Biomaterials (v.29, #32)

Strength and fluoride release characteristics of a calcium fluoride based dental nanocomposite by Hockin H.K. Xu; Jennifer L. Moreau; Limin Sun; Laurence C. Chow (4261-4267).
Secondary caries and restoration fracture remain the two most common problems in restorative dentistry. Release of fluoride ions (F) could be a substantial benefit because F could enrich neighboring enamel or dentin to combat caries. The objective of this study was to incorporate novel CaF2 nanoparticles into dental resin to develop stress-bearing, F-releasing nanocomposite. CaF2 nanoparticles, prepared in our laboratories for the first time, were combined with reinforcing whisker fillers in a resin. Flexural strength (mean ± sd; n  = 6) was 110 ± 11 MPa for the composite containing 30% CaF2 and 35% whiskers by mass. It matched the 108 ± 19 MPa of a stress-bearing, non-releasing commercial composite (Tukey's at 0.05). The composite containing 20% CaF2 had a cumulative F release of 2.34 ± 0.26 mmol/L at 10 weeks. The initial F release rate was 2 μg/(h cm2), and the sustained release rate after 10 weeks was 0.29 μg/(h cm2). These values exceeded the reported releases of traditional and resin-modified glass ionomer materials. In summary, nanocomposites were developed with relatively high strength as well as sustained release of fluoride ions, a combination not available in current materials. These strong and F-releasing composites may yield restorations that can reduce the occurrence of both secondary caries and restoration fracture.
Keywords: Dental nanocomposite; CaF2 nanoparticles; Fluoride release; Stress bearing; Tooth caries;

The behavior of aged regenerated Bombyx mori silk fibroin solutions studied by 1H NMR and rheology by Zainuddin; Tri T. Le; Yoosup Park; Traian V. Chirila; Peter J. Halley; Andrew K. Whittaker (4268-4274).
As part of a project to utilize the regenerated silk fibroin (RSF) membranes as a supporting matrix for the attachment and growth of corneal stem/progenitor cells in the development of tissue engineered constructs for the surgical restoration of the ocular surface, the behavior of the aged RSF solutions has been investigated. The solutions were produced from domesticated silkworm (Bombyx mori) cocoons according to a protocol involving successive dissolution steps, filtration and dialysis. The solutions were kept at 4 °C in a refrigerator for a certain period of time until near the gelation time. The changes in molecular conformation were studied by solution-state 1H NMR, while the flow of the solutions was characterized by rheological method. Upon ageing turbidity developed in solutions and the viscosity continuously decreased prior to a drastic increased near the gelation time. The 1H resonances of aged solutions showed a consistent downfield shift as compared to the 1H resonances of the fresh solution. Shear thinning with anomalous short recovery within a certain range of low shear rates occurred in both fresh and aged solutions. While the solutions behave as pseudo-plastic materials, the chain conformation in aged solutions adopted all secondary configurations with β-strand being predominant.
Keywords: Silk fibroin; Ageing; 1H NMR; Rheology; Chain conformation; Pseudo-plastic flow;

The surface properties of nanocrystalline diamond and nanoparticulate diamond powder and their suitability as cell growth support surfaces by Thomas Lechleitner; Frederik Klauser; Thomas Seppi; Judith Lechner; Paul Jennings; Paul Perco; Bernd Mayer; Doris Steinmüller-Nethl; Johannes Preiner; Peter Hinterdorfer; Martin Hermann; Erminald Bertel; Kristian Pfaller; Walter Pfaller (4275-4284).
Nanocrystalline diamond (NCD) films and nanoparticulate diamond powder (DP) are the two main representatives of diamond at the nanoscale. This study was designed to investigate the suitability of these biomaterials as cell growth supports and to determine surface characteristic properties best suited to cell attachment and proliferation. Surface topography, chemical termination and wetting properties of NCD- and DP-coated borosilicate glass substrates were correlated to attachment, proliferation and differentially regulated gene expression of human renal epithelial cells (HK-2 cell line) cultured on these surfaces.Hydrogen-terminated NCD (NCD-H) surfaces were shown to inhibit cell attachment, which indicates that the lack of functional polar groups prevents adherent cells from settling on a surface, whether nanostructured or not. In contrast to NCD-H, oxygen-terminated NCD (NCD-O) as well as DP surfaces demonstrated improved cell attachment, as compared to borosilicate glass, which is a commonly used material for cell growth supports. NCD-O not only revealed an increased cell attachment, but also a markedly increased proliferation rate. Finally, none of the investigated surface modifications appeared to cause adverse cellular reactions or markedly alter cellular phenotype.
Keywords: Surface treatment; Cell adhesion; Cell proliferation; Gene expression; Cytotoxicity; Hydrophilicity;

Blood compatibility of surfaces with superlow protein adsorption by Zheng Zhang; Min Zhang; Shengfu Chen; Thomas A. Horbett; Buddy D. Ratner; Shaoyi Jiang (4285-4291).
In this work, five self-assembled monolayers (SAMs) and three polymeric brushes with very low fibrinogen adsorption were prepared. The five SAMs are oligo(ethylene glycol) (OEG), phosphorylcholine (PC), oligo(phosphorylcholine) (OPC), and two mixed positively and negatively charged SAMs of SO3 /N+(CH3)3 (SA/TMA) and COO/N+(CH3)3 (CA/TMA). Three polymer brushes were prepared on gold surfaces via surface-initiated atom transfer radical polymerization (ATRP) using three monomers, sulfobetaine methacrylate (SBMA), carboxybetaine methacrylate (CBMA), and oligo(ethylene glycol) methyl ether methacrylate (OEGMA). Surface plasmon resonance (SPR) measurements show that although all of these surfaces are “nonfouling” to fibrinogen adsorption from buffer solution, their protein adsorption from undiluted human blood plasma varies widely. Polymer brushes exhibit much lower protein adsorption from plasma than any of the five SAMs tested. However, platelet adhesion measurements on plasma-preadsorbed surfaces show that all of these surfaces have very low platelet adhesion. Clotting time measurements using recalcified platelet poor plasma (PPP) incubation with the eight types of surfaces show that they do not shorten clotting times. Linear polymers of polySBMA and polyCBMA with similar molecular weights were also synthesized and characterized. In the presence of polyCBMA linear polymers, the clotting time of PPP was prolonged and increased with the concentration of the polymer, while no anticoagulant activity was observed for the polySBMA or PEG polymers. The unique anticoagulant activity of polyCBMA, as well as its high plasma protein adsorption resistance, makes polyCBMA a candidate for blood-contacting applications.
Keywords: Blood compatibility; Anticoagulant; Anti-adhesion; Self assembly; Protein adsorption;

The effect of devitalized trabecular bone on the formation of osteochondral tissue-engineered constructs by Eric G. Lima; Pen-hsiu Grace Chao; Gerard A. Ateshian; B. Sonny Bal; James L. Cook; Gordana Vunjak-Novakovic; Clark T. Hung (4292-4299).
In the current study, evidence is presented demonstrating that devitalized trabecular bone has an inhibitory effect on in vitro chondral tissue development when used as a base material for the tissue-engineering of osteochondral constructs for cartilage repair. Chondrocyte-seeded agarose hydrogel constructs were cultured alone or attached to an underlying bony base in a chemically defined medium formulation that has been shown to yield engineered cartilaginous tissue with native Young's modulus (E Y) and glycosaminoglycan (GAG) content. By day 42 in culture the incorporation of a bony base significantly reduced these properties (E Y  = 87 ± 12 kPa, GAG = 1.9 ± 0.8%ww) compared to the gel-alone group (E Y  = 642 ± 97 kPa, GAG = 4.6 ± 1.4%ww). Similarly, the mechanical and biochemical properties of chondrocyte-seeded agarose constructs were inhibited when co-cultured adjacent to bone (unattached), suggesting that soluble factors rather than direct cell–bone interactions mediate the chondro-inhibitory bone effects. Altering the method of bone preparation, including demineralization, or the timing of bone introduction in co-culture did not ameliorate the effects. In contrast, osteochondral constructs with native cartilage properties (E Y  = 730 ± 65 kPa, GAG = 5.2 ± 0.9%ww) were achieved when a porous tantalum metal base material was adopted instead of bone. This work suggests that devitalized bone may not be a suitable substrate for long-term cultivation of osteochondral grafts.
Keywords: Cartilage tissue-engineering; Biocompatibility; Bone;

Porous collagen-apatite nanocomposite foams as bone regeneration scaffolds by Y.S. Pek; Shujun Gao; M.S. Mohamed Arshad; Kwong-Joo Leck; Jackie Y. Ying (4300-4305).
We have created a porous bioresorbable nanocomposite bone scaffold that chemically, structurally and mechanically matched natural bone so that it could be recognized and remodeled by natural bone. Containing collagen fibers and synthetic apatite nanocrystals, our scaffold has high strength for supporting the surrounding tissue. The foam-like scaffold has a similar microstructure as trabecular bone, with nanometer-sized and micron-sized pores. The apatitic phase of the scaffold exhibited similar chemical composition, crystalline phase and grain size as the trabecular bone apatite. The nanocomposite scaffold demonstrated excellent bioactivity for promoting cell attachment and proliferation. It was osteoconductive and successfully healed a non-union fracture in rat femur as well as a critical-sized defect in pig tibia.
Keywords: Biomimetic material; Bone healing; Hydroxyapatite; Nanocomposite; Scaffold;

Endothelial cell colonization and angiogenic potential of combined nano- and micro-fibrous scaffolds for bone tissue engineering by Marina I. Santos; Kadriye Tuzlakoglu; Sabine Fuchs; Manuela E. Gomes; Kirsten Peters; Ronald E. Unger; Erhan Piskin; Rui L. Reis; C. James Kirkpatrick (4306-4313).
Presently the majority of tissue engineering approaches aimed at regenerating bone relies only on post-implantation vascularization. Strategies that include seeding endothelial cells (ECs) on biomaterials and promoting their adhesion, migration and functionality might be a solution for the formation of vascularized bone. Nano/micro-fiber-combined scaffolds have an innovative structure, inspired by extracellular matrix (ECM) that combines a nano-network, aimed to promote cell adhesion, with a micro-fiber mesh that provides the mechanical support. In this work we addressed the influence of this nano-network on growth pattern, morphology, inflammatory expression profile, expression of structural proteins, homotypic interactions and angiogenic potential of human EC cultured on a scaffold made of a blend of starch and poly(caprolactone). The nano-network allowed cells to span between individual micro-fibers and influenced cell morphology. Furthermore, on nano-fibers as well as on micro-fibers ECs maintained the physiological expression pattern of the structural protein vimentin and PECAM-1 between adjacent cells. In addition, ECs growing on the nano/micro-fiber-combined scaffold were sensitive to pro-inflammatory stimulus. Under pro-angiogenic conditions in vitro, the ECM-like nano-network provided the structural and organizational stability for ECs' migration and organization into capillary-like structures. The architecture of nano/micro-fiber-combined scaffolds elicited and guided the 3D distribution of ECs without compromising the structural requirements for bone regeneration.
Keywords: Starch-based scaffolds; Vascularization; Nano-fibers; Endothelial cells; Bone tissue engineering;

Electrospun biomimetic nanocomposite nanofibers of hydroxyapatite/chitosan for bone tissue engineering by Yanzhong Zhang; Jayarama Reddy Venugopal; Adel El-Turki; Seeram Ramakrishna; Bo Su; Chwee Teck Lim (4314-4322).
The development of bioinspired or biomimetic materials is essential and has formed one of the most important paradigms in today's tissue engineering research. This paper reports a novel biomimetic nanocomposite nanofibers of hydroxyapatite/chitosan (HAp/CTS) prepared by combining an in situ co-precipitation synthesis approach with an electrospinning process. A model HAp/CTS nanocomposite with the HAp mass ratio of 30 wt% was synthesized through the co-precipitation method so as to attain homogenous dispersion of the spindle-shaped HAp nanoparticles (ca. 100 × 30 nm) within the chitosan matrix. By using a small amount (10 wt%) of ultrahigh molecular weight poly(ethylene oxide) (UHMWPEO) as a fiber-forming facilitating additive, continuous HAp/CTS nanofibers with a diameters of 214 ± 25 nm had been produced successfully and the HAp nanoparticles with some aggregations were incorporated into the electrospun nanofibers. Further SAED and XRD analysis confirmed that the crystalline nature of HAp remains and had survived the acetic acid-dominant solvent system. Biological in vitro cell culture with human fetal osteoblast (hFOB) cells for up to 15 days demonstrated that the incorporation of HAp nanoparticles into chitosan nanofibrous scaffolds led to significant bone formation oriented outcomes compared to that of the pure electrospun CTS scaffolds. The electrospun nanocomposite nanofibers of HAp/CTS, with compositional and structural features close to the natural mineralized nanofibril counterparts, are of potential interest for bone tissue engineering applications.
Keywords: Nanofibers; Nanocomposite; Electrospinning; Hydroxyapatite; Chitosan; Bone tissue engineering;

Development of a chitosan-based wound dressing with improved hemostatic and antimicrobial properties by Shin-Yeu Ong; Jian Wu; Shabbir M. Moochhala; Mui-Hong Tan; Jia Lu (4323-4332).
Hemorrhage remains a leading cause of early death after trauma, and infectious complications in combat wounds continue to challenge caregivers. Although chitosan dressings have been developed to address these problems, they are not always effective in controlling bleeding or killing bacteria. We aimed to refine the chitosan dressing by incorporating a procoagulant (polyphosphate) and an antimicrobial (silver). Chitosan containing different amounts and types of polyphosphate polymers was fabricated, and their hemostatic efficacies evaluated in vitro. The optimal chitosan-polyphosphate formulation (ChiPP) accelerated blood clotting (p  = 0.011), increased platelet adhesion (p  = 0.002), generated thrombin faster (p  = 0.002), and absorbed more blood than chitosan (p  < 0.001). Silver-loaded ChiPP exhibited significantly greater bactericidal activity than ChiPP in vitro, achieving a complete kill of Pseudomonas aeruginosa and a >99.99% kill of Staphylococcus aureus consistently. The silver dressing also significantly reduced mortality from 90% to 14.3% in a P. aeruginosa wound infection model in mice. Although the dressing exerted severe cytotoxicity against cultured fibroblasts, wound healing was not inhibited. This study demonstrated for the first time, the application of polyphosphate as a hemostatic adjuvant, and developed a new chitosan-based composite with potent hemostatic and antimicrobial properties.
Keywords: Wound dressing; Chitosan; Hemostatic; Antimicrobial;

The effect of PEGylation on the aqueous solution properties and cell membrane disruptive activity of a pH-responsive pseudo-peptide, poly(l-lysine iso-phthalamide), has been investigated by dynamic light scattering, haemolysis and lactate dehydrogenase (LDH) assays. Intracellular trafficking of the polymers has been examined using confocal and fluorescence microscopy. With increasing degree of PEGylation, the modified polymers can form stabilised compact structures with reduced mean hydrodynamic diameters. Poly(l-lysine iso-phthalamide) with a low degree of PEGylation (17.4 wt%) retained pH-dependent solution behaviour and showed enhanced kinetic membrane disruptive activity compared to the parent polymer. It facilitated trafficking of endocytosed materials into the cytoplasm of HeLa cells. At levels of PEGylation in excess of 25.6 wt%, the modified polymers displayed a single particle size distribution unresponsive to pH, as well as a decrease in cell membrane lytic ability. The mechanism involved in membrane destabilisation was also investigated, and the potential applications of these modified polymers in drug delivery were discussed.
Keywords: Peptide; Polyethylene oxide; Hydrophilicity; Micelle; Haemolysis; Drug delivery;

Bioactive, luminescent and mesoporous europium-doped hydroxyapatite as a drug carrier by Piaoping Yang; Zewei Quan; Chunxia Li; Xiaojiao Kang; Hongzhou Lian; Jun Lin (4341-4347).
Bioactive, luminescent and mesoporous europium-doped hydroxyapatite (Eu:HAp) was successfully prepared through a simple one-step route using cationic surfactant as template. The obtained multifunctional hydroxyapatite was performed as a drug delivery carrier to investigate the drug storage/release properties using ibuprofen (IBU) as a model drug. The structural, morphological, textural and optical properties were well characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectra (XPS), Fourier transform infrared spectroscopy (FT-IR), N2 adsorption/desorption, and photoluminescence (PL) spectra, respectively. The results reveal that the multifunctional hydroxyapatites exhibit the typical ordered characteristics of the hexagonal mesostructure, and have rod-like morphology with the particle size of 20–40 nm in width and 100–200 nm in length. The drug storage/release test indicates that the luminescent HAp shows much similar drug loading amount and cumulative release rate to those of pure HAp. Interestingly, the IBU-loaded samples still show red luminescence of Eu3+ (5D07F1,2) under UV irradiation, and the emission intensities of Eu3+ in the drug carrier system vary with the released amount of IBU, thus making the drug release be easily tracked and monitored by the change of the luminescence intensity.
Keywords: Hydroxyapatite; Porosity; Luminescence; In vitro test; Drug delivery;

Self-assembled biodegradable micellar nanoparticles of amphiphilic and cationic block copolymer for siRNA delivery by Tian-Meng Sun; Jin-Zhi Du; Li-Feng Yan; Hai-Quan Mao; Jun Wang (4348-4355).
A novel amphiphilic and cationic triblock copolymer consisting of monomethoxy poly(ethylene glycol), poly(ɛ-caprolactone) (PCL) and poly(2-aminoethyl ethylene phosphate) denoted as mPEG45-b-PCL100-b-PPEEA12 was designed and synthesized for siRNA delivery. The copolymers were well characterized by 1H NMR spectroscopy and gel permeation chromatography. Micelle nanoparticles' (MNPs) formation of this amphiphilic copolymer in aqueous solution was studied by dynamic light scattering, transmission electron microscopy and fluorescence technique. MNPs took uniform spherical morphology with zeta potential of around 45 mV and were stabilized by hydrophobic–hydrophobic interaction in the PCL core, exhibiting the critical micelle concentration at 2.7 × 10−3  mg/mL. Such MNPs allowed siRNA loading post nanoparticle formation without change in uniformity. The average diameter of nanoparticles after siRNA binding ranged from 98 to 125 nm depending on N/P ratios. The siRNA loaded nanoparticles can be effectively internalized and subsequently release siRNA in HEK293 cells, resulting in significant gene knockdown activities, which was demonstrated by delivering two siRNAs targeting green fluorescence protein (GFP). It effectively silenced GFP expression in 40–70% GFP-expressed HEK293 cells and it was observed that higher N/P ratio resulted in more effective silence which was likely due to better cell internalization at higher N/P ratio. MTT assay demonstrated that neither MNPs themselves nor siRNA loaded MNPs showed cytotoxicity even at high concentrations. Such cationic MNPs made from biocompatible and biodegradable polymers are promising for siRNA delivery.
Keywords: siRNA delivery; Micellar nanoparticles; Biodegradable polymer; Self-assembly; Polyphosphoester;

The influence of RGD addition on the gene transfer characteristics of disulfide-containing polyethyleneimine/DNA complexes by Yun-Xia Sun; Xuan Zeng; Qing-Fei Meng; Xian-Zheng Zhang; Si-Xue Cheng; Ren-Xi Zhuo (4356-4365).
Arginine–glycine–aspartic acid (RGD) ligand is often chemically attached to polycation vector to improve the transfection efficiency. However, the chemical reaction may reduce or even inactivate the biological activities of peptides. In order to retain the targeting ability and biological activities, the RGD peptide was noncovalently introduced into polycations as gene delivery systems. In this paper, the tripeptide sequence RGD was added to disulfide-containing polyethyleneimine (SS-PEI)/DNA binary complexes to evaluate the influence of RGD addition for the particle size, zeta potential, morphology, and transfection efficiency. GelRed™ was used as a molecular probe to show the effect of RGD addition on the cellular uptake of complexes. In vitro transfection experiments showed that SS-PEI exhibited comparable transfection efficiency, but lower cytotoxicity in comparison with 25 kDa PEI. The transfection efficiency of complexes with RGD in HeLa cells was reduced statistically significantly with the increasing content of RGD peptide, but that in 293T cells was not altered significantly with the increasing content of RGD peptide. The reduced transfection efficiency of SS-PEI/DNA complexes with RGD in HeLa cells was attributed to the targeted binding interactions between the surplus RGD and the ανβ3 and ανβ5 integrins in HeLa cells, which would prevent the binding between RGD in complexes and integrin receptor on the surface of cells as well as nonspecific endocytosis of SS-PEI/DNA complexes mediated by proteoglycan in HeLa cells.
Keywords: Gene transfer; RGD peptide; Integrins; Polyethyleneimine;