vascular endothelial cells (ECs). Previous studies showed in situ fragmentation of HA by UV light to bioactivate hylan gels and elicit enhanced EC responses. Since fragmented HA can be pro-inflammatory, it is important to define an optimal size distribution of HA fragments on the hylan surface that will recruit and support normally functional ECs and limit ulterior responses. Related studies have shown that exogenous models of HA do not necessarily replicate cell responses to HA scaffolds. Since scaffolds cannot be created based on fragmented HA alone, we sought to determine size-specific responses of ECs to HA substrates of defined fragment sizes by creation of HA-tethered culture surfaces. HA (1000, 200, 20 kDa) and an oligomer mixture were tethered onto an aminosilane (APTMS)-treated glass surfaces using a carbodiimide reaction. MALDI–TOF showed the HA digests to contain HA 4–8mers with a 75±0.4% w/w of 4mers. Immuno-fluorescence, SEM, AFM and XPS analysis revealed homogeneous amine and HA surfaces. An amine s-SDTB assay and HA fluorophore-assisted carbohydrate electrophoresis (FACE) indicated surface densities of 9±3 amine groups/nm2 and 0.57±0.44 μg/cm2, respectively. HA/HA fragments/oligomers were stable over 21 days of incubation in serum-free culture media. EC proliferation on these surfaces resulted was limited, a possible effect of smooth surface topography, high anionicity, and in case of 4mers, non-interaction with primary HA cell–surface receptors (CD44). This work is significant in that it allows testing of cell responses to substrates composed of single-sized fragments of HA that cannot by themselves be cross-linked into a gel. Future work in our lab will use this model to assess the effects of other HA oligomer sizes on EC behavior.
Keywords: Hyaluronic acid; Oligomers; FACE; SEM; AFM; XPS;

Engineered matrix coatings to modulate the adhesion of CD133+ human hematopoietic progenitor cells by Katja Franke; Tilo Pompe; Martin Bornhäuser; Carsten Werner (836-843).
Interactions of hematopoietic progenitor cells (HPC) with their local microenvironments in the bone marrow are thought to control homing, differentiation, and self-renewal of the cells. To dissect the role of extracellular matrix (ECM) components of the niche microenvironment, a set of well-defined ECM coatings including fibronectin, heparin, heparan sulphate, hyaluronic acid, tropocollagen I, and co-fibrils of collagen I with heparin or hyaluronic acid was prepared and analysed with respect to the attachment of human CD133+ HPC in vitro. The extension of the adhesion areas of individual cells as well as the fraction of adherent cells were assessed by reflection interference contrast microscopy (RICM). Intense cell–matrix interactions were found on surfaces coated with fibronectin, heparin, heparan sulphate, and on the collagen I based co-fibrils. Insignificant adhesion was found for tropocollagen I and hyaluronic acid. The strongest adhesion of HPC was observed on fibronectin with contact areas of about 7 μm2. Interaction of HPC with coatings consisting of heparin, heparan sulphate, and co-fibrils result in small circular shaped contact zones of 3 μm2 pointing to another, less efficient, adhesion mechanism. Analysing the specificity of cell–matrix interaction by antibody blocking experiments suggests an integrin(α 5 β 1)-specific adhesion on fibronectin, while adhesion on heparin was shown to be mediated by selectins (CD62L). Taken together, our data provide a basis for the design of advanced culture carriers supporting site-specific proliferation or differentiation of HPC.
Keywords: Hematopoietic stem cell; Cell adhesion; Fibronectin; Heparin; Hyaluronic acid; Collagen;

Effects of sulfated hyaluronan on keratinocyte differentiation and Wnt and Notch gene expression by Tsutomu Nagira; Misao Nagahata-Ishiguro; Toshie Tsuchiya (844-850).
Sulfated hyaluronan (SHya), which is composed of a sulfated group and hyaluronan (Hya), has high activity on and biocompatibility with cells. When normal human epidermal keratinocytes (NHEKs) were incubated in dishes coated with SHya, cell proliferation was suppressed in a dose-dependent manner. The expression levels of keratin 1 and loricrin mRNAs, as detected by real-time RT-PCR, were increased significantly. The expressions of Wnt mRNAs, which play important roles in cell proliferation and differentiation, were modulated. Wnt4 and Wnt6 mRNA expressions were increased compared to controls, while expression of Wnt5a was similar to the control and that of Wnt7a mRNA was decreased. In addition, the expression of Notch mRNAs, which play a critical role in keratinocyte differentiation, were affected. Notch3 mRNA was increased significantly, while Notch1 mRNA was decreased compared to controls, and expression of Notch2 was similar to that of control. These results suggested that a SHya-coated scaffold might be useful for regulating cell activity in tissue engineering.
Keywords: Sulfated hyaluronan; Normal human epidermal keratinocyte; Differentiation; Wnt; Notch;

Fibronectin terminated multilayer films: Protein adsorption and cell attachment studies by Corinne R. Wittmer; Jennifer A. Phelps; W. Mark Saltzman; Paul R. Van Tassel (851-860).
Electrostatically driven layer-by-layer (LbL) assembly is a simple and robust method for producing structurally tailored thin film biomaterials, of thickness ca. 10 nm, containing biofunctional ligands. We investigate the LbL formation of multilayer films composed of polymers of biological origin (poly(l-lysine) (PLL) and dextran sulfate (DS)), the adsorption of fibronectin (Fn)—a matrix protein known to promote cell adhesion—onto these films, and the subsequent spreading behavior of human umbilical vein endothelial cells (HUVEC). We employ optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microgravimetry with dissipation (QCMD) to characterize multilayer assembly in situ, and find adsorbed Fn mass on PLL-terminated films to exceed that on DS terminated films by 40%, correlating with the positive charge and lower degree of hydration of PLL terminated films. The extent and initial rate of Fn adsorption to both PLL and DS-terminated films exceed those onto the bare substrate, indicating the important role of electrostatic complexation between negatively charged protein and positively charged PLL at or near the film surface. We use phase-contrast optical microscopy to investigate the time-dependent morphological changes of HUVEC as a function of layer number, charge of terminal layer, and the presence of Fn. We observe HUVEC to attach, spread, and lose circularity on all surfaces. Positively charged PLL-terminated films exhibit a greater extent of cell spreading than do (negatively charged) DS-terminated films, and spreading is enhanced while circularity loss is suppressed by the presence of adsorbed Fn. The number of layers plays a significant role only for DS-terminated films with Fn, where spreading on a bilayer greatly exceeds that on a multilayer, and PLL-terminated films without Fn, where initial spreading is significantly higher on a monolayer. We observe initial cell spreading to be followed by retraction (i.e. decreased cell area and circularity with time) for films without Fn, and for DS-terminated films with Fn. Overall, the Fn-coated PLL monolayer and the Fn-coated PLL-terminated multilayer are the best performing films in promoting cell spreading. We conclude the presence of Fn to be an important factor (more so than film charge or layer number) in controlling the interaction between multilayer films and living cells, and thus to represent a promising strategy toward in vivo applications such as tissue engineering.
Keywords: Fibronectin; Poly(l-lysine); Dextran sulfate; Protein adsorption; Endothelial cell; Layer-by-layer;

Esophageal epithelium regeneration on fibronectin grafted poly(l-lactide-co-caprolactone) (PLLC) nanofiber scaffold by Yabin Zhu; Meng Fatt Leong; Wey Feng Ong; Mary B. Chan-Park; Kerm Sin Chian (861-868).
In order to mimic normal epithelium regeneration on synthetic scaffold in vitro, biodegradable elastic poly(l-lactide-co-caprolactone) (PLLC) was processed into nanofibrous scaffold using electrospinning technology. An adhesive protein, fibronectin (Fn), was grafted onto the scaffold fiber surface via a two-step reaction: polyester aminolysis followed by Fn coupling via glutaraldehyde. Tensile testing was performed to measure the effect of aminolysis on the scaffold mechanical properties. The strain decreased but the tensile strength remained almost constant after aminolysis. However, no obvious difference of the nanofiber surface morphology was found after Fn grafting using scanning electron microscopy (SEM). Porcine esophageal epithelial cells were seeded on the Fn bonded scaffold to test the cell growth promotion against the control unmodified PLLC nanofiber scaffold using tissue culture polystyrene (TCPS) plate as a reference. Anti-cytokeratin AE1/AE3 was used as the primary antibody to confirm the esophageal epithelial phenotype. SEM observation, immunostaining and Western Blotting to compare the collagen type IV synthesis showed that the Fn grafted on PLLC scaffold greatly promotes epithelium regeneration. This modified scaffold is expected to be a good candidate for functional esophagus substitutes.
Keywords: Nanofiber; Electrospinning; Epithelial cell; Tissue engineering; Esophagus;

Formulation of functionalized PLGA–PEG nanoparticles for in vivo targeted drug delivery by Jianjun Cheng; Benjamin A. Teply; Ines Sherifi; Josephine Sung; Gaurav Luther; Frank X. Gu; Etgar Levy-Nissenbaum; Aleksandar F. Radovic-Moreno; Robert Langer; Omid C. Farokhzad (869-876).
Nanoparticle (NP) size has been shown to significantly affect the biodistribution of targeted and non-targeted NPs in an organ specific manner. Herein we have developed NPs from carboxy-terminated poly(d,l-lactide–co–glycolide)–block–poly(ethylene glycol) (PLGA–b–PEG–COOH) polymer and studied the effects of altering the following formulation parameters on the size of NPs: (1) polymer concentration, (2) drug loading, (3) water miscibility of solvent, and (4) the ratio of water to solvent. We found that NP mean volumetric size correlates linearly with polymer concentration for NPs between 70 and 250 nm in diameter (linear coefficient=0.99 for NPs formulated with solvents studied). NPs with desirable size, drug loading, and polydispersity were conjugated to the A10 RNA aptamer (Apt) that binds to the prostate specific membrane antigen (PSMA), and NP and NP-Apt biodistribution was evaluated in a LNCaP (PSMA+) xenograft mouse model of prostate cancer. The surface functionalization of NPs with the A10 PSMA Apt significantly enhanced delivery of NPs to tumors vs. equivalent NPs lacking the A10 PSMA Apt (a 3.77-fold increase at 24 h; NP-Apt 0.83%±0.21% vs. NP 0.22%±0.07% of injected dose per gram of tissue; mean±SD, n = 4 , p = 0.002 ). The ability to control NP size together with targeted delivery may result in favorable biodistribution and development of clinically relevant targeted therapies.
Keywords: Drug delivery; Nanoparticle; PLGA; Prostate cancer; Targeting; Aptamer;

Improvement of gentamicin poly(d,l-lactic-co-glycolic acid) microspheres for treatment of osteomyelitis induced by orthopedic procedures by Maria Rosa Virto; Begoña Elorza; Susana Torrado; Maria de Los Angeles Elorza; Gloria Frutos (877-885).
Poly(d,l-lactide-co-glycolide) (PLGA) biodegradable microspheres with gentamicin for local treatment of microbial bone infection were prepared and characterized. Gentamicin was assayed spectrophotometrically at 332 nm after derivation with the o-phthalaldehyde; biodegradable polymers studied did not interfere with this method of gentamicin analysis. PLGA microspheres were made by the double emulsion solvent evaporation method with modifications. The first W 1/O emulsion was obtained by ultrasonication or high-speed homogenization, and a large aqueous phase W 2 (200 ml) was used. The ultrasonication method increases the microsphere percentage observed in the 20–40 μm size range and, in all cases SEM-microphotographs revealed homogeneous and spherically shaped particles with smooth surfaces. The method including ultrasonication proposed in the present work improved the encapsulation efficiency of gentamicin by nearly 100% (97.94%). Several mathematical models based on heterogeneous hydrolytic degradation were applied to evaluate their suitability in describing gentamicin released from PLGA microspheres. Two models, one of them including an autocatalytic process, were finally proposed to contribute to understand the mass transport mechanism involved in drug release from these microspheres.
Keywords: PLGA microspheres; Gentamicin; Encapsulation efficiency; Autocatalytic release mechanism; Mathematical modeling;

In vivo performance of implantable biodegradable preparations delivering Paclitaxel and Etanidazole for the treatment of glioma by Pavan Kumar Naraharisetti; Benjamin Yung Sheng Ong; Jing Wei Xie; Timothy Kam Yiu Lee; Chi-Hwa Wang; Nikolaos V. Sahinidis (886-894).
Drug-releasing implants delivering chemotherapeutic and radio-sensitizing agents are beginning to play a major role in the post-surgical eradication of residual glioma in the brain. Benefits from early arresting of tumor growth and tumor recovery dynamics stress the impact of drug release profiles of the implants on the efficacy of the treatment. This paper examines responses of BALB/c nude mice, bearing C6 glioma tumors subcutaneously, to treatments by PLGA microspheres, microparticles and discs-delivering Paclitaxel and Etanidazole. The experimental results are used to correlate the efficacy of treatment to in vitro release profiles from the various formulations. Our study demonstrates that radio-sensitizing effects during irradiation could be achieved by double burst profiles from Etanidazole-loaded discs, when compared to controls 17 days after implantation despite the short half-life of Etanidazole (1.4 h) in vivo. These results also showed inhibited tumor growth on tumor volumes of 59%, 65% and 70% over the blank placebo groups after 21 days of tumor growth for spray-dried microspheres, electrohydrodynamic atomization microparticles and spray-dried discs, respectively.
Keywords: Glioma; Etanidazole; Paclitaxel; In vivo; Subcutaneous; Drug delivery;