Acta Biomaterialia

Editorial Board

2010-04-01

Passive and active microrheology for cross-linked F-actin networks in vitro

Hyungsuk Lee, Jorge M. Ferrer, Fumihiko Nakamura, Matthew J. Lang, Roger D. Kamm — 2009-11-02

Abstract: Actin filament (F-actin) is one of the dominant structural constituents in the cytoskeleton. Orchestrated by various actin-binding proteins (ABPs), F-actin is assembled into higher-order structures such as bundles and networks that provide mechanical support for the cell and play important roles in numerous cellular processes. Although mechanical properties of F-actin networks have been extensively studied, the underlying mechanisms for network elasticity are not fully understood, in part because different measurements probe different length and force scales. Here, we developed both passive and active microrheology techniques using optical tweezers to estimate the mechanical properties of F-actin networks at a length scale comparable to cells. For the passive approach we tracked the motion of a thermally fluctuating colloidal sphere to estimate the frequency-dependent complex shear modulus of the network. In the active approach, we used an optical trap to oscillate an embedded microsphere and monitored the response in order to obtain network viscoelasticity over a physiologically relevant force range. While both active and passive measurements exhibit similar results at low strain, the F-actin network subject to high strain exhibits non-linear behavior which is analogous to the strain-hardening observed in macroscale measurements. Using confocal and total internal reflection fluorescent microscopy, we also characterize the microstructure of reconstituted F-actin networks in terms of filament length, mesh size and degree of bundling. Finally, we propose a model of network connectivity by investigating the effect of filament length on the mechanical properties and structure.

Biodegradable fibrous scaffolds with diverse properties by electrospinning candidates from a combinatorial macromer library

2009-10-22

Abstract: The properties of electrospun fibrous scaffolds, including degradation, mechanics and cellular interactions, are important for their use in tissue engineering applications. Although some diversity has been obtained previously in fibrous scaffolds, optimization of scaffold properties relies on iterative techniques in both polymer synthesis and processing. Here, we electrospun candidates from a combinatorial library of biodegradable and photopolymerizable poly(β-amino ester)s (PBAEs) to show that the diversity in properties found in this library is retained when processed into fibrous scaffolds. Specifically, three PBAE macromers were electrospun into scaffolds and possessed similar initial mechanical properties, but exhibited mass loss ranging from rapid (complete degradation within ∼2weeks) to moderate (complete degradation within ∼3months) to slow (only partial degradation after 3months). These trends in mechanics and degradation mimicked what was previously observed in the bulk polymers. Although cellular adhesion was dependent on the polymer composition in films, adhesion to scaffolds that were electrospun with gelatin was similar on all formulations and controls. To further illustrate the diverse properties that are attainable in these systems, the fastest and slowest degrading polymers were electrospun together into one scaffold, but as distinct fiber populations. This dual-polymer scaffold exhibited behavior in mass loss and mechanics with time that fell between the single-polymer scaffolds. In general, this work indicates that combinatorial libraries may be an important source of information and specific polymer compositions for the fabrication of electrospun fibrous scaffolds with tunable properties.

Multiscale three-dimensional scaffolds for soft tissue engineering via multimodal electrospinning

2009-11-02

Abstract: A novel (scalable) electrospinning process was developed to fabricate bio-inspired multiscale three-dimensional scaffolds endowed with a controlled multimodal distribution of fiber diameters and geared towards soft tissue engineering. The resulting materials finely mingle nano- and microscale fibers together, rather than simply juxtaposing them, as is commonly found in the literature. A detailed proof of concept study was conducted on a simpler bimodal poly(ε-caprolactone) (PCL) scaffold with modes of fiber distribution at 600nm and 3.3μm. Three conventional unimodal scaffolds with mean diameters of 300nm and 2.6 and 5.2μm, respectively, were used as controls to evaluate the new materials. Characterization of the microstructure (i.e. porosity, fiber distribution and pore structure) and mechanical properties (i.e. stiffness, strength and failure mode) indicated that the multimodal scaffold had superior mechanical properties (Young’s modulus ∼40MPa and strength ∼1MPa) in comparison with the controls, despite the large porosity (∼90% on average). A biological assessment was conducted with bone marrow stromal cell type (mesenchymal stem cells, mTERT-MSCs). While the new material compared favorably with the controls with respect to cell viability (on the outer surface), it outperformed them in terms of cell colonization within the scaffold. The latter result, which could neither be practically achieved in the controls nor expected based on current models of pore size distribution, demonstrated the greater openness of the pore structure of the bimodal material, which remarkably did not come at the expense of its mechanical properties. Furthermore, nanofibers were seen to form a nanoweb bridging across neighboring microfibers, which boosted cell motility and survival. Lastly, standard adipogenic and osteogenic differentiation tests served to demonstrate that the new scaffold did not hinder the multilineage potential of stem cells.

Incorporation of biodegradable electrospun fibers into calcium phosphate cement for bone regeneration

Yi Zuo, Fang Yang, Joop G.C. Wolke, Yubao Li, John A. Jansen — 2009-10-26

Abstract: Inherent brittleness and slow degradation are the major drawbacks for the use of calcium phosphate cements (CPCs). To address these issues, biodegradable ultrafine fibers were incorporated into the CPC in this study. Four types of fibers made of poly(ε-caprolactone) (PCL) (PCL12: 1.1μm, PCL15: 1.4μm, PCL18: 1.9μm) and poly(l-lactic acid) (PLLA4: 1.4μm) were prepared by electrospinning using a special water pool technique, then mixed with the CPC at fiber weight fractions of 1%, 3%, 5% and 7%. After incubation of the composites in simulated body fluid for 7days, they were characterized by a gravimetric measurement for porosity evaluation, a three-point bending test for mechanical properties, microcomputer topography and scanning electron microscopy for morphological observation. The results indicated that the incorporation of ultrafine fibers increases the fracture resistance and porosity of CPCs. The toughness of the composites increased with the fiber fraction but was not affected by the fiber diameter. It was found that the incorporated fibers formed a channel-like porous structure in the CPCs. After degradation of the fibers, the created space and high porosity of the composite cement provides inter-connective channels for bone tissue in growth and facilitates cement resorption. Therefore, we concluded that this electrospun fiber-CPC composite may be beneficial to be used as bone fillers.

Electrospun microfiber meshes of silicon-doped vaterite/poly(lactic acid) hybrid for guided bone regeneration

Akiko Obata, Toshiki Hotta, Takashi Wakita, Yoshio Ota, Toshihiro Kasuga — 2009-11-12

Abstract: Silicon-releasable microfiber meshes consisting of silicon-doped vaterite (SiV) particles and poly(lactic acid) (PLA) hybrids were prepared by electrospinning. Due to their flexibility and porosity they formed ideal membranes or scaffolds for guided bone regeneration. In addition, a trace amount of silicon species has been reported to stimulate osteogenic cells to mineralize and enhance bone formation. We propose a new method of preparation of silicon-releasing microfiber meshes by electrospinning. Their structure and hydroxyapatite (HA)-forming abilities in simulated body fluid were examined. In addition, we studied their stimulatory effects on osteoblast-like cells in vitro and bone-forming ability in vivo, with a special emphasis on their ability to release silicon. The meshes consisted of a hybrid of carboxy groups in PLA and amino groups in siloxane, derived from aminopropyltriethoxysilane or calcium ions on the SiV surface. This hybrid exhibited an enhanced ability to form HA. The meshes coated with HA released 0.2–0.7mgl−1 silicon species into the culture medium over 7days. Enhanced proliferation of osteoblast-like cells was observed using the meshes and new bone formed on the meshes when implanted into the calvaria of rabbits. These meshes, therefore, provide an excellent substrate for bone regeneration and exhibit enhanced bone-forming ability under both in vitro and in vivo conditions.

Poly(lactic-co-glycolic acid) electrospun fibrous meshes for the controlled release of retinoic acid

2009-08-17

Abstract: Poly(lactic-co-glycolic acid) (PLGA) meshes loaded with retinoic acid (RA) were prepared by applying the electrospinning technique. The purpose of the present work was to combine the biological effects of RA and the advantages of electrospun meshes to enhancing the mass transfer features of controlled release systems and cell interaction with polymeric scaffolds. The processing conditions for the fabrication of three-dimensional meshes were optimized by studying their influence on mesh morphology. Tensile testing showed that RA loading influenced the meshes’ mechanical properties by increasing their strength and rigidity. Moreover, the drug release and degradation profiles of the electrospun systems were compared to analogous RA-loaded PLGA films prepared by solvent casting. The results of this study highlight that the electrospun meshes preserved their fibrous structure after 4months under in vitro physiological conditions and showed a sustained controlled release of the loaded agent in comparison to that observed for cast films. The bioactivity of the loaded RA was investigated on murine preosteoblasts cells by evaluating its influence on cell proliferation and morphology.

Flexible and elastic porous poly(trimethylene carbonate) structures for use in vascular tissue engineering

2009-10-08

Abstract: Biocompatible and elastic porous tubular structures based on poly(1,3-trimethylene carbonate), PTMC, were developed as scaffolds for tissue engineering of small-diameter blood vessels. High-molecular-weight PTMC (Mn=4.37×105) was cross-linked by gamma-irradiation in an inert nitrogen atmosphere. The resulting networks (50–70% gel content) were elastic and creep resistant. The PTMC materials were highly biocompatible as determined by cell adhesion and proliferation studies using various relevant cell types (human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) and mesenchymal stem cells (MSCs)). Dimensionally stable tubular scaffolds with an interconnected pore network were prepared by particulate leaching. Different cross-linked porous PTMC specimens with average pore sizes ranging between 55 and 116μm, and porosities ranging from 59% to 83% were prepared. These scaffolds were highly compliant and flexible, with high elongations at break. Furthermore, their resistance to creep was excellent and under cyclic loading conditions (20 deformation cycles to 30% elongation) no permanent deformation occurred. Seeding of SMCs into the wall of the tubular structures was done by carefully perfusing cell suspensions with syringes from the lumen through the wall. The cells were then cultured for 7days. Upon proliferation of the SMCs, the formed blood vessel constructs had excellent mechanical properties. Their radial tensile strengths had increased from 0.23 to 0.78MPa, which is close to those of natural blood vessels.

Highly porous bioresorbable scaffolds with controlled release of bioactive agents for tissue-regeneration applications

Orly Grinberg, Itzhak Binderman, Hila Bahar, Meital Zilberman — 2009-11-02

Abstract: Highly porous poly(dl-lactic-co-glycolic acid) films with controlled release of horseradish peroxidase (HRP) as a model protein have been successfully developed and studied. These films, which are prepared by freeze-drying inverted emulsions, are designed for use in tissue-regeneration applications. The effects of the emulsion’s formulation and host polymer’s characteristics on the film’s microstructure and HRP release profile over 4weeks were investigated. A dual pore size population is characteristic for most films, with large 12–18μm pores and small 1.5–7μm pores, and porosity in the range of 76–92%. An increase in the polymer content and its initial molecular weight, organic/aqueous (O:A) phase ratio and lactic acid content, or a decrease in the HRP content, all resulted in a decreased burst effect and a more moderate release profile. A simultaneous change in two or three of these formulation parameters (compared to a reference formulation) resulted in a synergistic effect on the HRP release profile. A constant HRP release rate was achieved when a composite film was used. Human gingival fibroblast adhesion to the films indicated good biocompatibility. Appropriate selection of the emulsion’s parameters can therefore yield highly porous films with the desired protein-release behavior which can serve as scaffolds for bioactive agents in tissue-regeneration applications.

An alternative technique to shape scaffolds with hierarchical porosity at physiological temperature

Juan Peña, Jesús Román, M. Victoria Cabañas, María Vallet-Regí — 2009-11-02

Abstract: The method described in this work, termed GELPOR3D, is characterised by its simplicity of use, low-cost equipment, compositional flexibility, and lack of aggressive or toxic solvents or other thermal treatment. This technique ensures the generation of a three-dimensional network of interconnected pores (300–900μm); in addition, a random and not necessarily connected porosity is generated, yielding a hierarchical porous architecture from the macro to the molecular scale. The interconnected pores, large enough to ensure an adequate vascularization and new tissue ingrowth, can be obtained by pouring a slurry containing a biodegradable thermogel (such as agarose and gellan) and a ceramic into a mold consisting of a three-dimensional network of rigid filaments. Additional pore distributions in the macropore region can be tailored as a function of the drying/preservation technology (10–100μm) or the interaction between the inorganic particles coated by the polymeric components (0.1–1μm). Moreover, porosity in the mesopore range can be created by shaping ceramics such as mesoporous silica or nanocrystalline carbonatehydroxyapatite. In addition to the various bioceramics that have been successfully shaped, this method is flexible enough to allow the introduction of certain substances whose controlled release may help to avoid some negative effects that usually appear with the implantation of a material, i.e. infection, inflammation, etc., or to simplify some of the many steps required for the successful integration of a graft.

Micro-structured smart hydrogels with enhanced protein loading and release efficiency

2009-11-13

Abstract: A series of temperature-responsive poly(N-isopropylacrylamide) (PNIPAAm) hydrogels with highly porous microstructures were successfully prepared by using hydrophobic polydimethylsiloxane (PDMS) and sodium dodecyl sulfate as liquid template and stabilizer, respectively. These newly prepared hydrogels possess highly porous structures. In contrast to the conventional PNIPAAm hydrogel, the swelling ratios of the porous gels at room temperature were higher, and their response rates were significantly faster as the temperature was raised above the lower critical solution temperature. For example, the novel hydrogel prepared with 40% PDMS template lost over 95% water within 5min, while the conventional PNIPAAm gel only lost approximately 14% water in the same time. The improved properties are achieved due to the presence of liquid PDMS templates in the reaction solutions, which lead to the formation of porous structures during the polymerization/crosslinking. Lysozyme and bovine serum albumin (BSA) as protein models were for the first time loaded into these micro-structured smart hydrogels through a physical absorption method. The experimental results show that the loading efficiency of BSA with a higher molecular weight is lower than that of lysozyme due to the size exclusion effect, and the loading efficiencies of both proteins in the porous hydrogel are much higher than those in the conventional PNIPAAm hydrogel. For example, the loading efficiency of BSA in porous hydrogel is 0.114, approximately 200% higher than that in conventional hydrogel (0.035). Both lysozyme and BSA were completely released from the porous hydrogel at 22°C. Furthermore, the release kinetics of the proteins from the porous hydrogel could be modulated by tuning the environmental temperature. These newly prepared porous materials provide an avenue to increase the loading efficiency and to control the release patterns of macromolecular drugs from hydrogels, and show great promise for application in protein or gene delivery.

Mechanical and swelling characterization of poly(N-isopropyl acrylamide -co- methoxy poly(ethylene glycol) methacrylate) sol–gels

Jacob F. Pollock, Kevin E. Healy — 2009-11-26

Abstract: The dimensional stability and rheological properties of a series of comb-like copolymers of N-isopropyl acrylamide (NIPAAm) and methoxy poly(ethylene glycol) methacrylate (mPEGMA), poly(NIPAAm–co-mPEGMA), with varying poly(ethylene glycol) (PEG) graft densities and molecular weights were studied. The thermoresponsive character of the copolymer solutions was investigated by kinetic and equilibrium swelling, as well as by static and dynamic mechanical analysis. Surface response mapping was employed to target particular compositions and concentrations with excellent dimensional stability and a relatively large change in dynamic mechanical properties upon thermoreversible gelation. The mechanical characteristics of the gels depended strongly upon concentration of total polymer and less so upon copolymer ratio. Increased PEG graft density was shown to slow the deswelling rate and increase the equilibrium water content of the gels. Upon gelation at sol concentrations of 1–20wt.% the materials underwent no deswelling or syneresis and maintained stable gels with a large elastic regime and high yield strain (i.e. elastic and soft but tough), even within the Pascal range of complex shear moduli. These materials are unique in that they maintained a physiologically useful lower critical solution temperature (∼33°C), despite having a high PEG content. Copolymers with a high PEG content and low polymer fraction were conveniently transparent in the gel phase, allowing visualization of cellular activity without disrupting the microenvironment. Mesenchymal stem cells showed good viability and proliferation in three-dimensional culture within the gels, despite the lack of ligand incorporation to promote cellular interaction. Multi-component matrices can be created through simple mixing of copolymer solutions and peptide-conjugated linear polymers and proteins to produce combinatorial microenvironments with the potential for use in cell biology, tissue engineering and medical applications.

Synthesis and recovery characteristics of branched and grafted PNIPAAm–PEG hydrogels for the development of an injectable load-bearing nucleus pulposus replacement

2009-10-19

Abstract: A family of injectable poly(N-isopropyl acrylamide) (PNIPAAm) copolymer hydrogels has been fabricated in order to tune mechanical properties to support load-bearing function and dimensional recovery for possible use as load-bearing medical devices, such as a nucleus pulposus replacement for the intervertebral disc. PNIPAAm–polyethylene glycol (PEG) copolymers were synthesized with varying hydrophilic PEG concentrations as grafted or branched structures to enhance dimensional recovery of the materials. Polymerizations were confirmed with attenuated total reflectance-Fourier transform infrared spectroscopy and proton nuclear magnetic resonance spectroscopy studies. Incorporation of PEG was effective in raising water content of pure PNIPAAm hydrogels (29.3% water for pure PNIPAAm vs. 47.7% for PEG branches and 39.5% for PEG grafts). PNIPAAm with 7% grafted as well as 7% branched PEG had significantly reduced compressive modulus compared to that of pure PNIPAAm. Initially recovered compressive strain was significantly increased for 7% PEG branches after pre-testing immersion in PBS for up to 33days, while 7% PEG grafts decreased this value. Sample height recovery for pure PNIPAAm was limited to 31.6%, while PNIPAAm with 7% branches was increased to 71.3%. When mechanically tested samples were allowed to recover without load over 30min, each composition was able to significantly recover height, indicating that the time to recovery is slower than the unloading rates typically used in testing. While the incorporation of hydrophilic PEG was expected to alter the mechanical behavior of the hydrogels, only the branched form was able to significantly enhance dimensional recovery.

Modified PHBV scaffolds by in situ UV polymerization: Structural characteristic, mechanical properties and bone mesenchymal stem cell compatibility

Y. Ke, Y.J. Wang, L. Ren, Q.C. Zhao, W. Huang — 2009-10-22

Abstract: An ideal scaffold provides an interface for cell adhesion and maintains enough biomechanical support during tissue regeneration. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) scaffolds with pore sizes ranging from 100 to 500μm and porosity ∼90% were prepared by the particulate-leaching method, and then modified by the introduction of polyacrylamide (PAM) on the inner surface of scaffolds using in situ UV polymerization, with the aim of enhancing the biological and mechanical properties of the PHBV scaffolds. The modified PHBV scaffolds had interconnected pores with porosity of 75.4–78.6% and pore sizes at peak volume from 20 to 50μm. The compressive load and modulus were up to 62.45N and 1.06MPa, respectively. The water swelling percentage (WSP) of the modified PHBV scaffolds increased notably compared with that of the PHBV scaffolds, with the maximum WSP at 537%. Sheep bone mesenchymal stem cells (BMSC) were cultured on the PHBV and modified PHBV. The hydrophilic PAM chains did not influence BMSC viability or proliferation index, but the initial cell adhesion at 1h of culture was enhanced significantly. Framing PHBV scaffold along with gel-like PAM chains inside is a novel model of inner surface modification for PHBV scaffolds, which shows potential in tissue engineering applications.

Fabrication and characterisation of protein fibril–elastomer composites

Tomas Oppenheim, Tuomas P.J. Knowles, Stéphanie P. Lacour, Mark E. Welland — 2009-10-15

Abstract: Protein fibrils are emerging as a novel class of functional bionanomaterials. In this paper we make use of their rigidity by combining lysozyme fibrils with a silicone elastomer and demonstrating that at a filling ratio of 10%, the protein fibril composite is at minimum 2 times stiffer than a CNT elastomeric composite of the same filling ratio. We also show that when the elastomer is patterned such that the lysozyme fibrils can be spatially modulated within the elastomer, anisotropic moduli varying by a factor of 2 is produced. By using shear mixing as the fabrication process, the modulus of a 2wt.% insulin fibril composite is equivalent to a CNT composite with the same filling ratio. In conclusion, we have presented the fabrication and mechanical characterisation of a class of elastomer/protein fibril composite material.

Manipulation of process parameters to achieve different ternary phase microparticle configurations

Wei Li Lee, Wan Ling Foo, Effendi Widjaja, Say Chye Joachim Loo — 2009-10-26

Abstract: Ternary phase microparticles of poly(d,l-lactide-co-glycolide) (50:50), poly(l-lactide) and poly(caprolactone) were fabricated through a one-step solvent evaporation technique. The purpose of this study was to examine the effects of various process parameters on the final configuration (i.e. polymer distribution and dimensions) of these composite microparticles and, subsequently, propose their mechanism of formation. Particle morphologies and configurations were determined using scanning electron microscopy, polymer dissolution tests and Raman mapping. It was found that a starting polymer solution prepared below the cloud point and an increased oil to water ratio will facilitate polymer configurations close to thermodynamic equilibrium, which is dictated by the interfacial energies of the components. By varying the polymer mass ratio or adjusting the precipitation rate, through stirring speed and oil to water ratio, a wide range of microparticles with different core–shell dimensions and embedded particulate sizes can also be fabricated. At the same time, lowering the polymer solution concentration and increasing the stirring speed may result in smaller microparticles. Correlation of these process parameters with the final composite particle morphology was thus established. This understanding should allow the controlled fabrication of ternary phase composite microparticles through a single step solvent evaporation technique.

Formation of a strong hydrogel–porous solid interface via the double-network principle

Takayuki Kurokawa, Hidemitsu Furukawa, Wei Wang, Yoshimi Tanaka, Jian Ping Gong — 2009-11-02

Abstract: A method for binding a tough double-network (DN) hydrogel and a porous solid utilizing the double-network principle is proposed. The effects of the pore size of the solid and the structure of the DN gel in the pores on bonding strength were investigated by a peeling test. Porous solids with pore sizes of the order of several microns afforded strong gel–substrate interfaces. Under optimal conditions a bonding strength as high as ∼1000Nm−1 was reached. The results obtained were compared with the strength of the bulk DN gel, and discussed in terms of the double-network principle at the bonding interface.

A study on partially biodegradable microparticles as carriers of active glycolipids

2009-11-12

Abstract: This paper describes a study on the preparation and characterisation of partially biodegradable microparticles of poly(ε-caprolactone)/poly(ethyl methacrylate) (PCL/PEMA) as carriers of synthetic glycolipids with antimitotic activity against brain tumour cells. Microparticles prepared by suspension polymerisation of methacrylate in the presence of already polymerised PCL showed a predominantly spherical but complex morphology, with segregation of PCL micro/nano-domains towards the surface. Small diameter discs were prepared by compression moulding of blends of microparticles and the active principle under mild conditions. The in vitro behaviour of the discs and release of the glycolipid were studied in different simulated fluid models. Ingress of fluids increased with increasing hydrophobicity of the medium. Release of the glycolipid was sustained in all fluids, the most prolonged profile being in human synovial fluid and phosphate-buffered saline modified with 20 vol.% dioxane. Slow disintegration of the discs and partial degradation of the microparticles was evident in accelerated studies. The antimitotic activity of glycolipid released from the discs was proved against a human glioblastoma line. This activity, along with selectivity against human fibroblasts, could be controlled by the amount of drug charged in the disc.

Biodegradable hybrid polymeric membranes for ocular drug delivery

Dharmendra Jain, Edmund Carvalho, R. Banerjee — 2009-11-09

Abstract: Ophthalmic delivery systems such as ocular inserts are useful strategies to improve the ocular bioavailability of topically administered drugs. In the present study polyvinyl alcohol and sodium carboxymethylcellulose based ocular inserts were prepared by solution casting for sustained drug delivery of ciprofloxacin for treatment of topical infections. The polymers were esterified and the formation of ester bonds was confirmed by Fourier transform infrared spectroscopy. The inserts had a smooth structure with a surface roughness of 7.3nm. Inserts were found to be wettable by simulated tear fluid with contact angle <45°. Mechanical testing results indicated that the tensile strength of polyvinyl alcohol–sodium carboxymethylcellulose (10:2wt.%) inserts was up to 8.9±1.9MPa, which is adequate to resist the pressure likely to be exerted during application. In vitro drug release kinetics showed sustained release of ciprofloxacin for up to 48h from the inserts. Sodium fluorescein-loaded inserts showed higher penetration of the dye in the posterior segment tissues of explanted goat eye balls as compared with an eye drop solution of sodium fluorescein. The inserts were non-toxic to corneal epithelial cells and showed no signs of acute ocular toxicity in in vivo studies in albino rabbits.

Water-insoluble silk films with silk I structure

2009-10-28

Abstract: Water-insoluble regenerated silk materials are normally produced by increasing the β-sheet content (silk II). In the present study water-insoluble silk films were prepared by controlling the very slow drying of Bombyx mori silk solutions, resulting in the formation of stable films with a predominant silk I instead of silk II structure. Wide angle X-ray scattering indicated that the silk films stabilized by slow drying were mainly composed of silk I rather than silk II, while water- and methanol-annealed silk films had a higher silk II content. The silk films prepared by slow drying had a globule-like structure at the core surrounded by nano-filaments. The core region was composed of silk I and silk II, surrounded by hydrophilic nano-filaments containing random turns and α-helix secondary structures. The insoluble silk films prepared by slow drying had unique thermal, mechanical and degradative properties. Differential scanning calorimetry results revealed that silk I crystals had stable thermal properties up to 250°C, without crystallization above the Tg, but degraded at lower temperatures than silk II structure. Compared with water- and methanol-annealed films the films prepared by slow drying had better mechanical ductility and were more rapidly enzymatically degraded, reflecting the differences in secondary structure achieved via differences in post processing of the cast silk films. Importantly, the silk I structure, a key intermediate secondary structure for the formation of mechanically robust natural silk fibers, was successfully generated by the present approach of very slow drying, mimicking the natural process. The results also point to a new mode of generating new types of silk biomaterials with enhanced mechanical properties and increased degradation rates, while maintaining water insolubility, along with a low β-sheet content.

Adhesion, migration and mechanics of human adipose-tissue-derived stem cells on silk fibroin–chitosan matrix

Andrew M. Altman, Vishal Gupta, Carmen N. Ríos, Eckhard U. Alt, Anshu B. Mathur — 2009-10-26

Abstract: Silk fibroin–chitosan (SFCS) scaffold is a naturally derived biocompatible matrix with potential reconstructive surgical applications. In this study, human adipose-derived mesenchymal stem cells (ASCs) were seeded on SFCS scaffolds and cell attachment was characterized by fluorescence, confocal, time-lapse, atomic force, and scanning electron microscopy (SEM) studies. Adhesion of ASCs on SFCS was 39.4±4.8% at 15min, increasing to 92.8±1.5% at 120min. ASC adhered at regions of architectural complexity and infiltrate into three-dimensional scaffold. Time-lapse confocal studies indicated a mean ASC speed on SFCS of 18.47±2.7μmh−1 and a mean persistence time of 41.4±9.3min over a 2.75h study period. Cytokinetic and SEM studies demonstrated ASC–ASC interaction via microvillus extensions. The apparent elastic modulus was significantly higher (p<0.0001) for ASCs seeded on SFCS (69.0±9.0kPa) than on glass (6.1±0.4kPa). Also, cytoskeleton F-actin fiber density was higher (p<0.05) for ASC seeded on SFCS (0.42±0.02 fibers μm−1) than on glass-seeded controls (0.24±0.03 fibers μm−1). Hence, SFCS scaffold facilitates mesenchymal stem cell attachment, migration, three-dimensional infiltration, and cell–cell interaction. This study showed the potential use of SFCS as a local carrier for autologous stem cells for reconstructive surgery application.

Cyclodextrin-functionalized biomaterials loaded with miconazole prevent Candida albicans biofilm formation in vitro

2009-10-28

Abstract: Polyethylene (PE) and polypropylene (PP) were functionalized at their surfaces with cyclodextrins (CDs) in order to prevent the adhesion and proliferation of Candida albicans on medical devices made from these polymers. The surface functionalization involved the grafting of glycidyl methacrylate (GMA) after oxidative γ-ray pre-irradiation, followed by the attachment of β-CD and HP-β-CD to PE-g-GMA and PP-g-GMA surfaces. The yield of CD functionalization directly depended on the amount of GMA grafted. The presence of CDs on the surface of the polymers did not compromise their cell compatibility, but remarkably changed their protein adsorption profile. In contrast to unmodified PE and PP that adsorb significant amounts of fibrinogen (∼0.047mgcm−2) but not albumin, the CD-modified polyethers promoted the adsorption of albumin (between 0.015 and 0.155mgcm−2) and reduced the adsorption of fibrinogen. Furthermore, functionalization with CDs provided PE and PP with the capability to incorporate the anti-fungal drug miconazole (up to 0.27mgcm−2), leading to reduced biofilm formation by C. albicans in an in vitro biofilm model system. Overall, the results of the work indicate that the novel approach for functionalization of PE and PP is potentially useful to reduce the likelihood of foreign body-related infections.

In vivo performance of absorbable collagen sponges with rosuvastatin in critical-size cortical bone defects

2009-10-07

Abstract: Rosuvastatin (RSV) is a synthetic statin with favourable pharmacologic properties, but its local effect in bone has yet to be investigated. The aim of this study was to evaluate the potential of absorbable collagen sponge (ACS) as a carrier for RSV to enhance bone formation in critical-size cortical bone defects adjacent to titanium implants. ACS, treated with different concentrations of RSV (R1=8.7±1.8μg; R2=52.0±4.4μg; R3=259.1±8.8μg) or phosphate-buffered saline alone, were placed into the bone marrow through a defect made in the proximal tibial cortical bone of New Zealand White rabbits. One empty defect (SHAM) served as an internal control in each animal. After a healing time of 4weeks, a concentration-dependent increase of alkaline phosphatase activity in ACS treated with RSV was detected in the bone fluid after removing the implants. In addition, a significant concentration-dependent increase in BMP-2 mRNA levels was found in the cortical bone tissue adjacent to the RSV-treated ACS. The cortical architecture of bone defects analysed by micro-computed tomography showed a trend towards higher bone volume in the ACS+R1 group compared with SHAM, which was accompanied by an increase in the bone mineral density. Evaluation of histological sections showed new bone formation in ACS treated with RSV but not in untreated ACS. These results indicate that RSV, when administered locally in bone, may have a potential effect in stimulating bone formation.

Evaluation of composition and crosslinking effects on collagen-based composite constructs

Krishna Madhavan, Dmitry Belchenko, Antonella Motta, Wei Tan — 2009-10-07

Abstract: Vascular grafts are widely used for a number of medical treatments. Strength, compliance, endothelialization and availability are issues of most concern for vascular graft materials. With current approaches, these requirements are difficult to satisfy simultaneously. To explore an alternative approach, the present study has engineered the collagen gel construct by incorporating mimetic components and crosslinking the construct with different crosslinkers. The effects of component additives, such as chitosan and elastin, have been evaluated in terms of their mechanical and biological properties. Results demonstrate that the incorporation of chitosan and/or elastin alter stress–strain curves in the low stress loading region, and significantly improve the stretching ratio and ultimate stress of gel constructs compared to collagen constructs. Electron microscopy results suggest that the mechanical improvements might be due to microstructural modifications by chitosan sheets and elastin fibers. The effects of crosslinkers, such as formaldehyde, genipin and ethyl-(dimethyl aminopropyl) and carbodiimide hydrochloride (EDAC) have also been evaluated. Results demonstrate that formaldehyde, EDAC and genipin employ different mechanisms to crosslink collagen-based constructs, and use of genipin as a construct crosslinker exhibits improved elongation and endothelial coverage as compared to formaldehyde and EDAC. In addition, extending gelation time increased the elastic modulus but not the ultimate strength. Therefore, this study suggests that the mimicry of natural vessel tissues with properly crosslinked biopolymer composites could be a potential material design strategy for vascular graft materials.

Novel pH-sensitive chitosan-based hydrogel for encapsulating poorly water-soluble drugs

Tse-Ying Liu, Yi-Ling Lin — 2009-10-12

Abstract: Carboxymethyl–hexanoyl chitosan (CHC) is an amphiphilic chitosan derivative with excellent swelling ability and water solubility under natural conditions. In this work, the influence of the degree of carboxymethyl and hexanoyl substitution on the pH-sensitive swelling behavior, drug release behavior, and antiadhesion behavior of CHC hydrogels (cross-linked with genipin) were studied. It was found that the pH sensitivity was more pronounced in CHC than in N,O-carboxymethyl chitosan because the hexanoyl group altered the state of water in CHC by inhibiting intermolecular hydrogen bonding. In addition, greater pH sensitivity was observed in samples bearing longer hydrophobic chains (carboxymethyl–palmityl chitosan). Interestingly, when used with ibuprofen (a poorly water-soluble therapeutic agent used here as a model drug), the bursting release of the drug was less prominent in the CHC samples having a high degree of carboxymethyl substitution. The CHC hydrogel also demonstrated good cell compatibility and its antiadhesive ability after grafting was altered by changes in the degree of hexanoyl substitution.

Effect of chitosan scaffold microstructure on mesenchymal stem cell chondrogenesis

2009-10-26

Abstract: Although numerous biomaterials have been investigated as scaffolds for cartilage tissue engineering, the effect of their microstructure on final construct characteristics remains unclear. The biocompatibility of chitosan and its similarity with glycosaminoglycans make it attractive as a scaffold for cartilage engineering. Our objective was to evaluate the effect of chitosan scaffold structure on mesenchymal stem cell proliferation and chondrogenesis. Chitosan fibrous scaffolds and chitosan sponges were seeded with mesenchymal stem cells in a chondrogenic medium. Constructs were analyzed 72h after seeding via scanning electron microscopy (SEM), weight measurements and DNA quantification. Constructs were cultured for 10 or 21days prior to confocal microscopy, SEM, histology, quantitative analysis (weight, DNA and glycosaminoglycan (GAG)), and quantitative real-time polymerase chain reaction. Mesenchymal stem cells maintained a viability above 90% on all chitosan scaffolds. The cell numbers in the constructs were similar at 72h, 10days and 21days. However, matrix production was improved in chitosan fibrous constructs based on the GAG quantification and collagen II mRNA expression. Chondrogenesis on chitosan scaffolds is superior on microfibers compared to macroporous sponges.

Polyelectrolyte multilayer films allow seeded human progenitor-derived endothelial cells to remain functional under shear stress in vitro

2009-11-13

Abstract: There is considerable interest in making multilayer films for various applications, among which are cell contacting biomaterials, allowing new opportunities to prepare functionalized biomaterials. In this study we have explored the capability of poly(sodium-4-styrene sulfonate)/poly(allylamine hydrochloride) polyelectrolyte multilayer films (PMFs) as functional coatings for human progenitor-derived endothelial cells (PDECs), since the latter are a potential source of endothelial-type cells to be used in bioartificial vascular substitutes. We performed investigations with PDECs derived from peripheral blood and characterized as endothelial cells. After forming a confluent monolayer on PMFs they were exposed to laminar pulsatile physiological shear stress. We investigated whether PDECs were able to withstand shear stress and to respond at the mRNA (microarray analysis) and protein levels (thrombomodulin and tissue factor functional activity), in comparison with collagen I and fibrin glue used as controls. After shear stress the PDECs remained spread on the substrates, with a resulting increase in the number of expressed genes. Considering the functional significance of our findings for the regulation of coagulation and fibrinolytic factors, mRNA tissue plasminogen activator and thrombomodulin, profibrinolytic and thrombin inhibiting respectively, were overexpressed in PDECs after 6h shear stress. von Willebrand factor showed down-regulation, while tissue factor was up-regulated. We can speculate that PMFs could favour anti-thrombogenic activity by PDECs because activated protein C generation, measuring thrombomodulin activity, was particularly high on PMFs, but unchanged after 6h shear stress. Thus, PMFs could represent suitable coatings able to provide functional surfaces for endothelialization with PDECs.

Control of cellular adhesiveness in an alginate-based hydrogel by varying peroxidase and H2O2 concentrations during gelation

S. Sakai, K. Hirose, K. Moriyama, K. Kawakami — 2009-10-09

Abstract: An aqueous solution of alginate possessing phenolic hydroxyl (Alg-Ph) groups is gellable via a horseradish peroxidase (HRP)-catalyzed oxidative crosslinking reaction between Ph groups, consuming H2O2 as an electron acceptor. This study evaluates the effect of H2O2 and HRP concentrations on cellular adhesiveness and proliferation on the resultant enzymatically crosslinked Alg-Ph gels. After 4h of seeding, 81.1% of L929 fibroblast cells adhere to an Alg-Ph hydrogel prepared with 1Uml−1 HRP and 1mM H2O2. Increasing the concentration of H2O2 to 15mM decreases the percentage of adhering cells to 28.4%. The cellular adhesion at this H2O2 concentration is increased to 82.6% by increasing the HRP concentration to 10Uml−1. The cells adhering to the Alg-Ph hydrogels with higher cellular adhesiveness establish a confluent monolayer during 168h of culture. A cell sheet can then be harvested within 5min of immersion in a medium containing alginate lyase at 1.0mgml−1. The harvested cell sheet re-adhere, and the cells contained in the sheet proliferate after being transferred to another cell culture dish.

Changes in stiffness of resin-infiltrated demineralized dentin after remineralization by a bottom-up biomimetic approach

2009-11-02

Abstract: This study examined changes in elastic modulus, mineral density and ultrastructure of resin-infiltrated dentin after biomimetic remineralization. Sixty demineralized dentin beams were infiltrated with Clearfil Tri-S Bond, One-Step or Prime&Bond NT. They were immersed in simulated body fluid (SBF) for 1week to maximize water sorption before determining the baseline elastic moduli. For each adhesive (N=20) half of the beams remained immersed in SBF (control). The rest were immersed in a biomimetic remineralization medium. The elastic moduli were measured weekly for 15 additional weeks. Representative remineralized specimens were evaluated by X-ray microtomography and transmission electron microscopy (TEM). The elastic moduli of control resin-infiltrated dentin remained consistently low, while those immersed in the biomimetic remineralization medium increased by 55–118% after 4months. X-ray microtomography of the remineralized specimens revealed decreases in mineral density from the beam surface to the beam core that were indicative of external mineral aggregation and internal mineral deposition. Interfibrillar and intrafibrillar remineralization of resin-sparse intertubular dentin were seen under TEM, together with remineralized peritubular dentin. Biomimetic remineralization occurs by diffusion of nanoprecursors and biomimetic analogs in completely demineralized resin-infiltrated dentin and proceeds without the contribution of materials released from a mineralized dentin base.

A novel three-dimensional aerogel biochip for molecular recognition of nucleotide acids

2009-10-08

Abstract: Mesoporous aerogel was produced under regular atmospheric conditions using the sol–gel polymerization of tetraethyl orthosilicate with an ionic liquid as both solvent and active agent. This was then used to build a three-dimensional structure to recognize nucleotide acids. Fourier transformation infrared spectroscopy, scanning electron microscopy, 29Si solid-state nuclear magnetic resonance, and Brunauer–Emmett–Teller instruments were used to characterize this 3D aerogel, demonstrating that it had high porosity and large internal networking surface area that could capture nucleotide acids. The functionality of molecular recognition on nucleotide acids was demonstrated by immobilizing an oligonucleotide to probe its DNA target and confirming the tagged fluorescent signals by confocal laser scanning microscopy. The results indicated that the as-prepared 3D bioaerogel was capable of providing a very large surface area to capture and recognize human gene ATP5O.

Development of a three-dimensional unit cell to model the micromechanical response of a collagen-based extracellular matrix

2009-11-13

Abstract: The three-dimensional microstructure and mechanical properties of the collagen fibrils within the extracellular matrix (ECM) is now being recognized as a primary factor in regulating cell proliferation and differentiation. Therefore, an appreciation of the mechanical aspects by which a cell interacts with its ECM is required for the development of engineered tissues. Ultimately, using these interactions to design tissue equivalents requires mathematical models with three-dimensional architecture. In this study, a three-dimensional model of a collagen fibril matrix undergoing uniaxial tensile stress was developed by making use of cellular solids. A structure consisting of thin struts was chosen to represent the arrangement of collagen fibrils within an engineered ECM. To account for the large deformation of tissues, the collagen fibrils were modeled as hyperelastic neo-Hookean or Mooney–Rivlin materials. The use of cellular solids allowed the fibril properties to be related to the ECM properties in closed form, which, in turn, allowed the estimation of fibril properties using ECM experimental data. A set of previously obtained experimental data consisting of simultaneous measures of the fibril microstructure and mechanical tests was used to evaluate the model’s capability to estimate collagen fibril mechanical property when given tissue-scale data and to predict the tissue-scale mechanical properties when given estimated fibril stiffness. The fibril tangent modulus was found to be 1.26±0.70 and 1.62±0.88MPa when the fibril was modeled as neo-Hookean and Mooney–Rivlin material, respectively. There was no statistical significance of the estimated fibril tangent modulus among the different groups. Sensitivity analysis showed that the fibril mechanical properties and volume fraction were the two input parameters which required accurate values. While the volume fraction was easily obtained from the initial image of the gel, the fibril mechanical properties were not readily available. Therefore the fibril mechanical properties were estimated in the leave-one-out cross-validation (LOOCV) analysis. The LOOCV analysis showed that the model was able to predict the ECM stress–stretch curve with an average mean squared error of 9.71kPa2. The three-dimensional architecture expands on previous continuum models and two-dimensional representations to provide a useful model for studying the hierarchical effects of ECM microstructure on cell function. This model can be used as a design tool to engineer the optimum microstructure for cells to function.

Mechanical modeling of a wrinkled fingertip immersed in water

Jie Yin, Gregory J. Gerling, Xi Chen — 2009-10-22

Abstract: Fingertips often wrinkle after extended exposure to water. The underlying mechanics issues, in particular the critical parameters governing the wrinkled morphology, are studied by using both finite element simulation and analytical modeling. The wrinkling behaviors, characterized by the wrinkle-to-wrinkle distance (wavelength), wrinkle depth (amplitude) and critical wrinkling stress/strain, are investigated as the geometry and material parameters of the fingertip are varied. A simple reduced model is employed to understand the effect of finger curvature and skin thickness, whereas a more refined full anatomical model provides the basis for analyzing the effect of a multilayered skin structure. The simulation results demonstrate that the stiffness of the stratum corneum and the dermal layer in the skin has a large effect on the wrinkling behavior, which agrees well with the analytical predictions. From the uncovered mechanical principles, potential ways for effectively slowing down and suppressing skin wrinkles are proposed; among them, increasing the modulus of the dermal layer in the skin appears to be the most effective.

Quantifying the attachment strength of climbing plants: A new approach

2009-10-09

Abstract: In order to grow vertically, it is essential for climbing plants to firmly attach to their supporting structures. In climbing plants, different strategies for permanent attachment can be distinguished. Besides twining stems and tendrils, many plants use attachment pads or attachment roots for this purpose. Using a novel custom-built tensile testing setup, the mechanical properties of different permanent attachment structures of self-clinging plant species were investigated, namely the attachment pads of Boston ivy (Parthenocissus tricuspidata), the attachment roots of ivy (Hedera helix) and the clustered attachment roots of trumpet creeper (Campsis radicans). Force–displacement measurements of individual attachment pads as well as of complete structures consisting of several pads or roots were conducted for both natural and laboratory growth conditions. The shapes of the curves and the maximum forces determined indicate clear differences in the detachment process for the different plants and structures tested. Based on these findings, it is argued that the attachment structures are displacement-optimized rather than force-optimized.

Mechanistic aspects of the fracture toughness of elk antler bone

M.E. Launey, P.-Y. Chen, J. McKittrick, R.O. Ritchie — 2009-11-26

Abstract: Bone is an adaptive material that is designed for different functional requirements; indeed, bones have a variety of properties depending on their role in the body. To understand the mechanical response of bone requires the elucidation of its structure–function relationships. Here, we examine the fracture toughness of compact bone of elk antler, which is an extremely fast-growing primary bone designed for a totally different function than human (secondary) bone. We find that antler in the transverse (breaking) orientation is one of the toughest biological materials known. Its resistance to fracture is achieved during crack growth (extrinsically) by a combination of gross crack deflection/twisting and crack bridging via uncracked “ligaments” in the crack wake, both mechanisms activated by microcracking primarily at lamellar boundaries. We present an assessment of the toughening mechanisms acting in antler as compared to human cortical bone, and identify an enhanced role of inelastic deformation in antler which further contributes to its (intrinsic) toughness.

NO-loaded Zn2+-exchanged zeolite materials: A potential bifunctional anti-bacterial strategy

2009-10-26

Abstract: Nitric oxide (NO) is important for the regulation of a number of diverse biological processes, including vascular tone, neurotransmission, inflammatory cell responsiveness, defence against invading pathogens and wound healing. Transition metal exchanged zeolites are nanoporous materials with high-capacity storage properties for gases such as NO. The NO stores are liberated upon contact with aqueous environments, thereby making them ideal candidates for use in biological and clinical settings. Here, we demonstrate the NO release capacity and powerful bactericidal properties of a novel NO-storing Zn2+-exchanged zeolite material at a 50wt.% composition in a polytetrafluoroethylene polymer. Further to our published data showing the anti-thrombotic effects of a similar NO-loaded zeolite, this study demonstrates the anti-bacterial properties of NO-releasing zeolites against clinically relevant strains of bacteria, namely Gram-negative Pseudomonas aeruginosa and Gram-positive methicillin-sensitive and methicillin-resistant Staphylococcus aureus and Clostridium difficile. Thus our study highlights the potential of NO-loaded zeolites as biocompatible medical device coatings with anti-infective properties.

Loading and release of doxycycline hyclate from strontium-substituted calcium phosphate cement

2009-11-02

Abstract: Novel Sr-substituted calcium phosphate cement (CPC) loaded with doxycycline hyclate (DOXY-h) was employed to elucidate the effect of strontium substitution on antibiotic delivery. The cement was prepared using as reactants Sr-substituted β-tricalcium phosphate (Sr-β-TCP) and acidic monocalcium phosphate monohydrate. Two different methods were used to load DOXY-h: (i) the adsorption on CPC by incubating the set cement in drug-containing solutions; and (ii) the use of antibiotic solution as the cement liquid phase. The results revealed that the Sr-substituted cement efficiently adsorbs the antibiotic, which is attributed to an enhanced accessibility to the drug-binding sites within this CPC. DOXY-h desorption is influenced by the initial adsorbed amount and the cement matrix type. Furthermore, the fraction of drug released from CPCs set with DOXY-h solution was higher, and the release rate was faster for the CPC prepared with 26.7% Sr-β-TCP. The analysis of releasing profiles points to Fickian diffusion as the mechanism responsible for antibiotic delivery. We can conclude that Sr substitution in secondary calcium phosphate cements improves their efficiency for DOXY-h adsorption and release. The antibiotic loading method provides a way to switch from rapid and complete to slower and prolonged drug release.

Phase composition, mechanical performance and in vitro biocompatibility of hydraulic setting calcium magnesium phosphate cement

2009-10-19

Abstract: Brushite (CaHPO4·2H2O)-forming calcium phosphate cements are of great interest as bone replacement materials because they are resorbable in physiological conditions. However, their short setting times and low mechanical strengths limit broad clinical application. In this study, we showed that a significant improvement of these properties of brushite cement could be achieved by the use of magnesium-substituted β-tricalcium phosphate with the general formula MgxCa(3–x)(PO4)2 with 0

Effect of phase composition and microstructure of calcium phosphate ceramic particles on protein adsorption

X.D. Zhu, H.J. Zhang, H.S. Fan, Wei Li, X.D. Zhang — 2009-10-26

Abstract: The biological performance of biomaterials is strongly influenced by their protein adsorption characteristics, which are related to the structures and properties of both the biomaterial and the protein. In the present study two groups of hydroxyapatite (HA) and biphasic calcium phosphate (BCP) ceramic powders were fabricated by different drying processes. The roles of the phase composition and microstructure of the powders in the adsorption of various model proteins were evaluated. The experimental results showed that BCP always had a higher ability to adsorb fibrinogen, insulin or type I collagen (Col-I) than HA. The microporosity and micropore size of the CaP particles also had a strong impact on their protein adsorption characteristics. HA and BCP particles with higher microporosities and/or more micropores >20nm in diameter could adsorb more fibrinogen or insulin. However, amounts of adsorbed Col-I were largely unaffected by the microstructure of HA and BCP particles.

Comparative study on bone regeneration by synthetic octacalcium phosphate with various granule sizes

2009-10-19

Abstract: The present study was designed to investigate whether the granule size of synthetic octacalcium phosphate (OCP) and the resultant intergranular spaces between the granules formed by the filling affect its osteoconductive and biodegradable characteristics in a mouse calvaria critical-sized defect up to 10weeks after implantation. Mercury intrusion porosimetry showed that OCP granules having distinct diameter sizes ranging from 53 to 300 (S-OCP), 300 to 500 (I-OCP) and 500 to 1000μm (L-OCP) produced distinct intergranular spaces between OCP granules ranging from 28.8 to 176.6μm. The dissolution rate of OCP, estimated by the phosphate concentration in the culture medium, was the highest in S-OCP, followed by I-OCP and L-OCP, while the specific surface area of OCP decreased. Histological and histomorphometric analyses showed that bone formation around the implanted granules increased significantly with increasing granule size coupled with activating the appearance of TRAP- and cathepsin K-positive osteoclastic cells. The rate of new bone formation formed with L-OCP was two times higher than that formed with S-OCP at 10weeks after implantation. The results indicated that the osteoconductive and biodegradable properties of OCP can be augmented by increasing the granule size, most probably by thus providing enough spaces between the granules, suggesting that the intergranular spaces formed by the granules may work similarly to pores, as reported in porous ceramic materials. It seems likely that the enhancement of bone formation by OCP is accompanied by simultaneous activation of osteoclastic resorption of OCP.

Polarization of hydroxyapatite: Influence on osteoblast cell proliferation

2009-11-16

Abstract: Hydroxyapatite (HA) has been used clinically to treat bone defects. However, modifications of the surface properties of HA could improve and control bone matrix deposition and localized host tissue integration. The aim of this study was to investigate the effect of developing a surface charge on HA discs with respect to osteoblast activity in vitro. HA discs (12mm×2mm) were sintered in either air or water vapour. The HA discs were then electrically polarized (positive and negative surfaces) or non-polarized (controls) and seeded with MC3T3-E1 cells. Polarized HA sintered in water vapour was shown to retain six times more charge than polarized HA sintered in air. Picogreen analysis demonstrated that at 4h cell number was significantly higher on the negatively and positively charged HA surface (water sintered) in comparison to the non-charged water and air-sintered HA controls. At 7days there was a significant increase in cell number on the negatively charged HA (air sintered) sample in comparison to the negatively charged water vapour sintered HA sample and the non-charged water vapour sintered control sample. Also at 7days, the picogreen data showed a significant increase in cell number on the positively charged water-treated HA sample in comparison to both the air- and water-treated HA non-charged control HA samples. An alamarBlue assay at 7days demonstrated significant cell metabolic activity on the charged surfaces (both positive and negative) in comparison to the non-charged HA and the tissue culture plastic controls. This study demonstrated that all of the HA discs tested supported cell viability/attachment. However, cell attachment/proliferation/metabolic activity was significantly increased as a result of developing a charge on the HA surface.

Cobalt, chromium and nickel affect hydroxyapatite crystal growth in vitro

G. Mabilleau, R. Filmon, P.K. Petrov, M.F. Baslé, A. Sabokbar, D. Chappard — 2009-10-26

Abstract: Metals are widely used in orthopaedics and recent studies have reported that patients with metal implants have a significant increase of metal levels in serum and synovial fluid. Femoral neck fracture occurred in some patients with metal-on-metal implants for unknown reasons. Recently, bone quality has emerged as an important factor of bone strength and few studies have investigated the effects of metal ions on hydroxyapatite properties. In the present study, we investigated the effects of Co2+, Cr3+ and Ni2+ on hydroxyapatite (HA) growth in vitro, using carboxymethylated poly(2-hydroxyethyl methacrylate) (pHEMA) as a biomaterial for calcification. We have demonstrated that metal ions reduced the quantity of mineral formed at the surface of the polymer and decreased the ratio Ca/P by 1.12-, 1.05- and 1.08-fold for Cr2+, Cr3+ and Ni2+ respectively. Furthermore, the size of calcospherites was significantly increased in the metal-doped HA compared to the controls, indicating a possible effect of metal ions on the crystal lattice. Indeed, the presence of metal ions increased the crystal size as well as the crystallinity of HA and reduce the lattice parameter c of the HA framework. The information obtained from this work suggests that the quality of the mineral around metallic implants could be altered. However, further investigation should be conducted to further elucidate the effects of metal incorporation on bone mineral and the functional consequences.

Carbonated apatites obtained by the hydrolysis of monetite: Influence of carbonate content on adhesion and proliferation of MC3T3-E1 osteoblastic cells

2009-11-09

Abstract: The influence of the carbonate content in apatites on the adhesion and the proliferation of MC3T3-E1 osteoblastic cells was investigated. B-type carbonated apatites (DCAps) were prepared by the hydrolysis of monetite (CaHPO4, DCP) in solutions with a carbonate concentration ranging from 0.001 to 0.075moll−1. Stoichiometric hydroxyapatite (DCAp0) was synthesized in carbonate-free solution. MC3T3-E1 cells were seeded on the compacted DCAps and cell adhesion and proliferation were analysed after 24h and 7days, respectively, using a MTS assay and fluorescence microscopy. Cell adhesion tends to increase with increasing carbonate content for carbonate contents between 0 and 6.9wt.% and levels out to an acceptable value (±50% compared to the control) for carbonate contents between 6.9 and 16.1wt.%. Only DCAps with a carbonate content equal to or higher than 11% support high cell proliferation comparable to the control. On the latter DCAps, the cells have a spread morphology and form a near-confluent layer. A decrease in charge density and crystallinity at the apatite surface, as well as the formation of more spheroidal crystals with increasing carbonate content, might attribute to changes in composition and three-dimensional structure of the protein adsorption layer and hence to the observed cell behaviour. Consequently, only DCAps with a high carbonate content, mimicking early in vivo mineralization, are possible candidates for bone regeneration.

Osteoinduction of hydroxyapatite/β-tricalcium phosphate bioceramics in mice with a fractured fibula

2009-11-06

Abstract: Many studies have shown that calcium phosphate ceramics can induce bone formation in non-osseous sites without the application of any osteoinductive biomolecules, but the mechanisms of this phenomenon (intrinsic osteoinduction of bioceramics) remain unclear. In this study, we compared the intrinsic osteoinduction of porous hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) implanted in mice at different sites. In 30 mice the left fibula was fractured and the right fibula was kept intact. A porous HA/β-TCP cylinder was implanted into both the left (group 1) and right (group 2) leg muscles of each animal. In addition, two HA/β-TCP cylinders were bilaterally implanted into leg subcutaneous pockets (group 3) in each of the remaining 15 mice. New bone formation was studied in the three groups by histology, histomorphometry and immunostaining. In group 1 new bone was observed at week 6 and bone marrow appeared at week 12. In group 2 new bone was observed at week 8 and bone marrow appeared at week 12. The new bone area percentage in group 1 was significantly higher than in group 2 at both weeks 8 and 12. In contrast, group 3 did not show any new bone within the period studied. These differences were explained based on the location of the implants and thus their proximity to the osteogenic environment of fracture healing. The results support the hypothesis that intrinsic osteoinduction by calcium phosphate ceramics is the result of adsorption of osteoinductive substances on the surface.

Biological responses of human osteoblasts and osteoclasts to flame-sprayed coatings of hydroxyapatite and fluorapatite blends

K.A. Bhadang, C.A. Holding, H. Thissen, K.M. McLean, J.S. Forsythe, D.R. Haynes — 2009-10-26

Abstract: The aim of this study was to determine how the activities of human osteoblastic cells and osteoclasts respond to substrates of thermal-sprayed mechanical blends of hydroxyapatite and fluorapatite with a view of determining an optimal blend ratio for osseointegration. Human osteoblastic cells and osteoclasts were grown on titanium alloy discs coated with blends of hydroxyapatite and fluorapatite, with concentrations ranging from 0 to 100% fluorapatite. Human osteoblastic cells attached in greater numbers and proliferated at a greater rate on blends containing 40% fluorapatite. Human osteoblastic cells grown on blends containing 40% fluorapatite for 7days also expressed the highest levels of mRNA for several proteins involved with regulating bone metabolism (osteoprotegerin and receptor activator nuclear factor kappa B ligand), and bone formation (osteopontin, osteonectin and bone sialoprotein 1). Osteoclasts resorbed the dentine but poorly resorbed the hydroxyapatite–fluorapatite blends, particularly at high levels of fluorapatite. This in vitro study demonstrates that thermal-sprayed hydroxyapatitecoatings containing 40% fluorapatite may promote optimal bone growth and improve osseointegration of implants.

Nanohydroxyapatite coating on a titanium–niobium alloy by a hydrothermal process

Jianyu Xiong, Yuncang Li, Peter D. Hodgson, Cui’e Wen — 2009-10-15

Abstract: A novel one-step hydrothermal coating process was used to produce nanohydroxyapatite (nano-HA) coating on a titanium–niobium (TiNb) alloy substrate in a newly designed solution containing calcium and phosphate ions. The morphology of the coating was studied using scanning electron microscopy. The phase identification of the coating was carried out using X-ray diffraction, attenuated total reflectance Fourier transform infrared spectroscopy and transmission electron microscopy. The reaction between the surface of TiNb alloy and the solution during the hydrothermal process was studied by X-ray photoelectron spectroscopy. Results show that the coating formed on the surface of TiNb alloy was composed of nano-HA particles. During the hydrothermal process, TiO2 and Nb2O5 formed on the TiNb alloy surface and hydrated to Ti(OH)4 and Nb(OH)5, respectively. Calcium phosphate nucleated and grew into a layer of nano-HA particles on the surface of TiNb alloy under the hydrothermal conditions. The crystallinity of the nano-HA coating was improved with the increase in hydrothermal treatment temperature and time duration. Nano-HA coating with good crystallinity was produced on the TiNb alloy via the hydrothermal process at a temperature of 200°C for 12h.

Biomineralized strontium-substituted apatite/titanium dioxide coating on titanium surfaces

2009-10-26

Abstract: Bone mineral is a multi-substituted calcium phosphate. One of these ion substitutions, strontium, has been proven to increase bone strength and decrease bone resorption. Biomimetics is a potential way to prepare surfaces that provide a favorable bone tissue response, thus enhancing the fixation between bone and implants. Here we prepared double-layered strontium-substituted apatite and titanium dioxide coatings on titanium substrates via mimicking bone mineralization. Morphology, crystallinity, surface chemistry and composition of Sr-substituted coatings formed via biomimetic coating deposition on crystalline titanium oxide substrates were studied as functions of soaking temperature and time in phosphate buffer solutions with different Sr ion concentration. The morphology of the biomimetic apatite changed from plate-like for the pure HA to sphere-like for the Sr ion substituted. Surface analysis results showed that 10–33% of Ca ions in the apatite have been substituted by Sr ions, and that the Sr ions were chemically bonded with apatite and successfully incorporated into the structure of apatite.

Improving the osteointegration and bone–implant interface by incorporation of bioactive particles in sol–gel coatings of stainless steel implants

2009-10-15

Abstract: In this study, we report a hybrid organic–inorganic TEOS–MTES (tetraethylorthosilicate–methyltriethoxysilane) sol–gel-made coating as a potential solution to improve the in vivo performance of AISI 316L stainless steel, which is used as permanent bone implant material. These coatings act as barriers for ion migration, promoting the bioactivity of the implant surface. The addition of SiO2 colloidal particles to the TEOS–MTES sol (10 or 30mol.%) leads to thicker films and also acts as a film reinforcement. Also, the addition of bioactive glass–ceramic particles is considered responsible for enhancing osseointegration. In vitro assays for bioactivity in simulated body fluid showed the presence of crystalline hydroxyapatite (HA) crystals on the surface of the double coating with 10mol.% SiO2 samples on stainless steel after 30days of immersion. The HA crystal lattice parameters are slightly different from stoichiometric HA. In vivo implantation experiments were carried out in a rat model to observe the osteointegration of the coated implants. The coatings promote the development of newly formed bone in the periphery of the implant, in both the remodellation zone and the marrow zone. The quality of the newly formed bone was assessed for mechanical and structural integrity by nanoindentation and small-angle X-ray scattering experiments. The different amount of colloidal silica present in the inner layer of the coating slightly affects the material quality of the newly formed bone but the nanoindentation results reveal that the lower amount of silica in the coating leads to mechanical properties similar to cortical bone.

Local application of fluvastatin improves peri-implant bone quantity and mechanical properties: A rodent study

2009-11-02

Abstract: Statins are known to stimulate osteoblast activity and bone formation. This study examines whether local application of fluvastatin enhances osteogenesis around titanium implants in vivo. Ten-week-old rats received a vehicle gel (propylene glycol alginate (PGA)) or PGA containing fluvastatin (3, 15, 75 or 300μg) in their tibiae just before insertion of the implants. For both histological and histomorphometric evaluations undecalcified ground sections were obtained and the bone–implant contact (BIC), peri-implant osteoid volume and mineralized bone volume (MBV) were calculated after 1, 2 and 4weeks. Using the same models mechanical push-in tests were also performed to evaluate the implant fixation strength. After 1week the MBV and push-in strength were significantly lower in the 300μg fluvastatin-treated group than in the other groups (P<0.01). At 2weeks, however, the BIC and MBV were both significantly higher in the 75μg fluvastatin-treated group than in the non-fluvastatin-treated groups (P<0.01). Similar tendencies were observed at week 4. Furthermore, the data showed a good correlation between the MBV and the push-in strength. These results demonstrate positive effects of locally applied fluvastatin on the bone around titanium implants and suggest that this improvement in osseointegration may be attributed to calcification of the peri-implant bone.

Diamond-like carbon coatings enhance the hardness and resilience of bearing surfaces for use in joint arthroplasty

M.E. Roy, L.A. Whiteside, J. Xu, B.J. Katerberg — 2009-10-26

Abstract: The purpose of this study was to evaluate the potential of a hard diamond-like carbon (DLC) coating to enhance the hardness and resilience of a bearing surface in joint replacement. The greater hardness of a magnesium-stabilized zirconium (Mg-PSZ) substrate was expected to provide a harder coating–substrate composite microhardness than the cobalt–chromium alloy (CoCr) also used in arthroplasty. Three femoral heads of each type (CoCr, Mg-PSZ, DLC–CoCr and DLC–Mg-PSZ) were examined. Baseline (non-coated) and composite coating/substrate hardness was measured by Vickers microhardness tests, while nanoindentation tests measured the hardness and elastic modulus of the DLC coating independent of the Mg-PSZ and CoCr substrates. Non-coated Mg-PSZ heads were considerably harder than non-coated CoCr heads, while DLC coating greatly increased the microhardness of the CoCr and Mg-PSZ substrates. On the nanoscale the non-coated heads were much harder than on the microscale, with CoCr exhibiting twice as much plastic deformation as Mg-PSZ. The mechanical properties of the DLC coatings were not significantly different for both the CoCr and Mg-PSZ substrates, producing similar moduli of resilience and plastic resistance ratios. DLC coatings greatly increased hardness on both the micro and nano levels and significantly improved resilience and resistance to plastic deformation compared with non-coated heads. Because Mg-PSZ allows less plastic deformation than CoCr and provides a greater composite microhardness, DLC–Mg-PSZ will likely be more durable for use as a bearing surface in vivo.

High resolution transmission electron microscopy study of the hardening mechanism through phase separation in a β-Ti–35Nb–7Zr–5Ta alloy for implant applications

Conrado R.M. Afonso, Peterson L. Ferrandini, Antonio J. Ramirez, Rubens Caram — 2009-11-13

Abstract: β-Ti alloys are highly attractive metallic materials for biomedical applications due to their high specific strength, high corrosion resistance and excellent biocompatibility, including low elastic modulus. This work aims to clarify the hardening mechanism of a β-Ti–Nb–Zr–Ta alloy using different characterization techniques. Ingots (50g) of Ti–35Nb–7Zr–5Ta (wt.%) alloy were arc furnace melted in an Ar(g) atmosphere, homogenized, hot rolled, solubilized and finally aged at several temperatures from 200 to 700°C for 4h. Microstructure characterization was performed using X-ray diffraction, optical microscopy, scanning and high resolution transmission electron microscopy (HR-TEM). The 4h aging showed that the highest hardness values were found when aged at 400°C and the HR-TEM images confirmed splitting of spots on the Fourier space map, which indicated the presence of a coherent interface between separated phases (β and β′) and explains the hardening mechanism of the alloy. Through geometric phase analysis analysis, using the HR-TEM image, the localized strain map showed 5–10nm domains of the β and β′ phases. The combination of suitable values of yield strength, hardness and low Young’s modulus makes Ti–35Nb–7Zr–5Ta alloy suitable for medical applications as a metallic orthopedic implant.

Effect of process control agent on the porous structure and mechanical properties of a biomedical Ti–Sn–Nb alloy produced by powder metallurgy

A. Nouri, P.D. Hodgson, C.E. Wen — 2009-10-07

Abstract: The influence of different amounts and types of process control agent (PCA), i.e., stearic acid and ethylene bis-stearamide, on the porous structure and mechanical properties of a biomedical Ti–16Sn–4Nb (wt.%) alloy was investigated. Alloy synthesis was performed on elemental metal powders using high-energy ball milling for 5h. Results indicated that varying the PCA content during ball milling led to a drastic change in morphology and particle-size distribution of the ball-milled powders. Porous titanium alloy samples sintered from the powders ball milled with the addition of various amounts of PCA also revealed different pore morphology and porosity. The Vickers hardness of the sintered titanium alloy samples exhibited a considerable increase with increasing PCA content. Moreover, the addition of larger amounts of PCA in the powder mixture resulted in a significant increase in the elastic modulus and peak stress for the sintered porous titanium alloy samples under compression. It should also be mentioned that the addition of PCA introduced contamination (mainly carbon and oxygen) into the sintered porous product.

Influence of porosity on mechanical properties and in vivo response of Ti6Al4V implants

2009-11-13

Abstract: Metallic biomaterials are widely used to restore the lost structure and functions of human bone. Due to the large number of joint replacements, there is a growing demand for new and improved orthopedic implants. More specifically, there is a need for novel load-bearing metallic implants with low effective modulus matching that of bone in order to reduce stress shielding and consequently increase the in vivo lifespan of the implant. In this study, we have fabricated porous Ti6Al4V alloy structures, using laser engineered net shaping (LENS™), to demonstrate that advanced manufacturing techniques such as LENS™ can be used to fabricate low-modulus, tailored porosity implants with a wide variety of metals/alloys, where the porosity can be designed in areas based on the patient’s need to enhance biological fixation and achieve long-term in vivo stability. The effective modulus of Ti6Al4V alloy structures has been tailored between 7 and 60GPa and porous Ti alloy structures containing 23–32vol.% porosity showed modulus equivalent to human cortical bone. In vivo behavior of porous Ti6Al4V alloy samples in male Sprague–Dawley rats for 16weeks demonstrated a significant increase in calcium within the implants, indicating excellent biological tissue ingrowth through interconnected porosity. In vivo results also showed that total amount of porosity plays an important role in tissue ingrowth.

Effects of micrometric titanium particles on osteoblast attachment and cytoskeleton architecture

Laura Saldaña, Nuria Vilaboa — 2009-10-26

Abstract: Titanium (Ti) and its alloys are widely used in biomedical devices as bone tissue replacements due to their advantageous bulk mechanical properties and biocompatibility. It is known that particles released from Ti-based implants impair essential functions of osteoblasts, which for survival require attachment to specific extracellular matrix proteins at the bone surface. This study investigates whether Ti particles of micrometric sizes affect the osteoblast attachment machinery. Exposure of human osteoblastic Saos-2 cells to Ti particles impaired their adhesion strength, migration and proliferation. Attenuation of these functions was associated with reduced cell spreading, cell membrane disruptions and loss of cell shape. Cell exposure to Ti particles led to changes in cytoskeletal structures, including reduced ventral stress fibers combined with a disorderly arrangement of β-tubulin and acetylated α-tubulin fibers. Cytoskeleton disassembly was associated with a reduction in overall cell adhesion area, characterized by fewer centrally localized focal adhesions and shorter focal contacts at the periphery. Paxillin adaptor protein redistributed to peripheral corner regions, colocalizing with poorly organized actin fibers at attachment sites. Total focal adhesion kinase (FAK) protein amounts, as well as its degree of phosphorylation on the active form p-FAK (Tyr-397), decreased, which was accompanied by a lesser extent of co-localization with paxillin in focal contacts. On the other hand, p-FAK (Tyr-407), an inhibitory form of FAK, accumulated in the focal contacts of Ti-treated cells. Pyk2 phosphorylated on Tyr-402 colocalized with paxillin in focal contacts of untreated cells, while it was barely detected upon exposure to particles. In summary, changes in the phosphorylation states of both FAK and Pyk2 tyrosine kinases at focal contacts underlie impaired bone-forming cell attachment after exposure to Ti particles of micrometric sizes.

Effects of phosphoric acid treatment of titanium surfaces on surface properties, osteoblast response and removal of torque forces

2009-10-12

Abstract: This study investigated the surface characteristics and biocompatibility of phosphate ion (P)-incorporated titanium (Ti) surfaces hydrothermally treated with various concentrations of phosphoric acid (H3PO4). The surface characteristics were evaluated by scanning electron microscopy, thin-film X-ray diffractometry, X-ray photoelectron spectroscopy, optical profilometry, contact angle and surface energy measurement and inductively coupled plasma mass spectroscopy (ICP-MS). MC3T3-E1 cell attachment, spreading, proliferation and osteoblastic gene expression on different surfaces were evaluated. The degree of bony integration was biomechanically evaluated by removal torque testing after 4weeks of healing in rabbit tibiae. The H3PO4 treatment produced micro-rough Ti surfaces with crystalline P-incorporated Ti oxide layers. High concentration H3PO4 treatment (1% and 2%) produced significantly higher hydrophilic surfaces compared with low H3PO4 treatment (0.5%) and untreated surfaces (P<0.01). ICP-MS analysis showed P ions were released from P-incorporated surfaces. Significant increased cell attachment (P<0.05) and notably higher mRNA expressions of Runx2, alkaline phosphatase, osteopontin and osteocalcin were observed in cells grown on P-incorporated surfaces compared with cells on untreated machined surfaces. P-incorporated surfaces showed significantly higher removal torque forces compared with untreated machined implants (P<0.05). Ti surfaces treated with 2% H3PO4 showed increasing tendencies in osteoblastic gene expression and removal torque forces compared with those treated with lower H3PO4 concentrations or untreated surfaces. These results demonstrate that H3PO4 treatment may improve the biocompatibility of Ti implants by enhancing osteoblast attachment, differentiation and biomechanical anchorage.

Radially arrayed nanopillar formation on metallic stent wire surface via radio-frequency plasma

Mariana C. Loya, Eunsung Park, Li Han Chen, Karla S. Brammer, Sungho Jin — 2009-11-18

Abstract: MP35N (Co–Ni–Cr–Mo alloy) is an important stent implant material for which many aspects, that include nanostructured surfaces, are yet to be understood. The present study provides the first creation of radially emanating metallic nanopillar structures on the surface of MP35N stent alloy wires; a novel textured surface structuring derived via controlled RF processing technique. The goal of this study was to characterize the newly found structures, identify evolution stages of nanopillar formations, as well as optimize RF process parameters for controlled surface texturing technique for stent wire materials. The exposure of a stent alloy wire, 250μm diameter Co–Ni–Cr–Mo alloy (MP35N), to parameter-controlled RF environment resulted in dense surface nanostructures consisting of high-aspect-ratio dendritic nanopillars/nanowires. Extensive surface characterization and local compositional analyses by Transmission Electron Microscopy (TEM), Energy Dispersive X-ray analysis (EDX) and X-ray photoelectron spectroscopy (XPS) show increased values of Mo contents on the outer edges of protruding nanopillars, indicating a possibility of the higher Mo content phase contributing to the differential plasma sputter etching on the MP35N surface and resultant nanowire formation. A comparative investigation on single phase alloy versus multi-phase alloy seems to point to the importance of phase segregation for successful nanowire formation by RF plasma treatment. In addition to MP35N, some specific single phased materials, such as Fe–Ni and Fe–Cr alloys or Pt metal wire, were exposed in same RF plasma conditions and results did not form the complex structures found on MP35N samples. For the purpose of this study, metallic stent wires that have nanostructured surfaces can be considered a “polymer-less” approach to surface modification, The creation and characterization of radially arrayed nanostructured surfaces has been demonstrated on MP35N stent alloy wires using this RF plasma process; where such nanostructured surfaces contribute to design concepts that may enhance endotheliazation of stent materials via surface texturing modification.