Acta Biomaterialia - Articles in Press

Ingrowth of Human Mesenchymal Stem Cells into Porous Silk Particle Reinforced Silk Composite Scaffolds: An In Vitro Study - Accepted Manuscript

2010-07-26

Abstract: Silk fibroin protein is biodegradable and biocompatible, exhibiting excellent mechanical properties for various biomedical applications. However, porous 3D silk fibroin scaffolds, or silk sponges, usually fall short in matching the initial mechanical requirements for bone tissue engineering. In the present study, silk sponge matrices were reinforced with silk microparticles to generate protein-protein composite scaffolds with desirable mechanical properties for in vitro osteogenic tissue formation. It was found that increasing the silk microparticle loading led to a substantial increase in the scaffold compressive modulus from 0.3 MPa (nonreinforced) to 1.9 MPa for 1:2 (matrix:particle) reinforcement loading by dry mass. Biochemical, gene expression, and histological assays were employed to study the possible effects of increasing composite scaffold stiffness, due to microparticle reinforcement, on in vitro osteogenic differentiation of human mesenchymal stem cells (hMSCs). Increasing silk microparticle loading increased the osteogenic capability of hMSCs in the presence of bone morphogenic protein-2 (BMP-2) and other osteogenic factors in static culture for up to six weeks. The calcium adsorption increased dramatically with increasing loading, as observed from biochemical assays, histological staining, and microCT (μCT) analysis. Specifically, calcium content in the scaffolds increased by 0.57, 0.71, and 1.27 mg (per μg of DNA) from 3 to 6 weeks for matrix to particle dry mass loading ratios of 1:0, 1:1 and 1:2, respectively. In addition, μCT imaging revealed that at 6 weeks, bone volume fraction increased from 0.78% for nonreinforced to 7.1% and 6.7% for 1:1 and 1:2 loading, respectively. Our results support the hypothesis that scaffold stiffness may strongly influence the 3D in vitro differentiation capabilities of hMSCs, providing a means to improve osteogenic outcomes.

Microelastic properties of lung cell-derived extracellular matrix - Accepted Manuscript

Patricia A. Soucy, Jeffery Werbin, William Heinz, Jan H. Hoh, Lewis H. Romer — 2010-07-26

Abstract: Mechanical properties of the extracellular microenvironment regulate cell behaviors including migration, proliferation, and morphogenesis. Although elastic moduli of synthetic materials have been studied, little is known about the properties of naturally produced extracellular matrix. Here, we utilized atomic force microscopy to characterize the microelastic properties of decellularized cell-derived matrix from human pulmonary fibroblasts. This heterogeneous three-dimensional matrix had an average thickness of 5±0.4 μm and a Young’s modulus of 105±14 Pa. Ascorbate treatment of the lung fibroblasts prior to extraction produced a two-fold increase in collagen I content, but did not affect the stiffness of the matrices compared to matrices produced in standard medium. However, fibroblast-derived matrices that were crosslinked with glutaraldehyde demonstrated a 67% increase in stiffness. This work provides a microscale characterization of fibroblast-derived matrix mechanical properties. An accurate understanding of native three-dimensional extracellular microenvironments will be essential for controlling cell responses in tissue engineering applications.

A multilayered synthetic human elastin/polycaprolactone hybrid vascular graft with tailored mechanical properties - Accepted Manuscript

2010-07-26

Abstract: Small-diameter synthetic vascular graft materials fail to match the patency of human tissue conduits used in vascular bypass surgery. The foreign surface retards endothelialization and is highly thrombogenic, while the mismatch in mechanical properties induces intimal hyperplasia. Using recombinant human tropoelastin, we have developed a synthetic vascular conduit for small-diameter applications. We show that tropoelastin enhances endothelial cell attachment (3-fold vs control) and proliferation by 54.7 ± 1.1% (3 days vs control). Tropoelastin when presented as a monomer and when cross-linked into synthetic elastin for biomaterials applications had low thrombogenicity. Activation of the intrinsic pathway of coagulation, measured by plasma clotting time was reduced for tropoelastin (60.4 ± 8.2% vs control). Platelet attachment was also reduced compared to collagen. Reductions in platelet interactions were mirrored on cross-linked synthetic elastin scaffolds. Tropoelastin was subsequently incorporated into a synthetic elastin/polycaprolactone conduit with mechanical properties optimized to mimic the human internal mammary artery, including permeability, compliance, elastic modulus and burst pressure. Further, this multilayered conduit presented a synthetic elastin internal lamina to circulating blood and demonstrated suturability and mechanical durability in a small scale rabbit carotid interposition model.

Properties of tooth enamel in great apes - Accepted Manuscript

2010-07-26

Abstract: A comparative study is made of human and great ape molar tooth enamel. Nanoindentation techniques are used to map profiles of elastic modulus and hardness across sections from the enamel–dentin junction to the outer tooth surface. The measured data profiles overlap between species, suggesting a degree of commonality in material properties. Using established deformation and fracture relations, critical loads to produce function-threatening damage in the enamel of each species are calculated for characteristic tooth size and enamel thickness. The results suggest that differences in load-bearing capacity of molar teeth in primates are less a function of underlying material properties than of morphology.

Silver-Polysaccharide Nanocomposite Antimicrobial Coatings for Methacrylic Thermosets - Accepted Manuscript

2010-07-26

Abstract: Bisphenol A glycidylmethacrylate (BisGMA)/triethyleneglycol dimethacrylate (TEGDMA) thermosets are receiving a growing interest as biomaterials for dental and orthopaedic applications; for both these fields, bacterial adhesion to the surface of the implant represents a major issue for the outcome of the surgical procedure. Moreover, the biological behavior of these materials is influenced by their ability to establish proper interactions between their surface and eukaryotic cells of the surrounding tissues which accounts for a good implant integration. The aim of this work was to develop an antimicrobial non-cytotoxic coating for methacrylic thermosets by means of a nanocomposite material based on a lactose-modified chitosan and antibacterial silver nanoparticles. The coating was characterized by UV-Visible spectrophotometry, optical microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). In vitro tests were employed for a biological characterization of the material: antimicrobial efficacy tests were carried out with both Gram+ and Gram- strains. Osteoblasts-like cell lines, primary human fibroblasts and adipose-derived stem cells were used for LDH cytotoxicity assays and Alamar Blue cell proliferation assays. Cell morphology and distribution were evaluated by SEM and Confocal Laser Scanning Microscopy. In vitro results showed that the nanocomposite coating is effective in killing both bacterial strains and that this material does not exert any significant cytotoxic effect towards tested cells, which are able to firmly attach and proliferate on the surface of the coating. Such biocompatible antimicrobial polymeric films containing silver nanoparticles may have good potentials for surface modification of medical devices, especially for prosthetic applications in orthopaedics and dentistry.

Influence of hydrothermal and mechanical conditions on the strength of zirconia - Accepted Manuscript

2010-07-26

Abstract: Low temperature degradation, and mechanical and thermal cycling may decrease the strength of zirconia and jeopardize the long term success of dental restorations made of this material. The objective of this study is to reveal the influence of different environmental and loading conditions on the strength of 3Y-TZP zirconia. From each of two 3Y-TZP materials, 180 disk specimens were produced and subjected to one of the following conditions: (A) no further treatment (control), (B and C) 106 and 5∙106 mechanical cycles, respectively, with an upper load limit of 100N, (D) 104 thermal cycles between 5°C and 55°C, (E) 200 days storage in water at 36°C, (F) a successive combination of conditions B, D and E, (G) storage in water at 80°C for 64 days, (H) storage in an autoclave at 134°C for eight hours. Monoclinic phase content was evaluated following XRD analysis. Specimen strength was determined in the biaxial bending test. The two ceramics exhibited average strengths of 995MPa and 1239MPa, respectively. No statistically significant influence of any treatment on strength was demonstrated with either material. However, XRD measurements revealed a substantial increase in monoclinic phase content, from initially 2% (control) to up to 10%, according to storage conditions. As a consequence of hydrothermal loading, the tetragonal-to-monoclinic phase transformation takes place at the surface of the 3Y-TZP materials investigated, but, like thermal and mechanical cycling, does not lead to significant changes in bulk strength.

Corrosion fatigue behaviors of two biomedical Mg alloys-AZ91D and WE43 in simulated body fluid - Accepted Manuscript

2010-07-26

Abstract: Magnesium alloys have been recently developed as biodegradable implant materials, and yet there is no study concerning their corrosion fatigue properties under cyclic loading. In this study the die-cast AZ91D (A for aluminum 9%, Z for zinc 1% and D for 4th stage) and extruded WE43 (W for yttrium 4%, E for rare earth mischmetal 3%) alloys were chosen to evaluate their fatigue and corrosion fatigue behaviors in simulated body fluid (SBF). Die-cast AZ91D alloy indicated a fatigue limit of 50MPa at 107 cycles in air compared to 20MPa at 106 cycles tested in SBF at 37°C. A fatigue limit of 110MPa at 107 cycles in air was observed for extruded WE43 alloy compared to 40MPa at 107 cycles tested in SBF at 37°C. The fatigue cracks initiated from the micro-pores when tested in air and from corrosion pits when tested in SBF, respectively. The overload zone of the extruded WE43 alloy exhibited the ductile fracture mode with deep dimples in comparison with brittle fracture mode for die-cast AZ91D. The corrosion rate of the two experimental alloys increased under cyclic loading compared to that in the static immersion test.

Hemocompatible pullulan-polyethyleneimine conjugates for liver cell gene delivery: In vitro evaluation of cellular uptake, intracellular trafficking and transfection efficiency - Accepted Manuscript

M.R. Rekha, Chandra P. Sharma — 2010-07-26

Abstract: Polyethyleneimine (25KDa) conjugated pullulan (PPE1, PPE2 and PPE3) were developed and investigated towards gene delivery applications. The cytotoxicity and blood component interactions such as Red blood cell (RBC)/White Blood Cell (WBC) aggregation, platelet and complement activation and protein interaction of the pullulan conjugated PEI was drastically reduced in comparison to PEI based nanocomplexes. Based on the blood compatibility studies PPE1 was selected for further studies. Buffering capacity of this derivative was similar to PEI, which plays an important role in efficient gene transfection. The particle size, zeta potential, stability in presence of plasma and resistance to nuclease degradation was evaluated. In addition, cellular uptake and localization of plasmid, as well as transgene expression, were evaluated following in vitro transfection of Hep G 2 cells. Endocytosis inhibitors, confocal laser scanning microscopy (CLSM) and fluorescent labeling techniques were used to visualize the nanoplex uptake mechanism, cellular distribution and nuclear localization. The results from inhibitor experiments in presence of asialofetuin indicated that the asialoglycoprotein receptor is involved in transfection of hepatocytes with pullulan-PEI complexes. The conjugation of pullulan with PEI did not hinder the plasmid nuclear localization ability of PEI. The transfection efficiency of pullulan conjugate was similar to PEI with the added advantage of hemocompatibility and noncytotoxicity. The transfection efficiency of PEI and PPE 1 was 1.6 and 2 fold respectively in presence of serum than that in the absence of serum. Therefore the pullulan-PEI conjugate seems to be a promising gene delivery vector with good hemocompatibility and low toxicity but without compromising the transfection efficacy of PEI.

Liquid-liquid two phase systems for the production of porous hydrogels and hydrogel microspheres for biomedical applications: A tutorial review - Accepted Manuscript

Donald L. Elbert — 2010-07-26

Abstract: Macroporous hydrogels may have direct applications in regenerative medicine as scaffolds to support tissue formation. Hydrogel microspheres may be used as drug delivery vehicles or as building blocks to assemble modular scaffolds. A variety of techniques exist to produce macroporous hydrogels and hydrogel microspheres. A subset of these relies on liquid-liquid two phase systems. Within this subset, vastly different types of polymerization processes are found. In this review, the history, terminology and classification of liquid-liquid two phase polymerization and crosslinking are described. Instructive examples of hydrogel microsphere and macroporous scaffold formation by precipitation/dispersion, emulsion and suspension polymerizations are used to illustrate the nature of these processes. The role of the kinetics of phase separation in determining the morphology of scaffolds and microspheres is also delineated. Brief descriptions of miniemulsion, microemulsion polymerization and ionotropic gelation are also included.

Characterization of human fibroblast-derived extracellular matrix components for human pluripotent stem cell propagation - Accepted Manuscript

Sheena Abraham, Marion J. Riggs, Kristina Nelson, Vladimir Lee, Raj R. Rao — 2010-07-26

Abstract: Recent studies from our laboratory have shown that acellular substrates generated from human fibroblasts successfully maintained human pluripotent stem cells (hPSCs) in their undifferentiated state for extended periods. Towards better characterization, we conducted proteomic analyses to identify the extracellular matrix (ECM) proteins in mouse embryonic and two human fibroblasts derived acellular substrates. Our studies identified heparan sulfate proteoglycan (HSPG) as a core component of these substrates and immunocytochemical analyses confirmed the presence of HSPG as well as other ECM proteins identified through proteomic analyses. In our attempt to develop surfaces that mimic fibroblast-deposited ECM and their self-renewal capabilities, substrates comprising of HSPG and other core ECM proteins were formulated and assessed for the function of hPSC self-renewal. WA09 and BG01v hPSCs maintained on these substrates exhibit multiple characteristics of pluripotency that include (a) tight colony formation with typical stem cell morphology (b) positive expression of alkaline phosphatase (c) positive expression of SSEA3, SSEA4 and Oct4 based on immunocytochemical analyses (d) POU5F1, Nanog and SOX2 mRNA expression and (e) in-vitro differentiation and expression of germ-layer specific markers. Our studies also reveal that although HSPG by itself does not support hPSC self-renewal, a substrate that combines HSPG and fibronectin is sufficient for undifferentiated propagation of hPSCs. These studies form the basis for identification of appropriate ECM components in a substrate that synergistically promotes activation of adhesion and signaling pathways responsible for hPSC self-renewal.

Ciprofloxacin-modified electrosynthesised hydrogel coatings to prevent titanium implant-associated infections - Accepted Manuscript

2010-07-26

Abstract: New promising and versatile materials for the development of in situ sustained release systems consisting of thin films of either poly(2-hydroxyethyl methacrylate) (PHEMA) or a copolymer based on poly(ethylene-glycol diacrylate) (PEGDA) and acrylic acid (AA) have been investigated. These polymers have been electrosynthesised directly on titanium substrates and loaded with ciprofloxacin (CIP) either during or after the synthesis step. X-ray Photoelectron Spectroscopy (XPS) was used to check CIP entrapment efficiency as well as its surface availability in the hydrogel films, while High Performance Liquid Chromatography (HPLC) was employed to assess release property of the films and to quantify the amount of CIP released by the coatings. These systems have then been tested to evaluate the in vitro inhibition of Methicillin-resistant Staphylococcus aureus (MRSA) growth. Moreover, a model equation has been proposed which can easily correlate the diameter of the inhibition haloes with the amount of the antibiotic released. Finally, MG63 human osteoblast-like cells were employed to assess the CIP-modified hydrogel coatings biocompatibility.

A Review of the mechanical behavior of CaP and CaP/polymer composites for applications in bone replacement and repair - Accepted Manuscript

Amy J. Wagoner Johnson, Brad A. Herschler — 2010-07-23

Abstract: Repair of load-bearing defects resulting from disease or trauma remains a critical barrier for bone tissue engineering. Calcium phosphate (CaP) scaffolds are among the most extensively studied for this application. However, CaPs are reportedly too weak for use in such defects and therefore have been limited to non load-bearing applications. This paper reviews the compression, flexural, and tensile properties of CaPs and CaP/polymer composites for applications in bone replacement and repair. This review reveals interesting trends that have not previously been reported, to our knowledge. Data are classified as bulk, scaffolds and composites, then organized in order of decreasing strength, allowing for general comparisons of magnitudes of strength both within and across classifications. Bulk and scaffold strength and porosity overlap significantly and scaffold data are comparable to bone both in strength and porosity. Further, for compression, all composite data fall below that of the bulk and most of the scaffold. Another interesting trend revealed is that strength decreases with increasing β-tricalcium phosphate (β-TCP) content for CaP scaffolds and with increasing CaP content for CaP/polymer composites. The real limitation for CaPs appears to be not strength, but the toughness and reliability, which are rarely characterized. We propose that research should focus on novel ways of toughening CaPs and discuss several potential strategies.

Effects of strontium ranelate administration on bisphosphonate-altered hydroxyapatite: Matrix incorporation of strontium is accompanied by changes in mineralization and microstructure - Accepted Manuscript

2010-07-23

Abstract: Strontium ranelate (SR) is one therapeutic option to reduce fracture risk in osteoporosis. The effects of SR treatment on hydroxyapatite previously altered by bisphosphonate (BP) administration remain to be established. Patients who have received long-term BP treatment and present with persistent high fracture risk are of particular interest. Paired iliac crest biopsies from 15 patients post BP therapy were subjected to a baseline biopsy and a follow-up biopsy after treatment with 2 g SR/d after either 6 months (n = 5) or 12 months (n = 10). DXA scans, serum parameters, and biochemical markers were obtained. Quantitative backscattered electron imaging and energy-dispersive X-ray analyses combined with μ-X-ray fluorescence determinations were performed to observe any mineralization changes. Static 2-D histomorphometry was carried out to evaluate cellular and structural indices. After 6 months of SR treatment, increases in osteoid surface and strontium content were observed, but no other indices showed significant change. After 12 months of SR treatment, there was a significant increase in bone volume and trabecular thickness as well as further increases in strontium content and backscattered signal intensity. These structural changes were accompanied by increased numbers of osteoblasts, and increased osteoid surface and volume. Additionally, low bone resorption, as measured by ß-cross-laps, and a low number of osteoclasts were observed. SR treatment led to increased strontium content within the bisphosphonate-hydroxyapatite nanocomposites as well as to increased osteoid indices and bone volume, which is indicative of newly formed bone, while osteoclasts were still suppressed. These data points suggest that SR might be considered as a therapeutic option for patients following long-term BP treatment.

Microencapsulation of islets within alginate/poly(ethylene glycol) gels cross-linked via Staudinger ligation - Accepted Manuscript

Kristina K. Hall, Kerim M. Gattás-Asfura, Cherie L. Stabler — 2010-07-22

Abstract: Functionalized alginate and PEG polymers were used to generate covalently linked alginate-PEG (XAlgPEG) microbeads of high stability. The cell-compatible Staudinger ligation scheme was used to chemoselectively cross-link phosphine-terminated poly(ethylene glycol) (PEG) to azide-functionalized alginate, resulting in XAlgPEG hydrogels. XAlgPEG microbeads were formed by co-incubation of the two polymers, followed by ionic cross-linking of the alginate using barium ions. The enhanced stability and gel properties of the resulting XAlgPEG microbeads, as well as the compatibility of these polymers for the encapsulation of islets and beta cells lines, were investigated. Our data show that XAlgPEG microbeads exhibit superior resistance to osmotic swelling compared to traditional barium cross-linked alginate (Ba-Alg) beads, with a 5-fold reduction in observed swelling, as well as resistance to dissolution via chelation solution. Diffusion and porosity studies found XAlgPEG beads to exhibit properties comparable to standard Ba-Alg. Our data found XAlgPEG microbeads to be highly cell compatible with insulinoma cell lines, as well as rat and human pancreatic islets, where the viability and functional assessment of cells within XAlgPEG were comparable to Ba-Alg controls. The remarkable improved stability, as well as demonstrated cellular compatibility, of XAlgPEG hydrogels makes them an appealing option for a wide variety of tissue engineering applications.

Cell-adhesive and mechanically tunable glucose-based biodegradable hydrogels - Accepted Manuscript

Hyeongho Shin, Jason W. Nichol, Ali Khademhosseini — 2010-07-21

Abstract: The development of materials with biomimetic mechanical and biological properties is of great interest for regenerative medicine applications. In particular, hydrogels are a promising class of biomaterials due to their high water content, which mimic that of natural tissues. We have synthesized a hydrophilic biodegradable polymer, designated poly(glucose malate)methacrylate (PGMma), that is composed of glucose and malic acid which are commonly found in the human metabolic system. This polymer is made photocrosslinkable by the incorporation of methacrylate groups. The resulting properties of the hydrogels can be tuned by altering the reacting ratio of the starting materials, the degree of methacrylation, and the polymer concentration of the resultant hydrogel. Hydrogels exhibited compressive moduli ranging from 1.8 ± 0.4kPa to 172.7 ± 36kPa with compressive strain at failure from 37.5 ± 0.9% to 61.2 ± 1.1%, and hydration by mass ranging from 18.7 ± 0.5% to 114.1 ± 1.3%. PGMma hydrogels also showed a broad range of degradation rates and were cell-adhesive, enabling the spreading of adherent cells. Overall, this work introduces a class of cell adhesive, mechanically tunable and biodegradable glucose-based hydrogels that may be useful for various tissue engineering and cell culture applications.

Ab initio study of thermodynamic, structural, and elastic properties of Mg-substituted crystalline calcite - Accepted Manuscript

2010-07-21

Abstract: Arthropoda, that represent nearly 80 % of all known animal species, are protected by an exoskeleton formed by their cuticle. The cuticle represents a hierarchically structured multifunctional bio-composite based on chitin and proteins. Some groups like Crustacea reinforce the load-bearing parts of their cuticle with calcite. As the calcite sometimes contains Mg it was speculated that Mg may have a stiffening impact on the mechanical properties of the cuticle (Becker et al., 2005[22]). Motivated by these facts, we present a theoretical parameter-free quantum-mechanical study of phase stability and structural and elastic properties of Mg-substituted calcite crystals. The Mg-substitutions were chosen as examples of states that occur in complex chemical environments typical for biological systems in which calcite crystals contain impurities the role of which is still the topic of debates. Density functional theory calculations of bulk (Ca,Mg)CO3 were performed employing 30-atomic supercells within the generalized gradient approximation (GGA) as implemented in the Vienna Ab-initio Simulation Package (VASP). Based on the calculated thermodynamical results, low concentrations of Mg atoms are predicted to be stable in calcite crystals in agreement with experimental findings. Examining the structural characteristics, Mg additions nearly linearly decrease the volume of substituted crystals. The predicted elastic bulk modulus results reveal that the Mg substitution nearly linearly stiffens the calcite crystals. Due to the quite large size-mismatch of Mg and Ca atoms a Mg substitution results in local distortions such as off-planar tilting of the CO32- group.

Corrigendum to “Polarization of hydroxyapatite: Influence on osteoblast cell proliferation” [Acta Biomaterialia 6 (2010) 1549–1554] - Uncorrected Proof

2010-07-20

The authors regret the misspelling of L.A. Hidalgo-Bastida on the author line. It is now listed correctly above.

In vitro Microbial Inhibition and Cellular Response to Novel Biodegradable Composite Wound Dressings with Controlled Release of Antibiotics - Accepted Manuscript

Jonathan J. Elsner, Israela Berdicevsky, Meital Zilberman — 2010-07-20

Abstract: About 70% of all people with severe burns die from related infections, despite advances in treatment regimens and the best efforts of nurses and doctors. Although silver-eluting wound dressings are available for addressing this problem, there is growing evidence of the deleterious effects of such dressings in delaying the healing process due to cellular toxicity. Our new concept of antibiotic-eluting composite wound dressings is described here. These dressings are based on a polyglyconate mesh, coated with a porous poly-(DL-lactic-co-glycolic acid) matrix loaded with antibiotic drugs. The effect of antibiotic release on bacterial inhibition was studied and cell cytotoxicity was examined. The dressings resulted in a 99.99% decrease in the viable counts of P. aeruginosa and S. albus at very high initial inoculations of 107-108 CFU/mL after only one day, while such a decrease in S. aureus was obtained within 3 days. Bacterial inhibition zones around the dressing material were found to persist for 2 weeks, indicating a long-lasting antimicrobial effect. Despite severe toxicity to bacteria, the dressing material was found to have no toxic effect on cultured fibroblasts, indicating that our new antibiotic-eluting wound dressings represent an effective option for selective treatment of bacterial infections.

Polymer-Conjugated Albumin and Fibrinogen Composite Hydrogels as Cell Scaffolds Designed with Affinity-Based Drug Delivery - Accepted Manuscript

Liat Oss-Ronen, Dror Seliktar — 2010-07-20

Abstract: Serum albumin was conjugated to Poly-(ethylene glycol) (PEG) and cross-linked to form mono-PEGylated albumin hydrogels. These hydrogels were used as a basis for drug carrying tissue engineered scaffold materials, based on the natural affinity of various drugs and compounds to the tethered albumin in the polymer network. The results of the drug release validation experiments showed that the release kinetics of the drugs from the mono-PEGylated albumin hydrogels were controlled by the molecular weight (MW) of PEG conjugated to the albumin protein, the drug MW and its inherent affinity to albumin. Composite hydrogels containing both mono-PEGylated albumin and PEGylated fibrinogen were used specifically for 3D cell culture scaffolds, with inherent bioactivity, proteolytic biodegradability, and controlled drug release properties. The specific characteristics of these complex hydrogels were controlled by the ratio between the concentrations of each protein, the addition of free PEG diacrylate (PEG-DA) molecules into the hydrogel matrix, and the MW of the PEG conjugated to each protein. Comprehensive characterization of the drug release and degradation properties, as well as three-dimensional (3-D) cell culture experiments using these composite materials demonstrated the effectiveness of this combined approach for creating a tissue engineered scaffold material with controlled drug release features.

A novel controlled drug delivery system based on pH-responsive hydrogels included in soft gelatin capsules - Accepted Manuscript

G. Frutos, A. Prior-Cabanillas, R. París, I. Quijada-Garrido — 2010-07-20

Abstract: pH-sensitive hydrogels based on methacrylic acid (MAA) and poly(ethylene glycol) macromonomer (PEGMEMA) entrapping diltiazem hydrochloride (DIL·HCl), were synthesized inside soft gelatin capsules for using as a new dosage form for drug oral administration. Thus, different monomer compositions were used for the evaluation of their swelling and release behavior in two media: at low pH, simulating the acid pH of the stomach, and at pH 7, simulating the higher pH environment of the intestine. Both, the swelling process and the DIL·HCl release strongly depend on pH and monomer composition. Therefore, hydrogels with intermediate composition showed a diminished amount of DIL·HCl released at pH 1.2. This fact was related to the formation of an impermeable outer skin, experimentally observed by magnetic resonance imaging (MRI). At pH 7 similar shapes of the release profiles were found for the four hydrogel compositions under investigation. At this neutral pH, the slow protonation of the carboxylic groups of MAA leads to a swelling front and a dry core, also observed by MRI. As a consequence of this anomalous swelling, release curves exhibited a long period of zero-order kinetics. This fact shows that the system could be a suitable candidate to develop a zero-order release dosage form for drug oral administration of DIL·HCl. The swelling and dissolution processes were analyzed by different mathematical approaches.

Enhancement of adhesion strength and cellular stiffness of osteoblasts on mirror-polished titanium surface by UV-photofunctionalization - Uncorrected Proof

2010-07-14

Abstract: Ultraviolet (UV)-photofunctionalization of titanium substantially enhances the strength and quality of osseointegration by promoting osteogenic cellular attachment and proliferation. However, the mechanism underlying the initial interaction between the cells and the surface of the material remains to be elucidated, especially where the influence of surface roughness is excluded as a factor. The effect of UV-photofunctionalization on the adhesive strength and cellular stiffness of a single osteoblast and its association with the extent of cell spread, cytoskeletal development and focal adhesion assembly on a very smooth titanium surface was evaluated. Rat bone marrow-derived osteoblasts were cultured on UV-treated or untreated mirror-polished titanium disks. The mean critical shear force required to initiate detachment of a single osteoblast (n=10) was >2000nN on a UV-treated surface at 3h incubation, which was 17 times greater than that on an untreated surface. The mean total energy required to complete the detachment of osteoblasts (n=10) was consistently >60pJ on a UV-treated titanium surface after 24h culture, which was up to 42 times greater than that on an untreated surface. Cellular shear modulus, which represents cellular stiffness, was consistently greater on a UV-treated surface than on an untreated surface after 24h incubation (n=10). This strengthening of cell adhesion and cellular mechanical properties on UV-treated titanium was accompanied by enhanced cell spread and actin fiber development and increased levels of vinculin expression. These results indicate that UV-photofunctionalization substantially strengthens osteoblast retention on titanium bulk material with no topographical features, and that this is associated with enhancement of intracellular structural development during the cell adhesion process.

Wear rate evaluation of a novel polycarbonate-urethane cushion form bearing for artificial hip joints - Uncorrected Proof

2010-07-14

Abstract: There is growing interest in the use of compliant materials as an alternative to hard bearing materials such as polyethylene, metal and ceramics in artificial joints. Cushion form bearings based on polycarbonate-urethane (PCU) mimic the natural synovial joint more closely by promoting fluid-film lubrication. In the current study, we used a physiological simulator to evaluate the wear characteristics of a compliant PCU acetabular buffer, coupled against a cobalt–chrome femoral head. The wear rate was evaluated over 8 million cycles gravimetrically, as well as by wear particle isolation using filtration and bio-ferrography (BF). The gravimetric and BF methods showed a wear rate of 9.9–12.5mg per million cycles, whereas filtration resulted in a lower wear rate of 5.8mg per million cycles. Bio-ferrography was proven to be an effective method for the determination of wear characteristics of the PCU acetabular buffer. Specifically, it was found to be more sensitive towards the detection of wear particles compared to the conventional filtration method, and less prone to environmental fluctuations than the gravimetric method. PCU demonstrated a low particle generation rate (1–5×106 particles per million cycles), with the majority (96.6%) of wear particle mass lying above the biologically active range, 0.2–10μm. Thus, PCU offers a substantial advantage over traditional bearing materials, not only in its low wear rate, but also in its osteolytic potential.

Mitral valvular interstitial cell responses to substrate stiffness depend on age and anatomic region - Uncorrected Proof

2010-07-12

Abstract: The material properties of heart valves depend on the subject’s age, the state of the disease and the complex valvular microarchitecture. Furthermore, valvular interstitial cells (VICs) are mechanosensitive, and their synthesis of extracellular matrix not only determines the valve’s material properties but also provides an adhesive substrate for VICs. However, the interrelationship between substrate stiffness and VIC phenotype and synthetic properties is poorly understood. Given that the local mechanical environment (substrate stiffness) surrounding VICs differs among different age groups and different anatomic regions of the valve, it was hypothesized that there may be an age- and valve-region-specific response of VICs to substrate stiffness. Therefore, 6-week-, 6-month- and 6-year-old porcine VICs from the center of the mitral valve anterior leaflet (MVAC) and posterior leaflet (PML) were seeded onto poly(ethylene) glycol hydrogels of different stiffnesses and stained for markers of VIC activation (smooth muscle alpha-actin (SMaA)) and collagen synthesis (heat shock protein-47 (HSP47), prolyl 4-hydroxylase (P4H)). Six-week-old MVAC demonstrated decreased SMaA, P4H and HSP47 on stiffer gels, while 6-week-old PML only demonstrated decreased HSP47. Six-month-old MVAC demonstrated no difference between substrates, while 6-month-old PML demonstrated decreased SMaA, P4H and HSP47. Six-year-old MVAC demonstrated decreased P4H and HSP47, while 6-year-old PML demonstrated decreased P4H and increased HSP47. In conclusion, the age-specific and valve-region-specific responses of VICs to substrate stiffness link VIC phenotype to the leaflet regional matrix in which the VICs reside. These data provide further rationale for investigating the role of substrate stiffness in VIC remodeling within diseased and tissue engineered valves.

Zirconia nanoparticles prepared by laser vaporization as fillers for dental adhesives - Uncorrected Proof

2010-07-12

Abstract: Zirconia nanoparticles prepared by laser vaporization were incorporated into the primer or into the adhesive of a commercial adhesive system in order to evaluate its effect on bond strength to dentin. Zirconia nanoparticles (20–50nm) were prepared using a particular laser vaporization technique and incorporated into the primer (P) or into the adhesive (A) of the Adper Scotchbond Multi-Purpose (SBMP) system at 5, 10, 15 and 20wt.% by means of mechanical mixing (stirring) and ultrasonication. Control (unfilled) and experimental groups (filled) were applied, according to the manufacturer’s instructions, onto flat mid-coronal human dentin. Composite crowns were built up, stored in distilled water for 24h at 37°C and cut into 0.65±0.05mm2 beams following a non-trimming microtensile technique. Specimens were fractured in tension using a universal testing machine (Zwick) and examined by scanning electron microscopy for fractographic analysis. Microtensile bond strength (μTBS) data were analyzed using a two-way ANOVA and modified LSD test at α=0.05. Analysis of the nanofiller distribution and ultramorphological characterization of the interface were performed by transmission electron microscopy (TEM). Zirconia nanoparticle incorporation into the primer or into the adhesive of SBMP significantly increased μTBS to dentin. Filler concentration only affected μTBS significantly in the P group. Statistically significant differences between groups P and A occurred only at 20wt.% filler content, with a significantly higher μTBS in group P. TEM micrographs revealed nanoparticle deposition on top of a hybrid layer when incorporated into the primer, whereas they remained dispersed through the adhesive layer in group A. Zirconia nanoparticles incorporation into SBMP increased bond strength to dentin by reinforcing the interface adhesive layer. Nanofiller incorporation into the primer solution showed a tendency of increasing bond strength with increasing concentration. At high concentrations (20wt.%) nanofiller incorporation was more efficient in increasing bond strength if incorporated in the primer solution. Adding nanofillers to the primer and to the adhesive solutions resulted in different particle distributions at the interface.

Influence of β-tricalcium phosphate granule size and morphology on tissue reaction in vivo - Uncorrected Proof

2010-07-12

Abstract: In this study the tissue reaction to five different β-tricalcium phosphate (β-TCP)-based bone substitute materials differing only in size, shape and porosity was analyzed over 60days, at 3, 10, 15, 30 and 60days after implantation. Using the subcutaneous implantation model in Wistar rats both the inflammatory response within the implantation bed and the resulting vascularization of the biomaterials were qualitatively and quantitatively assessed by means of standard and special histological staining methods. The data from this study showed that all investigated β-TCP bone substitutes induced the formation of multinucleated giant cells. Changes in size, shape and porosity influenced the integration of the biomaterials within the implantation bed and the formation of tartrate-resistant acid phosphatase (TRAP)-positive and TRAP-negative multinucleated giant cells, as well as the rate of vascularization. While a high porosity generally allowed cell and fiber in-growth within the center of the bone substitute, a lower porosity resulted in a mosaic-like integration of the materials, with the granules serving as place holders. The number of multinucleated giant cells located in the implantation bed positively correlated with the vascularization rate. These data emphasize that all biomaterials investigated were capable of inducing the formation of TRAP-positive multinucleated giant cells as a sign of biomaterial stability. Furthermore, these cells directly influenced vascularization by secretion of vascular endothelial growth factor (VEGF), as well as other chemokines. Based on these findings, the role of multinucleated giant cells in the foreign body reaction to biomaterials might need to be reconsidered. This study demonstrates that variations in the physical properties of a bone substitute material clearly influence the (extent of the) inflammatory reaction and its consequences.

Plasma-induced nanopillars on bare metal coronary stent surface for enhanced endothelialization - Uncorrected Proof

Mariana C. Loya, Karla S. Brammer, Chulmin Choi, Li-Han Chen, Sungho Jin — 2010-07-12

Abstract: An increased risk of late stent thrombosis associated with polymer carriers on the surface of drug-eluting stents remains one of the challenges in cardiovascular stent technology, which has instigated a renewed interest in the polymer-less, bare metal stent approach. As thrombus formation is most likely augmented by the lack of endothelial cell coverage at the exposed stented site, an improved stent surface that enhances cell coverage is essential for viable polymer-less all metal stents. We demonstrate superior endothelial cell growth, more continuous monolayer formation and overall improved endothelialization with nanopillar arrays created via radio frequency plasma surface texturing on our all metallic stent surface of MP35N stent alloy. It is shown that the nanotextured surface significantly up-regulates primary bovine aortic endothelial cell (BAEC) functionality when compared with unprocessed, smooth MP35N surfaces without a nanopillar topography. The desirable presence of transmembrane tight junctions and highly organized monolayer formation was induced by the presence of the nanopillar surface texture. The nanopillar structure also produced a reduced level of oxidative stress in the BAECs. These findings may contribute to new nanotechnology-based surface design concepts for bare metal stents producing advanced cardiovascular implants which mitigate late stent thrombosis.

Tuning adhesion failure strength for tissue-specific applications - Uncorrected Proof

2010-07-12

Abstract: Soft tissue adhesives are employed to repair and seal many different organs, which range in both tissue surface chemistry and mechanical challenges during organ function. This complexity motivates the development of tunable adhesive materials with high resistance to uniaxial or multiaxial loads dictated by a specific organ environment. Co-polymeric hydrogels comprising aminated star polyethylene glycol and dextran aldehyde (PEG:dextran) are materials exhibiting physico-chemical properties that can be modified to achieve this organ- and tissue-specific adhesion performance. Here we report that resistance to failure under specific loading conditions, as well as tissue response at the adhesive material–tissue interface, can be modulated through regulation of the number and density of adhesive aldehyde groups. We find that atomic force microscopy (AFM) can characterize the material aldehyde density available for tissue interaction, and in this way enable rapid, informed material choice. Further, the correlation between AFM quantification of nanoscale unbinding forces with macroscale measurements of adhesion strength by uniaxial tension or multiaxial burst pressure allows the design of materials with specific cohesion and adhesion strengths. However, failure strength alone does not predict optimal in vivo reactivity. Thus, we demonstrate that the development of adhesive materials is significantly enabled when experiments are integrated along length scales to consider organ chemistry and mechanical loading states concurrently with adhesive material properties and tissue response.

Differential distribution of structural components and hydration in aortic and pulmonary heart valve conduits: Impact of detergent-based cell removal - Uncorrected Proof

2010-07-09

Abstract: Evaluation of the physiological performance of biological scaffolds for tissue engineering applications has been mostly based on biophysical and morphological methods, with limited attention paid to the quantitative contribution of the main structural components to native and/or treated valve assemblies. In the present study quantitation addressed the porcine leaflet, sinus and adjacent wall of aortic and pulmonary valved conduits before and after detergent-based cell removal. Collagen, elastin, glycosaminoglycan, lipid and water contents were expressed in terms of relative concentration and volume fraction in order to assess their effective contribution to the native tissue and to changes following decellularization procedures. The main findings were recognition of unexpectedly large water and underestimated collagen contents, differential distribution of elastin between the sectors and of glycosaminoglycan along the conduits and pulmonary scaffold destabilization upon cell removal, not found in the aortic case. Simultaneous investigations allowed consistent comparisons between native and decellularized tissues and added analytical knowledge crucial for designing realistic constitutive models. We have provided a quantitative structural foundation for earlier biomechanical findings in pulmonary leaflets and the basis for validation of theoretical assumptions still lacking the support of experimental evidence in both conduits. Future insights into the distribution of load-bearing components in human conduits are likely to provide indications important to optimize the surgical positioning of valvular grafts.

Three-dimensional scaffold of electrosprayed fibers with large pore size for tissue regeneration - Uncorrected Proof

Jong Kyu Hong, Sundararajan V. Madihally — 2010-07-09

Abstract: The regeneration of tissues using biodegradable porous scaffolds has been intensely investigated. Since electrospinning can produce scaffolds mimicking nanofibrous architecture found in the body, it has recently gained widespread attention. However, a major problem is the lack of pore size necessary for infiltration of cells into the layers below the surface, restricting cell colonization to the surfaces only. This study describes a novel twist to the traditional electrospinning technology: specifically, collector plates are designed which allow the formation of very thin layers with pore sizes suitable for cell infiltration. The thin samples could be handled without mechanically damaging the structure and could be transferred into cell culture. These thin layers were stacked layer-by-layer to develop thick structures. Thirty day cultures of fibroblasts show attachment and spreading of cells in every layer. This concept is useful in regenerating thick tissues with uniformly distributed cells and others in in vitro cell culture.

Predicting bulk mechanical properties of cellularized collagen gels using multiphoton microscopy - Uncorrected Proof

C.B. Raub, A.J. Putnam, B.J. Tromberg, S.C. George — 2010-07-09

Abstract: Cellularized collagen gels are a common model in tissue engineering, but the relationship between the microstructure and bulk mechanical properties is only partially understood. Multiphoton microscopy (MPM) is an ideal non-invasive tool for examining collagen microstructure, cellularity and crosslink content in these gels. In order to identify robust image parameters that characterize microstructural determinants of the bulk elastic modulus, we performed serial MPM and mechanical tests on acellular and cellularized (normal human lung fibroblasts) collagen hydrogels, before and after glutaraldehyde crosslinking. Following gel contraction over 16days, cellularized collagen gel content approached that of native connective tissues (∼200mgml–1). Young’s modulus (E) measurements from acellular collagen gels (range 0.5–12kPa) exhibited a power-law concentration dependence (range 3–9mgml–1) with exponents from 2.1 to 2.2, similar to other semiflexible biopolymer networks such as fibrin and actin. In contrast, cellularized collagen gel stiffness (range 0.5–27kPa) produced concentration-dependent exponents of 0.7 uncrosslinked and 1.1 crosslinked (range ∼5–200mgml–1). The variation in E of cellularized collagen hydrogels can be explained by a power-law dependence on robust image parameters: either the second-harmonic generation (SHG) and two-photon fluorescence (TPF) (matrix component) skewness (R2=0.75, exponents of -1.0 and -0.6, respectively); or alternatively the SHG and TPF (matrix component) speckle contrast (R2=0.83, exponents of −0.7 and −1.8, respectively). Image parameters based on the cellular component of TPF signal did not improve the fits. The concentration dependence of E suggests enhanced stress relaxation in cellularized vs. acellular gels. SHG and TPF image skewness and speckle contrast from cellularized collagen gels can predict E by capturing mechanically relevant information on collagen fiber, cell and crosslink density.

Controlled fabrication of triple layered and molecularly defined collagen/elastin vascular grafts resembling the native blood vessel - Uncorrected Proof

2010-07-08

Abstract: There is a consistent need for a suitable natural biomaterial to function as an arterial prosthesis in achieving arterial regeneration. Natural grafts are generally obtained by decellularization of native blood vessels, but batch to batch variations may occur and the nature/content of remaining contaminants is generally unknown. In this study we fabricated a molecularly defined natural arterial graft from scratch resembling the native three layered architecture from the fibrillar extracellular matrix components collagen and elastin. Using casting, moulding, freezing and lyophilization techniques, a triple layered construct was prepared consisting of an inner layer of elastin fibres, a middle (porous) film layer of collagen fibrils and an outer scaffold layer of collagen fibrils. The construct was carbodiimide cross-linked and heparinized. Characterization included biochemical/biophysical analyses, scanning electron microscopy, micro-computed tomography, (immuno)histology and haemocompatibility. Burst pressures were up to 400mm Hg and largely conferred by the intermediate porous collagen film layer. The highly purified type I collagen fibrils and elastin fibres used did not evoke platelet aggregation in vitro. Suturability of the graft in end to side anastomosis was successful and considered adequate for in vivo application.

Intra-myocardial biomaterial injection therapy in the treatment of heart failure: Materials, outcomes and challenges - Uncorrected Proof

2010-07-08

Abstract: Heart failure initiated by coronary artery disease and myocardial infarction (MI) is a widespread, debilitating condition for which there are a limited number of options to prevent disease progression. Intra-myocardial biomaterial injection following MI theoretically provides a means to reduce the stresses experienced by the infarcted ventricular wall, which may alter the pathological remodeling process in a positive manner. Furthermore, biomaterial injection provides an opportunity to concurrently introduce cellular components and depots of bioactive agents. Biologically derived, synthetic and hybrid materials have been applied, as well as materials designed expressly for this purpose, although optimal design parameters, including degradation rate and profile, injectability, elastic modulus and various possible bioactivities, largely remain to be elucidated. This review seeks to summarize the current body of growing literature where biomaterial injection, with and without concurrent pharmaceutical or cellular delivery, has been pursued to improve functional outcomes following MI. The literature to date generally demonstrates acute functional benefits associated with biomaterial injection therapy across a broad variety of animal models and material compositions. Further functional improvements have been reported when cellular or pharmaceutical agents have been incorporated into the delivery system. Despite these encouraging early results, the specific mechanisms behind the observed functional improvements remain to be fully explored and future studies employing hypothesis-driven material design and selection may increase the potential of this approach to alleviate the morbidity and mortality of heart failure.

Bioactivation of biomorphous silicon carbide bone implants - Uncorrected Proof

2010-07-07

Abstract: Wood-derived silicon carbide (SiC) offers a specific biomorphous microstructure similar to the cellular pore microstructure of bone. Compared with bioactive ceramics such as calcium phosphate, however, silicon carbide is considered not to induce spontaneous interface bonding to living bone. Bioactivation by chemical treatment of biomorphous silicon carbide was investigated in order to accelerate osseointegration and improve bone bonding ability. Biomorphous SiC was processed from sipo (Entrandrophragma utile) wood by heating in an inert atmosphere and infiltrating the resulting carbon replica with liquid silicon melt at 1450°C. After removing excess silicon by leaching in HF/HNO3 the biomorphous preform consisted of β-SiC with a small amount (approximately 6wt.%) of unreacted carbon. The preform was again leached in HCl/HNO3 and finally exposed to CaCl2 solution. X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared analyses proved that oxidation of the residual carbon at the surface induced formation of carboxyl [COO−] groups, which triggered adsorption of Ca2+, as confirmed by XPS and inductively coupled plasma optical emission spectroscopy measurements. A local increase in Ca2+ concentration stimulated in vitro precipitation of Ca5(PO4)3OH (HAP) on the silicon carbide preform surface during exposure to simulated body fluid, which indicates a significantly increased bone bonding activity compared with SiC.

Biofunctionalization of materials for implants using engineered peptides - Corrected Proof

2010-07-05

Abstract: Uncontrolled interactions between synthetic materials and human tissues are a major concern for implants and tissue engineering. The most successful approaches to circumvent this issue involve the modification of the implant or scaffold surfaces with various functional molecules, such as anti-fouling polymers or cell growth factors. To date, such techniques have relied on surface immobilization methods that are often applicable only to a limited range of materials and require the presence of specific functional groups, synthetic pathways or biologically hostile environments. In this study we have used peptide motifs that have been selected to bind to gold, platinum, glass and titanium to modify surfaces with poly(ethylene glycol) anti-fouling polymer and the integrin-binding RGD sequence. The peptides have several advantages over conventional molecular immobilization techniques; they require no biologically hostile environments to bind, are specific to their substrates and could be adapted to carry various active entities. We successfully imparted cell-resistant properties to gold and platinum surfaces using gold- and platinum-binding peptides, respectively, in conjunction with PEG. We also induced a several-fold increase in the number and spreading of fibroblast cells on glass and titanium surfaces using quartz and titanium-binding peptides in conjunction with the integrin ligand RGD. The results presented here indicate that control over the extent of cell–material interactions can be achieved by relatively simple and biocompatible surface modification procedures using inorganic binding peptides as linker molecules.

Amorphous calcium (ortho)phosphates - Uncorrected Proof

Sergey V. Dorozhkin — 2010-07-05

Abstract: Amorphous calcium phosphates (ACPs) represent a unique class of biomedically relevant calcium orthophosphate salts, having variable chemical but essentially identical glass-like physical properties, in which there is neither translational nor orientational long-range ordering of the atomic positions. Normally, ACPs are the first solid phases, precipitated after a rapid mixing of aqueous solutions containing ions of Ca2+ and ; however, other production techniques are known. Interestingly, ACPs prepared by wet-chemical techniques were found to have a relatively constant chemical composition over a relatively wide range of preparation conditions, which suggests the presence of a well-defined local structural unit, presumably with the structure of Ca9(PO4)6 – so-called Posner cluster. However, the presence of similar clusters in ACPs produced by other techniques remains uncertain. All ACPs are thermodynamically unstable compounds and, unless stored in dry conditions or doped by stabilizers, spontaneously tend to transform to crystalline calcium orthophosphates, mainly to calcium apatites. This solution instability of ACPs and their easy transformation to crystalline phases are of a great biological relevance. Specifically, the initiating role ACPs play in matrix vesicle biomineralization raises the importance of ACPs from a mere laboratory curiosity to that of a key intermediate in skeletal calcification. In addition, due to significant chemical and structural similarities with calcified mammalian tissues, as well as excellent biocompatibility and bioresorbability, all types of ACPs are very promising candidates for the manufacture of artificial bone grafts. This review summarizes the current knowledge on the occurrence, preparation, composition, structure, major properties and biomedical applications of ACPs. To assist readers in looking for the specific details on ACPs, a great number of references have been collected and systematized.

Constitutive response of brain tissue - Uncorrected Proof

Thibault P. Prevost, Asha Balakrishnan, Subra Suresh, Simona Socrate — 2010-07-05

Abstract: The dynamic behavior of porcine brain tissue, obtained from a series of in vitro observations and experiments, is analyzed and described here with the aid of a large strain, nonlinear, viscoelastic constitutive model. Mixed gray and white matter samples excised from the superior cortex were tested in unconfined uniaxial compression within 15h post mortem. The test sequence consisted of three successive load–unload segments at strain rates of 1, 0.1 and 0.01s−1, followed by stress relaxation (n=25). The volumetric compliance of the tissue was assessed for a subset of specimens (n=7) using video extensometry techniques. The tissue response exhibited moderate compressibility, substantial nonlinearity, hysteresis, conditioning and rate dependence. A large strain kinematics nonlinear viscoelastic model was developed to account for the essential features of the tissue response over the entire deformation history. The corresponding material parameters were obtained by fitting the model to the measured conditioned response (axial and volumetric) via a numerical optimization scheme. The model successfully captures the observed complexities of the material response in loading, unloading and relaxation over the entire range of strain rates. The accuracy of the model was further verified by comparing model predictions with the tissue response in unconfined compression at higher strain rate (10s−1) and with literature data in uniaxial tension. The proposed constitutive framework was also found to be adequate to model the loading response of brain tissue in uniaxial compression over a wider range of strain rates (0.01–3000s−1), thereby providing a valuable tool for simulations of dynamic transients (impact, blast/shock wave propagation) leading to traumatic brain injury.

A matrix micropatterning platform for cell localization and stem cell fate determination - Corrected Proof

2010-07-02

Abstract: To study the role of cell–extracellular matrix (ECM) interactions, microscale approaches provide the potential to perform high throughput assessment of the effect of the ECM microenvironment on cellular function and phenotype. Using a microscale direct writing (MDW) technique, we characterized the generation of multicomponent ECM microarrays for cellular micropatterning, localization and stem cell fate determination. ECMs and other biomolecules of various geometries and sizes were printed onto epoxide-modified glass substrates to evaluate cell attachment by human endothelial cells. The endothelial cells displayed strong preferential attachment to the ECM patterned regions and aligned their cytoskeleton along the direction of the micropatterns. We next generated ECM microarrays that contained one or more ECM components (namely gelatin, collagen IV and fibronectin) and then cultured murine embryonic stem cell (ESCs) on the microarrays. The ESCs selectively attached to the micropatterned features and expressed markers associated with a pluripotent phenotype, such as E-cadherin and alkaline phosphatase, when maintained in growth medium containing leukemia inhibitory factor. In the presence of the soluble factors retinoic acid and bone morphogenetic protein-4 the ESCs differentiated towards the ectodermal lineage on the ECM microarray with differential ECM effects. The ESCs cultured on gelatin showed significantly higher levels of pan cytokeratin expression, when compared with cells cultured on collagen IV or fibronectin, suggesting that gelatin preferentially promotes ectodermal differentiation. In summary, our results demonstrate that MDW is a versatile approach to print ECMs of diverse geometries and compositions onto surfaces, and it is amenable to the generation of multicomponent ECM microarrays for stem cell fate determination.

Three-dimensional nanocomposite scaffolds fabricated via selective laser sintering for bone tissue engineering - Corrected Proof

Bin Duan, Min Wang, Wen You Zhou, Wai Lam Cheung, Zhao Yang Li, William W. Lu — 2010-06-30

Abstract: Bionanocomposites formed by combining biodegradable polymers and nanosized osteoconductive inorganic solids have been regarded as promising biomimetic systems which possess much improved structural and functional properties for bone tissue regeneration. In this study three-dimensional nanocomposite scaffolds based on calcium phosphate (Ca-P)/poly(hydroxybutyrate–co-hydroxyvalerate) (PHBV) and carbonated hydroxyapatite (CHAp)/poly(l-lactic acid) (PLLA) nanocomposite microspheres were successfully fabricated using selective laser sintering, which is a rapid prototyping technology. The sintered scaffolds had controlled material microstructure, totally interconnected porous structure and high porosity. The morphology and mechanical properties of Ca-P/PHBV and CHAp/PLLA nanocomposite scaffolds as well as PHBV and PLLA polymer scaffolds were studied. In vitro biological evaluation showed that SaOS-2 cells had high cell viability and normal morphology and phenotype after 3 and 7days culture on all scaffolds. The incorporation of Ca-P nanoparticles significantly improved cell proliferation and alkaline phosphatase activity for Ca-P/PHBV scaffolds, whereas CHAp/PLLA nanocomposite scaffolds exhibited a similar level of cell response compared with PLLA polymer scaffolds. The nanocomposite scaffolds provide a biomimetic environment for osteoblastic cell attachment, proliferation and differentiation and have great potential for bone tissue engineering applications.

Mechanical evaluation of poly(vinyl alcohol)-based fibrous composites as biomaterials for meniscal tissue replacement - Corrected Proof

Julianne L. Holloway, Anthony M. Lowman, Giuseppe R. Palmese — 2010-06-30

Abstract: In this study, poly(vinyl alcohol) (PVA) hydrogels were reinforced with ultrahigh molecular weight polyethylene (UHMWPE) and PP fibers and evaluated as potential nondegradable meniscal replacements. An investigation of hydrogel and composite mechanical properties indicates that fiber-reinforced PVA hydrogels could replicate the unique anisotropic modulus distribution present in the native meniscus; the most commonly damaged orthopedic tissue. More specifically, fibrous reinforcement successfully increased the tensile modulus of the biomaterial from 0.23±0.02MPa without any reinforcement to 258.1±40.1MPa at 29vol.% UHMWPE. Additionally, the molecular weight between cross-links, bound water and the microstructure of the PVA hydrogels were evaluated as a function of freeze–thaw cycles and polymer concentration to lend insight into the processes occurring during synthesis. These results suggest the presence of multiple mechanisms as causes for increasing hydrogel modulus with freeze–thaw cycling, including hydrogen bonding between amorphous and/or crystalline regions, and the formation of highly concentrated regions of mostly amorphous PVA chains. It is possible that the formation of regions with highly concentrated amounts of PVA increases the load-bearing ability of the hydrogels.

Influence of cooling rate on zirconia/veneer interfacial adhesion - Corrected Proof

2010-06-30

Abstract: Slow cooling firing schedules have recently been introduced by some manufacturers to reduce chipping complications in zirconia-based core/veneer composites. The aim of this study was to test the hypothesis that these firing schedules may influence the bond strength between the zirconia core and veneering ceramic. Four different veneering ceramics recommended for zirconia (Lava Ceram, Triceram, VM9 and Zirox) were fired onto rectangular shaped Y-TZP specimens (Lava Frame) and cooled using a rapid or a slow cooling rate. The resulting bilayer specimens were notched, loaded in a four-point bending test and load–displacement curves were recorded. The critical load to induce stable crack extension at the core/veneer interface was determined, in order to calculate the strain energy release rate (G, Jm−2). Additionally, dilatometric measurements of the veneering ceramics were performed to determine the coefficient of thermal expansion (α, ppm.K−1) between 50 and 450°C (α1) and in the temperature region above the glass transition temperature (α2). Discrepancies between α2 and α1 (Δα) were calculated. For all core/veneer compositions G values were lower for the slowly cooled specimens than for the rapidly cooled specimens. Significant differences with respect to the firing schedule were found in the Triceram and VM9 groups (P<0.05). The reductions in G values correlated with Δα. The bond strength between the zirconia core and the veneer decreased with the slow cooling rate. These results indicate that slow cooling of zirconia restorations may increase the risk of adhesive delamination failures between the core and veneer.

Hierarchically structured titanium foams for tissue scaffold applications - Corrected Proof

2010-06-30

Abstract: We present a novel route for producing a new class of titanium foams for use in biomedical implant applications. These foams are hierarchically porous, with both the traditional large (>300μm) highly interconnected pores and, uniquely, wall struts also containing micron scale (0.5–5μm) interconnected porosities. The fabrication method consists of first producing a porous oxide precursor via a gel casting method, followed by electrochemical reduction to produce a metallic foam. This method offers the unique ability to tailor the porosity at several scales independently, unlike traditional space-holder techniques. Reducing the pressure during foam setting increased the macro-pore size. The intra-strut pore size (and percentage) can be controlled independently of macro-pore size by altering the ceramic loading and sintering temperature during precursor production. Typical properties for an 80% porous Ti foam were a modulus of ∼1GPa, a yield strength of 8MPa and a permeability of 350 Darcies, all of which are in the range required for biomedical implant applications. We also demonstrate that the micron scale intra-strut porosities can be exploited to allow infiltration of bioactive materials using a novel bioactive silica–polymer composite, resulting in a metal–bioactive silica–polymer composite.

Correlation of mineral density and elastic modulus of natural enamel white spot lesions using X-ray microtomography and nanoindentation - Corrected Proof

Tiffany T.Y. Huang, Li-Hong He, M. Ali Darendeliler, Michael V. Swain — 2010-06-30

Abstract: Our objectives were to correlate the mineral density (MD) and elastic modulus (E) of natural white spot lesions (WSLs) and compare them with analytical and numerical models. Five natural WSLs from four extracted sound premolar teeth were scanned at a voxel size of 7.6μm using a desktop X-ray microtomography (XRMT) system. Five hydroxyapatite phantoms with densities ranging from 1.52 to 3.14gcm−3 were used as calibration standards for each scan. MD throughout the WSLs was quantified using an MD calibration equation derived from hydroxyapatite phantoms. Subsequently, teeth were cross-sectioned and the E modulus was measured systematically across the WSLs at intervals of 25 and 50μm using nanoindentation. The MD and E modulus of WSLs correlated well. The relationship may be expressed as E=E0exp−bP (R2=0.952) with E0 the elastic modulus of the fully dense material, P the porosity and b a constant. The results for sound enamel were compared with Spears model. The limitation of Spears model to the WSLs is discussed and an alternative model developed by Rice for porous materials is proposed. Clinical implications of this work for quantifying de-/remineralization of teeth are pointed out. We conclude that XRMT can be utilized to extrapolate the E modulus of WSLs. This provides a basis for non-destructive, longitudinal analysis of WSLs in de-/remineralization studies of enamel.

Design principles for cytokine-neutralizing gels: Cross-linking effects - Uncorrected Proof

2010-06-30

Abstract: Constructs composed of cytokine-neutralizing antibodies conjugated to high-molecular-weight hyaluronic acid have been shown to be effective at controlling inflammatory responses in vivo. A critical question in the development of this new class of biomaterial is whether crosslinked conjugates have similar anti-inflammatory effects, which would open up a broad range of tissue engineering applications in which the material would have intrinsic inflammation-controlling function. To test this, high-molecular-weight hyaluronic acid was conjugated with monoclonal antibodies to the pro-inflammatory cytokines interleukin-1β and tumor necrosis factor-α in two forms of the material: viscous, non-crosslinked polymer–antibody conjugates and crosslinked, elastomeric polymer–antibody conjugates. The cytokine affinities of both constructs were validated using molecular characterization methods, and the biological activities were tested through subcutaneous implantation in Sprague–Dawley rats. In vitro, both forms of these constructs are capable of binding cytokines, but in vivo only the non-crosslinked polymer significantly reduces markers of acute inflammation compared to controls that lack the antibodies. We propose that these materials function by retarding cytokine diffusion, with the non-crosslinked polymers being capable of retarding the diffusion of cytokines in the extracellular matrix and preventing engagement with receptors. In contrast, crosslinked materials have long diffusion lengths into the gel compared with those between cells on the surface of the material, which may make them ineffective at sequestering pro-inflammatory cytokines on biologically relevant timescales. These results suggest an important design principle for preparing cytokine-regulating materials based on consideration of transport phenomena.

Magnetic mesoporous silica spheres for hyperthermia therapy - Corrected Proof

2010-06-30

Abstract: Magnetic nanoparticles coated with materials having unique properties, such as ordered pore structures and large surface areas, hold great potential for multimodal therapies. This study reports on the biocompatibility of composites of maghemite nanoparticles embedded in an ordered mesoporous silica-matrix to form magnetic microspheres (MMS), and on their ability to conduct magnetic hyperthermia upon exposure to a low-frequency alternating magnetic field (AMF). MMS particles were efficiently internalized by human A549, Saos-2 and HepG2 cells, and were excluded from the nuclear compartment. MMS treatment did not interfere with morphological features or metabolic activities of the cells, indicating good biocompatibility of the material. MMS did not affect the endogenous heat-shock response of a HeLa-derived cell line that precisely reports the intensity of thermal stresses through changes in the activities of a stably integrated hsp70B promoter and a constitutive viral promoter. Maximum temperature in MMS suspensions increased to a range above 42°C as a function of the amounts of particles exposed to AMF. Cell culture experiments showed that, by adjusting the amount of MMS and the time of exposure to AMF, heat treatments of mild to very high intensities could be achieved. Cell viability dropped as a function of the intensity of the heat treatment achieved by MMS and AMF exposures. The possibility of fine-tuning the heating power output, together with efficient uptake by tumor cells in vitro, makes MMS a promising agent by which to provide hyperthermia treatments aimed toward remission of solid tumors.

Composite alginate hydrogels: An innovative approach for the controlled release of hydrophobic drugs - Corrected Proof

Elinor Josef, Meital Zilberman, Havazelet Bianco-Peled — 2010-06-30

Abstract: We present an innovative methodology for the sustained delivery of hydrophobic drugs using composite hydrogels, prepared by embedding oil-in-water microemulsions in hydrophilic hydrogels. The hydrophobic nature of the microemulsion core enhances the solubilization of hydrophobic drugs, while the crosslinked matrix could be readily used as a solid controlled delivery vehicle. A microemulsion was formulated from pharmaceutical accepted components; the droplets diameter was shown to be about 10nm by dynamic light scattering, cryo-transmission electron microscopy and small-angle X-ray scattering (SAXS). Combining the microemulsion with alginate solution and crosslinking with calcium ions resulted in a clear hydrogel. A model hydrophobic drug, Ketoprofen, precipitated from the alginate hydrogel, but the drug–containing composite hydrogel was clear and macroscopically homogeneous. The nanostructure was investigated by SAXS; scattering plots indicate that oil droplets exist in the composite hydrogel. Release profiles of the drug from the composite hydrogel with various concentrations of polymer and crosslinker demonstrate the applicability of this system as a controlled delivery vehicle, and suggest that the release rate is governed not by the microemulsion structure but, rather, by the network properties. Furthermore, it was demonstrated that the release rate could be tailored for a specific application utilizing different alginate and calcium concentrations. The generalization of the methodology of including hydrophobic drugs in composite gels is discussed.

Multifunctional nature of UV-irradiated nanocrystalline anatase thin films for biomedical applications - Corrected Proof

2010-06-28

Abstract: Anatase is known to decompose organic material by photocatalysis and to enhance surface wettability once irradiated by ultraviolet (UV) light. In this study, pulse magnetron-sputtered anatase thin films were investigated for their suitability with respect to specific biomedical applications, namely superhydrophilic and biofilm degrading implant surfaces. UV-induced hydrophilicity was quantified by static and dynamic contact angle analysis. Photocatalytic protein decomposition was analyzed by quartz crystal microbalance with dissipation. The surfaces were characterized by X-ray diffraction, atomic force microscopy, scanning electron microscopy and X-ray photoelectron spectroscopy. The radical formation on anatase, responsible for photocatalytic effects, was analyzed by electron spin resonance spectroscopy. Results have shown that the nanocrystalline anatase films, in contrast to reference titanium surfaces, were sensitive to UV irradiation and showed rapid switching towards superhydrophilicity. The observed decrease in carbon adsorbents and the increase in the fraction of surface hydroxyl groups upon UV irradiation might contribute to this hydrophilic behavior. UV irradiation of anatase pre-conditioned with albumin protein layers induces the photocatalytic decomposition of these model biofilms. The observed degradation is mainly caused by hydroxyl radicals. It is concluded that nanocrystalline anatase films offer different functions at implant interfaces, e.g. bedside hydrophilization of anatase-coated implants for improved osseointegration or the in situ decomposition of conditioning films forming the basal layer of biofilms in the oral cavity.

Histology and research at the hard tissue–implant interface using Technovit 9100 New embedding technique - Corrected Proof

Elmar Willbold, Frank Witte — 2010-06-28

Abstract: Calcified tissues, like bones and teeth, are among the most challenging tissues for histological research. However, especially with respect to dental or orthopaedic research, powerful histological techniques are necessary to study pathological conditions or traumatic injuries, and to investigate the molecular and cellular mechanisms of regeneration processes and functional recovery. The situation is even more complicated in orthopaedic research because here metallic implants or other devices made of various materials are often present, and the hard tissue–implant interface is of crucial interest in both biocompatibility and functional recovery research. After the cutting–grinding technique, embedding in technical resins is the most promising approach. Here we describe an optimized and standardized embedding and cutting technique using Technovit 9100 New. Using this technique, we are able to perform enzyme histochemistry, immunohistochemistry, a great variety of classical histological stains and even in situ hybridization.

Fibrillar superstructure formation of hemoglobin A and its conductive, photodynamic and photovoltaic effects - Corrected Proof

2010-06-28

Abstract: The fabrication of biomaterials which serve as functional scaffolds exhibiting diversified effects has been valued. We report here a unique strategy to fibrillate hemoglobin A (HbA), which exhibits multiple photoelectrochemical properties, and a subsequent specific defibrillation procedure. A subtle structural rearrangement of the α/β-subunits within the quaternary structure of HbA is responsible for the HbA fibril formation in the presence of 0.5% CHCl3. The narrow pH dependence of the suprastructure formation around pH 7.4 illustrates the highly sensitive nature of the structural alteration. The CHCl3-induced fibrils become disintegrated by ascorbic acid, indicating that the oxidation–reduction process of the iron within the heme moiety could be involved in stabilization of the fibrillar structures. The electron-transferring property of the iron allows the fibrils to exhibit not only their conductive behavior but also a photodynamic effect generating hydroxyl radicals in the presence of H2O2 with light illumination. A photovoltaic effect is also demonstrated with the HbA fibrils, which generate an electric current on the fibril-coated microelectrode upon irradiation at 405nm. Taken together, the multiple effects of HbA fibrils and the selective fibrillation/defibrillation procedures could qualify the fibrils to be employed for various future applications in biotechnology, including bio-machine interfaces.

In vivo short-term and long-term host reaction to starch-based scaffolds - Corrected Proof

2010-06-24

Abstract: The implantation of biomaterials may elicit a host response to this foreign body, and the magnitude of that reaction depends on the host and on the implanted material. The aim of this study was to compare the inflammatory response induced by the implantation of starch-based (SPCL) scaffolds in two implantation rat models: subcutaneous (SC) and intramuscular (IM). Moreover, two methodologies, wet spinning (WS) and fibre-bonding (FB), were used to prepare the scaffolds. The short-term inflammatory/immune host reaction was assessed by SC and IM implantations in rats after 1 and 2weeks, and the long-term host response was addressed after 8 and 12weeks of SC implantation of both types of SPCL scaffolds in rats. After each time period, the scaffolds, surrounding tissue and nearby lymph nodes were explanted, and used for histological analysis and molecular biology evaluation. The results showed that SPCL-WS scaffolds seem to induce a slight lower inflammatory/immune reaction in both types of implantation models. Nonetheless, comparing the two models, the IM implantation resulted in a slightly higher inflammatory response than the SC implantation with early activation of the lymph nodes. The overall data suggests a good integration of the materials in the host, independently of the tissue location with a normal progress of the reaction for all the conditions.

Genetic profiling of osteoblast-like cells cultured on a novel bone reconstructive material, consisting of poly-l-lactide, carbon nanotubes and microhydroxyapatite, in the presence of bone morphogenetic protein-2 - Corrected Proof

2010-06-21

Abstract: In bone tissue engineering composite materials have been introduced, combining a degradable polymer matrix with, for instance, carbon nanotubes (CNTs) to improve mechanical properties or with microhydroxyapatite (μHA) to improve osteoconduction. The addition of bone morphogenetic protein-2 (BMP-2) can further improve the biological response to the material. However, the influence of such an elaborate composite formation on osteoprogenitor cells is unknown.To examine this, rat bone marrow (RBM) cells were cultured on porous poly-l-lactic acid and composite scaffolds, with or without added BMP-2. Cell proliferation and differentiation were studied using DNA, alkaline phosphatase and scanning electron microscopic analysis. Further, genetic profiles were examined by microarray investigation. Results showed that the composite scaffold had no significant effect on the proliferation of RBM cells, but indicated a negative effect on cell differentiation. The addition of BMP-2 also had no significant effect on the proliferation of RBM cells, but differentiation towards the osteogenic lineage was confirmed. In the arrays results, the addition of BMP-2 alone led to the expression of genes involved in (minor) inflammation. The composite scaffold, and even more distinctly the combination of the composite scaffold with BMP-2, led to the expression of genes, based on gene ontology, connected to tumorigenesis. Therefore, CNT- and μHA-containing composite materials are not recommended as a bone restorative material.