Acta Biomaterialia

Editorial Board

2010-09-01

Recipients of the 2009 Acta Student Awards

2010-09-01

Amorphous calcium phosphates: Synthesis, properties and uses in biomaterials

C. Combes, C. Rey — 2010-02-17

Abstract: This review paper on amorphous calcium phosphates (ACPs) provides an update on several aspects of these compounds which have led to many studies and some controversy since the 1970s, particularly because of the lack of irrefutable proof of the occurrence of an ACP phase in mineralised tissues of vertebrates. The various synthesis routes of ACPs with different compositions are reported and the techniques used to characterise this phase are reviewed. We focus on the various physico-chemical properties of ACPs, especially the reactivity in aqueous media, which have been exploited to prepare bioactive bone substitutes, particularly in the form of coatings and cements for orthopaedic applications and composites for dental applications.

Octacalcium phosphate: Osteoconductivity and crystal chemistry

O. Suzuki — 2010-04-05

Abstract: Octacalcium phosphate (OCP), which is structurally similar to hydroxyapatite (HA), is a possible precursor of bone apatite crystals. Although disagreement remains as to whether OCP comprises the initial mineral crystals in the early stage of bone mineralization, the results of recent biomaterial studies using synthetic OCP indicate the potential role of OCP as a bone substitute material, owing to its highly osteoconductive and biodegradable characteristics. OCP tends to convert to HA not only in an in vitro environment, but also as an implant in bone defects. Several lines of evidence from both in vivo and in vitro studies suggest that the conversion process could be involved in the stimulatory capacity of OCP for osteoblastic differentiation and osteoclast formation. However, the osteoconductivity of OCP cannot always be secured if an OCP with distinct crystal characteristics is used, because the stoichiometry and microstructure of OCP crystals greatly affect bone-regenerative properties. Osteoconductivity and stimulatory capabilities may be caused by the chemical characteristics of OCP, which allows the release or exchange of calcium and phosphate ions with the surrounding of this salt, and its tendency to grow towards specific crystal faces, which could be a variable of the synthesis condition. This paper reviews the effect of calcium phosphates on osteoblastic activity and bone regeneration, with a special emphasis on OCP, since OCP seems to be performing better than other calcium phosphates in vivo.

Molecular resurfacing of cartilage with proteoglycan 4

K. Chawla, H.O. Ham, T. Nguyen, P.B. Messersmith — 2010-03-24

Abstract: Early loss of proteoglycan 4 (PRG4), a lubricating glycoprotein implicated in boundary lubrication, from the cartilage surface has been associated with degeneration of cartilage and early onset of osteoarthritis. Viscosupplementation with hyaluronic acid and other macromolecules has been proposed as a treatment of osteoarthritis. However, the efficacy of viscosupplementation is variable and may be influenced by the short residence time of lubricant in the knee joint after injection. Recent studies have demonstrated the use of aldehyde (CHO) modified extracellular matrix proteins for targeted adherence to a biological tissue surface. It is hypothesized that CHO could be exploited to enhance the binding of lubricating proteoglycans to the surface of PRG4-depleted cartilage. The objective of this study was to determine the feasibility of molecular resurfacing of cartilage with CHO-modified PRG4. PRG4 was chemically functionalized with aldehyde (PRG4-CHO) and aldehyde plus Oregon Green (OG) fluorophore (PRG4-OG-CHO) to allow for differentiation of endogenous and exogenous PRG4. Cartilage disks depleted of native PRG4 were then treated with solutions of PRG4, PRG4-CHO, or PRG4-OG-CHO and then assayed for the presence of PRG4 by immunohistochemistry, ELISA, and fluorescence imaging. Repletion of cartilage surfaces was significantly enhanced with the inclusion of CHO compared with repletion with unmodified PRG4. These findings suggest a generalized approach which may be used for molecular resurfacing of tissue surfaces with PRG4 and other lubricating biomolecules, perhaps leading in the future to a convenient method for overcoming loss of lubrication during the early stages of osteoarthritis.

Vascular differentiation of bone marrow stem cells is directed by a tunable three-dimensional matrix

Ge Zhang, Charles T. Drinnan, Laura R. Geuss, Laura J. Suggs — 2010-03-18

Abstract: Microenvironmental cues are critical in regulating cell behavior and fate. The roles that matrix mechanical signals play in regulating cell behavior have recently been elucidated. An artificial matrix that can maintain the appropriate characteristics for transplanted stem cells is therefore needed to achieve a desired cell phenotype. The objective of this study was to develop a three-dimensional (3-D) matrix with tunable physical and mechanical properties and investigate their effects on mesenchymal stem cell (MSC) differentiation towards vascular cell types. In this study we developed an extracellular microenvironment by modifying fibrinogen with various polyethylene glycol (PEG) derivatives. We hypothesized that adjusting the type of PEG derivative to modify the resultant physical and mechanical characteristics of fibrin would allow us to create a tunable system for use in culture or in vivo in conjunction with a regenerative medicine strategy. Human MSC (hMSC) were entrapped into PEGylated fibrin matrices at a density of 50,000cellsml−1. Cell phenotypes were confirmed by immunofluorescent staining as well as the use of oligonucleotide arrays. Vascular phenotypes were correlated with measured mechanical properties and fiber diameters of the PEGylated fibrin matrices. Blocking studies were performed to identify mechanistic factors controlling MSC differentiation through selected blocking of matrix degradation or cell contraction. Cell–matrix interactions were also examined in vivo. Our results demonstrate that transdifferentiation of MSC towards an endothelial cell phenotype is profoundly affected by the 3-D matrix microenvironment. Our work provides a predictive road map for the creation of fibrin-based matrices that support robust endothelial cell gene expression and tubulogenesis.

Influence of cell-adhesive peptide ligands on poly(ethylene glycol) hydrogel physical, mechanical and transport properties

Silviya P. Zustiak, Rohan Durbal, Jennie B. Leach — 2010-04-12

Abstract: Synthetic three-dimensional scaffolds for cell and tissue engineering routinely utilize peptide ligands to provide sites for cell adhesion and to promote cellular activity. Given the fact that recent studies have dedicated great attention to the mechanisms by which cell behavior is influenced by various ligands and scaffold material properties, it is surprising that little work to date has been carried out to investigate the influence of covalently bound ligands on hydrogel material properties. Herein we report the influence of three common ligands utilized in tissue engineering, namely RGD, YIGSR and IKVAV, on the mechanical properties of cross-linked poly(ethylene glycol) (PEG) hydrogels. The effect of the ligands on hydrogel storage modulus, swelling ratio, mesh size and also on the diffusivity of bovine serum albumin through the hydrogel were investigated in detail. We identified conditions under which these ligands strikingly influence the properties of the material. The extent of influence and whether the ligand increases or decreases a specific property is linked to ligand type and concentration. Further, we pinpoint mechanisms by which the ligands interact with the PEG network. This work thus provides specific evidence for interactions between peptide ligands and cross-linked PEG hydrogels that have a significant impact on hydrogel material and transport properties. As a result, this work may have important implications for interpreting cell experiments carried out with ligand-modified hydrogels, because the addition of ligand may affect not only the scaffold’s biological properties, but also key physical properties of the system.

Regulation of keratinocyte signaling and function via changes in epidermal growth factor presentation

Tracy J. Puccinelli, Paul J. Bertics, Kristyn S. Masters — 2010-04-15

Abstract: Motivated by the need for bioactive materials that can accelerate dermal wound healing, this work describes the responses of keratinocytes to covalently immobilized epidermal growth factor (EGF) and how differences in the physical presentation of this growth factor affect cell function. Specifically, human keratinocytes were cultured with EGF delivered in soluble form, immobilized in a homogeneous pattern or immobilized in a gradient pattern, followed by analysis of cellular signaling, proliferation and migration. By changing the manner in which EGF was presented, keratinocyte behavior was dramatically altered. Keratinocytes responded to immobilized EGF patterns with high EGF receptor (EGFR) but low ERK1/2 and Akt phosphorylation, accompanied by low proliferation, high migratory activity and coordinated cell alignment. In contrast, keratinocytes treated with soluble EGF experienced lower EGFR but higher ERK1/2 and Akt phosphorylation and displayed a highly proliferative, rather than migratory, phenotype. Keratinocytes also responded to differences in immobilized EGF patterns, as migration was fastest upon immobilized gradients of EGF. A better understanding the interaction of cells with soluble vs. immobilized growth factors can help elucidate native healing events and achieve greater control over cell function, which may be useful in the development of wound repair treatments for many types of tissues.

Sustained plasmid DNA release from dissolving mineral coatings

Siyoung Choi, William L. Murphy — 2010-03-22

Abstract: Calcium phosphate (CaP) minerals such as hydroxyapatite are able to bind a diverse range of biological molecules due to the presence of anions and cations in their crystal structure. The well-characterized ability of CaP minerals to bind and release plasmid DNA, coupled with the ability of biodegradable CaP coatings to form on the surface of common biomaterials, provides a potential mechanism for controlled release of plasmid DNA from various biomaterials. In this study we hypothesized that the release of plasmid DNA from CaP coatings formed on poly(lactide–co-glycolide) (PLG) substrates would be dependent on both the intrinsic properties of the CaP mineral coating and the surrounding solution conditions. Experiments were designed to consider two general parameters: (i) the stability of various CaP mineral coatings in solution environments that are relevant to physiological conditions; (ii) the relationship between mineral stability and sustained plasmid DNA release. Our results corroborate previous studies that have demonstrated a direct relationship between intrinsic mineral composition and mineral stability. In addition, we further demonstrate that ion composition and pH of the surrounding solution environment can significantly influence mineral stability. In turn, mineral stability significantly influenced release of plasmid DNA from mineral coatings in vitro, and the DNA release efficiency could be tuned by controlling the mineral properties in various solution environments. These CaP mineral coatings may be a useful platform for plasmid DNA delivery applications using various biomaterial platforms.

Differential uptake of DNA–poly(ethylenimine) polyplexes in cells cultured on collagen and fibronectin surfaces

Anandika Dhaliwal, Maricela Maldonado, Zenas Han, Tatiana Segura — 2010-04-05

Abstract: Genetically modified bone marrow-derived mesenchymal stem cells (MSCs) have proven to be efficient cell carriers for local or systemic delivery of therapeutics as well as growth factors to augment tissue formation. However, efficient non-viral gene transfer to these cells is limiting their applicability. Although most studies have focused on designing more efficient condensation agents for DNA, our focus in this manuscript is to study the role of two extracellular matrix (ECM) proteins, collagen I (Col I) and fibronectin (Fn), on the ability of MSCs to become transfected. Here we report that plating MSCs on Col I-coated surfaces inhibits transfection, while plating MSCs on Fn-coated surfaces enhances transfection. The mechanism by which these ECM proteins affect non-viral gene transfer involves the endocytosis pathway used for polyplex uptake and intracellular tension. We found that Fn promoted internalization through clathrin-mediated endocytosis and that this pathway resulted in more efficient transfection than caveolae-mediated endocytosis and macropinocytosis. Further, the disruption of actin–myosin interactions resulted in an enhancement of gene transfer for cells plated on Fn-coated surfaces, but not for cells plated on Col I. We believe that the cellular microenvironment can be engineered to enhance the ability of cells to become transfected and that through understanding the mechanisms by which the ECM affects non-viral gene transfer better materials and transfection protocols can be realized.

Shaping the micromechanical behavior of multi-phase composites for bone tissue engineering

2010-03-25

Abstract: Mechanical stiffness is a fundamental parameter in the rational design of composites for bone tissue engineering in that it affects both the mechanical stability and the osteo-regeneration process at the fracture site. A mathematical model is presented for predicting the effective Young’s modulus (E) and shear modulus (G) of a multi-phase biocomposite as a function of the geometry, material properties and volume concentration of each individual phase. It is demonstrated that the shape of the reinforcing particles may dramatically affect the mechanical stiffness: E and G can be maximized by employing particles with large geometrical anisotropy, such as thin platelet-like or long fibrillar-like particles. For a porous poly(propylene fumarate) (60% porosity) scaffold reinforced with silicon particles (10% volume concentration) the Young’s (shear) modulus could be increased by more than 10 times by just using thin platelet-like as opposed to classical spherical particles, achieving an effective modulus E ∼ 8GPa (G ∼ 3.5GPa). The mathematical model proposed provides results in good agreement with several experimental test cases and could help in identifying the proper formulation of bone scaffolds, reducing the development time and guiding the experimental testing.

Chitosan–poly(lactide-co-glycolide) microsphere-based scaffolds for bone tissue engineering: In vitro degradation and in vivo bone regeneration studies

2010-03-22

Abstract: Natural polymer chitosan and synthetic polymer poly(lactide-co-glycolide) (PLAGA) have been investigated for a variety of tissue engineering applications. We have previously reported the fabrication and in vitro evaluation of a novel chitosan/PLAGA sintered microsphere scaffold for load-bearing bone tissue engineering applications. In this study, the in vitro degradation characteristics of the chitosan/PLAGA scaffold and the in vivo bone formation capacity of the chitosan/PLAGA-based scaffolds in a rabbit ulnar critical-sized-defect model were investigated. The chitosan/PLAGA scaffold showed slower degradation than the PLAGA scaffold in vitro. Although chitosan/PLAGA scaffold showed a gradual decrease in compressive properties during the 12-week degradation period, the compressive strength and compressive modulus remained in the range of human trabecular bone. Chitosan/PLAGA-based scaffolds were able to guide bone formation in a rabbit ulnar critical-sized-defect model. Microcomputed tomography analysis demonstrated that successful bridging of the critical-sized defect on the sides both adjacent to and away from the radius occurred using chitosan/PLAGA-based scaffolds. Immobilization of heparin and recombinant human bone morphogenetic protein-2 on the chitosan/PLAGA scaffold surface promoted early bone formation as evidenced by complete bridging of the defect along the radius and significantly enhanced mechanical properties when compared to the chitosan/PLAGA scaffold. Furthermore, histological analysis suggested that chitosan/PLAGA-based scaffolds supported normal bone formation via intramembranous formation.

Biodegradable and injectable cure-on-demand polyurethane scaffolds for regeneration of articular cartilage

2010-03-08

Abstract: This paper describes the synthesis and characterization of an injectable methacrylate functionalized urethane-based photopolymerizable prepolymer to form biodegradable hydrogels. The tetramethacrylate prepolymer was based on the reaction between two synthesized compounds, diisocyanato poly(ethylene glycol) and monohydroxy dimethacrylate poly(ε-caprolactone) triol. The final prepolymer was hydrated with phosphate-buffered saline (pH 7.4) to yield a biocompatible hydrogel containing up to 86% water. The methacrylate functionalized prepolymer was polymerized using blue light (450nm) with an initiator, camphorquinone and a photosensitizer, N,N-dimethylaminoethyl methacrylate. The polymer was stable in vitro in culture media over the 28days tested (1.9% mass loss); in the presence of lipase, around 56% mass loss occurred over the 28days in vitro. Very little degradation occurred in vivo in rats over the same time period. The polymer was well tolerated with very little capsule formation and a moderate host tissue response. Human chondrocytes, seeded onto Cultispher-S beads, were viable in the tetramethacrylate prepolymer and remained viable during and after polymerization. Chondrocyte–bead–polymer constructs were maintained in static and spinner culture for 8weeks. During this time, cells remained viable, proliferated and migrated from the beads through the polymer towards the edge of the polymer. New extracellular matrix (ECM) was visualized with Masson’s trichrome (collagen) and Alcian blue (glycosaminoglycan) staining. Further, the composition of the ECM was typical for articular cartilage with prominent collagen type II and type VI and moderate keratin sulphate, particularly for tissue constructs cultured under dynamic conditions.

Polyurethane anionomers containing metal ions with antimicrobial properties: Thermal, mechanical and biological characterization

I. Francolini, L. D’Ilario, E. Guaglianone, G. Donelli, A. Martinelli, A. Piozzi — 2010-04-05

Abstract: In recent years the employment of implantable medical devices has increased remarkably, notwithstanding that microbial infections are a frequent complication associated with their use. Different strategies have been attempted to overcome this problem, including the incorporation of antimicrobial agents into the device itself. In this study a new approach to obtain intrinsically antimicrobial materials was developed. Polymer anionomers containing Ag(I), Cu(II), Zn(II), Al(III) and Fe(III) were prepared by neutralization of a carboxylated polyurethane. In the case of the PEUA-Ag, PEUA-Fe and PEUA-Cu ionomers the ion aggregates behaved as reinforcing filler particles, increasing the mechanical properties of the systems in terms of hardness and strength at break over the pristine carboxylated polymer. With the exception of the Al-containing polymer, all the other experimented ionomers showed satisfactory antimicrobial properties. The best antibacterial effect was obtained with the silver ion-containing polymer, which inhibited Staphylococcus epidermidis growth for up to 16days. Ciprofloxacin was also adsorbed onto the above mentioned ionomers. A synergistic effect of the antibiotic and silver ions on bacterial growth inhibition was observed for at least 25days.

Intravital microscopy imaging of macrophage localization to immunogenic particles and co-localized tissue oxygen saturation

Se-woon Choe, Abhinav P. Acharya, Benjamin G. Keselowsky, Brian S. Sorg — 2010-03-12

Abstract: Well-designed biomaterial polymer particle-based vaccines will optimally promote immune cell antigen-presenting behavior while minimizing adverse inflammatory responses to the particles and encapsulated drugs or adjuvants. It is important in the design of particle-based vaccines to consider possible harmful effects of immune response on tissue at the vaccination site. Intravital microscopy with rodent dorsal skin window chambers enables in vivo serial observations in the same animal, and such models which have been used to study angiogenesis and macrophage response to implanted biomaterials may also be useful for the development of particle-based vaccines. To our knowledge there have been no reports where intravital microscopy has documented real-time immune cell localization and potentially harmful co-localized tissue effects. In this proof-of-principle study we used fluorescence and spectral imaging intravital microscopy of mouse window chambers to measure macrophage localization and co-localized tissue microvessel hemoglobin saturation changes in response to an immunogenic stimulus from polymer particles loaded with lipopolysaccharide (LPS) serving as a model vaccine/adjuvant system. We observed greater and faster macrophage localization to stronger inflammatory stimuli from LPS-loaded particle doses, a trend of decreased microvessel oxygenation with increased macrophage accumulation and, in an extreme case, complete microvessel collapse accompanied by tissue necrosis. Our technique may be useful for optimizing design of particle-based vaccines and may give insight into the use of hemoglobin saturation as a biomarker of tissue inflammation for clinical investigations of particle-based vaccines.

Analysing protein competition on self-assembled mono-layers studied with quartz crystal microbalance

Johan Benesch, João F. Mano, Rui L. Reis — 2010-03-22

Abstract: The mechanisms by which proteins adsorb to surfaces of biomaterials have long been of interest. The present work started with the premise that small/hard and large/soft proteins will yield different sets of normalized frequency shift and dissipation signals when studied with a quartz crystal microbalance. The aim was to evaluate the usefulness of these raw data to study protein competition using protein incubations in sequence and from mixtures of albumin (BSA) and gamma-globulin (BGG) at various ratios. Increasing the concentration of BSA decreases the adsorption of subsequently incubated BGG. For BSA/BGG mixtures the dissipation is similar for all logarithmic molar ratios BGG/BSA below 1 but soon decreases when the molar ratio of BSA/BGG (and opposite for the normalized frequency shift) is above 1, indicating preferential binding of BGG. Modelling indicated that differences in the film shear modulus and viscosity depend more on the properties of the self-assembling mono-layers (SAMs) than on the proteins. Films high in BSA tentatively differ in film shear modulus and viscosity from that of films high in BGG but only on the hydrophobic surfaces. The results were encouraging as the raw data were deemed to be able to point at protein adsorption competition.

Films of dextran-graft-polybutylmethacrylate to enhance endothelialization of materials

2010-04-05

Abstract: We have synthesized new structures obtained from amphiphilic copolymers of dextran and polybutylmethacrylate with the aim of endothelialization of biomaterials. Grafting of butylmethacrylate onto dextran has been carried out using ceric ammonium nitrate as initiator. Three copolymers were obtained (11, 30 and 37 wt.% dextran) and homogeneous thin films were successfully prepared. In contrast to dextran, the resulting films were stable in water, and copolymers characterized by Fourier transform infrared spectroscopy, differential scanning calorimetry and dynamic mechanical analysis showed evidence of hybrid properties between the parent homopolymers. Surfaces of films were smooth when analyzed by atomic force microscopy (roughness 2±1nm) but greatly differed in their hydrophilicity by increasing the dextran content (water contact angle from 99° to 57°). In contrast to polybutylmethacrylate, where the proliferation of vascular smooth muscle cells (VSMCs) was excellent but that of endothelial cells was very low, the copolymer containing 11% of dextran was excellent for endothelial cells but very limited for VSMCs. An in vitro wound assay demonstrated that copolymer with 11% dextran is even more favorable for endothelial cell migration than tissue-culture polystyrene. Increasing the dextran content in the copolymers decreased the proliferation for both vascular cell types. Altogether, these results show that transparent and water-insoluble films made from copolymers of dextran and butylmethacrylate copolymers with an appropriate composition could enhance endothelial cell proliferation and migration. Therefore, a potential benefit of this approach is the availability of surfaces with tunable properties for the endothelialization of materials.

Geometric microenvironment directs cell morphology on topographically patterned hydrogel substrates

2010-04-05

Abstract: Cell behavior is influenced by numerous factors in the physical environment, and a deep understanding of these interactions can lead to the design of better scaffolds for tissue engineering. In vitro substrates can be used to evaluate a wide range of factors, such as topography, and identify which show promise for further evaluating in vivo. Polyacrylamide hydrogels featuring a combinatorial, micropatterned array of posts with varied shape, width, and spacing were produced using a one-step technique. Substrates were covalently modified with collagen and seeded with D1 ORL UVA mesenchymal stem cells. Patterning was shown to direct several quantitative measures of cell morphology. Cell bodies tended to be located in gaps 15μm and wider, but on top of posts when gaps were 5μm and smaller. Cells on substrates with square posts and narrow gaps tended to elongate in the direction of gaps. Finally, smaller gaps on all substrates were also shown to influence the placement of cell extensions. The parameters identified may be incorporated into substrates to direct specific aspects of cell morphology.

Boron nitride nanotube reinforced polylactide–polycaprolactone copolymer composite: Mechanical properties and cytocompatibility with osteoblasts and macrophages in vitro

2010-03-11

Abstract: Biodegradable polylactide–polycaprolactone copolymer (PLC) has been reinforced with 0, 2 and 5wt.% boron nitride nanotubes (BNNTs) for orthopedic scaffold application. Elastic modulus of the PLC–5wt.% BNNT composite, evaluated through nanoindentation technique, shows a 1370% increase. The same amount of BNNT addition to PLC enhances the tensile strength by 109%, without any adverse effect on the ductility up to 240% elongation. Interactions of the osteoblasts and macrophages with bare BNNTs prove them to be non-cytotoxic. PLC–BNNT composites displayed increased osteoblast cell viability as compared to the PLC matrix. The addition of BNNTs also resulted in an increase in the expression levels of the Runx2 gene, the main regulator of osteoblast differentiation. These results indicate that BNNT is a potential reinforcement for composites for orthopedic applications.

Extracellular microbial synthesis of biocompatible CdTe quantum dots

2010-03-29

Abstract: An efficient bacterial synthesis method to harvest cadmium telluride (CdTe) quantum dots (QDs) with tunable fluorescence emission using Escherichia coli is demonstrated. Ultraviolet–visible, photoluminescence, X-ray diffraction and transmission electron microscopy analysis confirmed the superior size-tunable optical properties, with fluorescence emission from 488 to 551nm, and the good crystallinity of the as synthesized QDs. A surface protein capping layer was confirmed by hydrodynamic size, ζ potential and Fourier transform infrared spectroscopy measurements, which could maintain the viability (92.9%) of cells in an environment with a QD concentration as high as 2μM. After functionalization with folic acid the QDs were used to image cultured cervical cancer cells in vitro. Investigations of bacterial growth and morphology and the biosynthesis of CdTe QDs in Luria–Bertani medium containing E. coli-secreted proteins showed that extracellular synthesis directly relied on the E. coli-secreted proteins, and a mechanism for protein-assisted biosynthesis of QDs is proposed. This work provides an economical approach to fabricate highly fluorescent biocompatible CdTe QDs via an environmentally friendly production process. The biosynthesized QDs may have great potential in broad bio-imaging and bio-labeling applications.

Novel ultrasound contrast agent based on microbubbles generated from surfactant mixtures of Span 60 and polyoxyethylene 40 stearate

Zhanwen Xing, Hengte Ke, Jinrui Wang, Bo Zhao, Xiuli Yue, Zhifei Dai, Jibin Liu — 2010-03-11

Abstract: In this study, novel perfluorocarbon-filled microbubbles as ultrasound contrast agent were fabricated using ultrasonication of a surfactant mixture of sorbitan monostearate (Span 60) and polyoxyethylene 40 stearate (PEG40S) in aqueous media. The microbubbles generated from a 1:9 mixture of PEG40S/Span 60 exhibited an average diameter of 2.08±1.27μm. More than 99% of the microbubbles had a mean particle diameter less than 8μm, indicating that they were appropriately sized for intravenous administration as ultrasound contrast agent. The stabilization mechanism of the microbubbles was investigated by the Langmuir–Blodgett technique including the measurements of surface pressure–area (π–A) isotherms and compression–decompression cycles with a two-dimensional monolayer of Span 60 and PEG40S. The dependence on molar fraction of PEG40S in π–A isotherms of mixed monolayers provided a strong evidence of interactions between the two microbubble-forming materials. It is suggested that the monolayer shell imparts good stability to the microbubbles by three means: (1) a low surface tension monolayer hinders dissolution through the reduction of surface tension, which introduces a mechanical surface pressure that counters the Laplace pressure; (2) the presence of a monolayer shell imparts a significant barrier to gas escaping from the core into the aqueous medium; and (3) encapsulation elasticity stabilizes microbubbles against diffusion-driven dissolution and explains the long shelf-life of microbubble contrast agent. The preliminary in vivo ultrasound imaging study showed that such stabilized microbubbles demonstrated excellent enhancement under grey-scale pulse inversion harmonic imaging and power Doppler imaging.

Characterization and in vitro cytocompatibility of piezoelectric electrospun scaffolds

N. Weber, Y.-S. Lee, S. Shanmugasundaram, M. Jaffe, T.L. Arinzeh — 2010-04-05

Abstract: Previous studies have shown that electrical charges influence cell behavior (e.g. enhancement of nerve regeneration, cell adhesion, cell morphology). Thus, piezoelectric scaffolds might be useful for various tissue engineering applications. Fibrous scaffolds were successfully fabricated from permanent piezoelectric poly(vinylidene fluoride–trifluoroethylene) (PVDF-TrFE) by the electrospinning technique. Scanning electron microscopy and capillary flow analyses verified that the fiber mats had an average fiber diameter of 970±480nm and a mean pore diameter of 1.7μm, respectively. Thermally stimulated depolarization current spectroscopy measurements confirmed the piezoelectric property of the PVDF-TrFE fibrous scaffolds by the generation of a spontaneous current with the increase in temperature in the absence of an electric field, which was not detected in the unprocessed PVDF-TrFE powder. Differential scanning calorimetry, thermogravimetric analysis, X-ray diffraction and Fourier transform infrared spectroscopy results showed that the electrospinning process increased the crystallinity and presence of the polar, beta-phase crystal compared with the unprocessed powder. Confocal fluorescence microscopy and a cell proliferation assay demonstrated spreading and increased cell numbers (human skin fibroblasts) over time on PVDF-TrFE scaffolds, which was comparable with tissue culture polystyrene. The relative quantity of gene expression for focal adhesion proteins (measured by real-time RT-PCR) increased in the following order: paxillin

Nanostructured poly(ε-caprolactone)–silica xerogel fibrous membrane for guided bone regeneration

2010-03-22

Abstract: A novel fibrous membrane was developed for guided bone regeneration (GBR) through electrospinning a uniform poly(ε-caprolactone) (PCL)–silica hybrid sol. The membrane was composed of fibers with a mean diameter of approximately 400nm. The hybrid fibers were nano-sized with uniform patterns throughout the fibers, in contrast to the homogeneous structure of pure PCL fibers. The tensile strengths and elastic moduli of the membranes were significantly enhanced with increasing silica content up to 40%. The surfaces of the hybrid membranes were highly hydrophilic with a water contact angle of almost zero. The hybrid membranes possessed excellent in vitro cellular responses in terms of proliferation and differentiation of pre-osteoblast cells. The in vivo animal tests not only confirmed excellent biocompatibility but also revealed bioresorbability of the membranes. These mechanical and biomedical properties make the hybrid membranes very attractive as GBR applications.

Evaluation of mesoporous silicon/polycaprolactone composites as ophthalmic implants

2010-03-29

Abstract: The suitability of porous silicon (pSi) encapsulated in microfibers of the biodegradable polymer polycaprolactone (PCL) for ophthalmic applications was evaluated, using both a cell attachment assay with epithelial cells and an in vivo assessment of biocompatibility in rats. Microfibers of PCL containing encapsulated pSi particles at two different concentrations (6 and 20wt.%) were fabricated as non-woven fabrics. Given the dependence of Si particle dissolution kinetics on pSi surface chemistry, two different types of pSi particles (hydride-terminated and surface-oxidized) were evaluated for each of the two particle concentrations. Significant attachment of a human lens epithelial cell line (SRA 01/04) to all four types of scaffolds within a 24h period was observed. Implantation of Si fabric samples beneath the conjunctiva of rat eyes for 8weeks demonstrated that the composite materials did not cause visible infection or inflammation, and did not erode the ocular surface. We suggest that these novel composite materials hold considerable promise as scaffolds in tissue engineering with controlled release applications.

Nano-controlled molecular interaction at adhesive interfaces for hard tissue reconstruction

2010-03-25

Abstract: Although decayed/fractured teeth can be reconstructed minimally invasively and nearly invisibly using adhesive technology, the clinical longevity of dental composite restorations is still too short. Water sorption is thought to be the principal cause of destabilization of the biomaterial–tooth bond. However, the actual mechanisms of interfacial degradation are far from understood. Here we report how nano-controlled molecular interaction at the biomaterial–hard tissue interface can improve bond durability. The use of functional monomers with a strong chemical affinity for the calcium in hydroxyapatite is essential for long-term durability. Correlative X-ray diffraction and solid-state nuclear magnetic resonance disclosed a time-dependent molecular interaction at the interface with stable ionic bond formation of the monomer to hydroxyapatite competing in time with the deposition of less stable calcium phosphate salts. The advanced tooth–biomaterial interaction model gives not only an insight into the mechanisms of bond degradation, but also provides a basis to develop functional monomers for more durable tooth reconstruction.

Raman tensor analysis of ultra-high molecular weight polyethylene and its application to study retrieved hip joint components

Yasuhito Takahashi, Leonardo Puppulin, Wenliang Zhu, Giuseppe Pezzotti — 2010-03-08

Abstract: The angular dependences of the polarized Raman intensity of Ag, B1g, B2g, and B3g modes have been preliminary investigated on a model fiber sample of ultra-high molecular weight polyethylene (UHMWPE) in order to retrieve the Raman tensor elements, i.e. the intrinsic parameters governing the vibrational behavior of the orthorhombic structure of polyethylene. Based on this Raman analysis, a method is proposed for determining unknown crystallographic orientation patterns in UHMWPE biomedical components concurrently with the orientation distribution functions for orthorhombic lamellae. An application of the method is shown, in which we quantitatively examined the molecular orientation patterns developed on the surface of four in vivo exposed UHMWPE acetabular cups vs. an unused cup. Interesting findings were: (i) a clear bimodal distribution of orientation angles was observed on worn surfaces; and (ii) a definite and systematic increase in both molecular orientation and crystallinity in main wear zones vs. non-wear zones was found in all retrieved acetabular cups. The present crystallographic analysis is an extension of our previous Raman studies of UHMWPE acetabular cups related to assessments of oxidation and residual strain and suggests a viable path to track back wear-history information from the surface of UHMWPE, thus unfolding the in vivo kinematics of the bearing surfaces in hip joints on the microscopic scale.

Distribution of polyethylene wear particles and bone fragments in periprosthetic tissue around total hip joint replacements

2010-04-23

Abstract: Ultra-high molecular weight polyethylene (UHMWPE) wear particles play a significant role in failures of total joint replacements (TJRs). In this work, we investigated the distribution of these wear particles in periprosthetic tissues obtained from nine revisions of hip TJR. In the first step, all periprosthetic tissues were combined and mechanically separated into granuloma tissue (containing hard granules visible to the naked eye) and surrounding tissue (without visible granules). In the second step, the tissues were hydrolyzed by protease from Streptomyces griseus and granules were separated by filtration; this divided the sample into four groups: (i) lyzate and (ii) non-degraded large granules from the granuloma tissue plus (iii) lyzate and (iv) non-degraded small granules from the surrounding tissue. In the third step, the large as well as small granules were hydrolyzed by collagenase from Clostridium histolyticum. In the last step, the UHMWPE wear particles from all four groups were purified by HNO3 digestion and weighed. The purity of the isolated particles was verified by scanning electron microscopy, infrared spectroscopy and energy-dispersive X-ray analysis. Of the total amount of polyethylene particles in the whole granuloma tissue, 72% of particles in the size range 0.1–10μm and 68% of those larger than 10μm were found in granules. Therefore, the formation of granules significantly lowers the effective amount of wear particles available for interaction with reactive cells and seems to be a natural defense mechanism.

Biotribology of alternative bearing materials for unicompartmental knee arthroplasty

2010-04-05

Abstract: The objective of our wear simulator study was to evaluate the suitability of two different carbon fibre-reinforced poly-ether-ether-ketone (CFR-PEEK) materials for fixed bearing unicompartmental knee articulations with low congruency. In vitro wear simulation was performed according to ISO 14243-1:2002 (E) with the clinically introduced Univation® F fixed bearing unicompartmental knee design (Aesculap AG, Tuttlingen, Germany) made of UHMWPE/CoCr29Mo6 in a direct comparison to experimental gliding surfaces made of CFR-PEEK pitch and CFR-PEEK PAN. Gliding surfaces of each bearing material (n=6+2) were γ-irradiated, artificially aged and tested for 5 million cycles with a customized four-station knee wear simulator (EndoLab, Thansau, Germany). Volumetric wear assessment, optical surface characterization and an estimation of particle size and morphology were performed.The volumetric wear rate of the reference PE1–6 was 8.6±2.17mm3 per million cycles, compared to 5.1±2.29mm3 per million cycles for PITCH1–6 and 5.2±6.92mm3 per million cycles for PAN1–6; these differences were not statistically significant. From our observations, we conclude that CFR-PEEK PAN is obviously unsuitable as a bearing material for fixed bearing knee articulations with low congruency, and CFR-PEEK pitch also cannot be recommended as it remains doubtful wether it reduces wear compared to polyethylene. In the fixed bearing unicompartmental knee arthroplasty examined, application threshold conditions for the biotribological behaviour of CFR-PEEK bearing materials have been established. Further in vitro wear simulations are necessary to establish knee design criteria in order to take advantage of the biotribological properties of CFR-PEEK pitch for its beneficial use to patients.

Tailoring the morphology of high molecular weight PLLA scaffolds through bioglass addition

N. Barroca, A.L. Daniel-da-Silva, P.M. Vilarinho, M.H.V. Fernandes — 2010-03-29

Abstract: Thermally induced phase separation (TIPS) has proven to be a suitable method for the preparation of porous structures for tissue engineering applications, and particular attention has been paid to increasing the pore size without the use of possible toxic surfactants. Within this context, an alternative method to control the porosity of polymeric scaffolds via the combination with a bioglass is proposed in this work. The addition of a bioactive glass from the 3CaO·P2O5–MgO–SiO2 system enables the porous structure of high molecular weight poly(l-lactic) acid (PLLA) scaffolds prepared by TIPS to be tailored. Bioglass acts as a nucleating catalyst agent of the PLLA matrix, promoting its crystallization, and the glass solubility controls the pore size. A significant increase in the pore size is observed as the bioglass content increases and scaffolds with large pore size (∼150μm) can be prepared. In addition, the bioactive character of the scaffolds is proved by in vitro tests in synthetic plasma. The importance of this approach resides on the combination of the ability to tailor the porosity of polymeric scaffolds via the tunable solubility of bioglasses, without the use of toxic surfactants, leading to a composite structure with suitable properties for bone tissue engineering applications.

Spatial control of cells, peptide delivery and dynamic monitoring of cellular physiology with chitosan-assisted dual color quantum dot FRET peptides

Ru-Huei Fu, Shih-Ping Liu, Chen-Wei Ou, Chin-Mao Huang, Yu-Chi Wang — 2010-03-08

Abstract: Cell-based assays have become important tools in the pharmaceutical and biotechnology industries. However, observing and monitoring molecules in cells that mimic the physiological environment is often difficult. Dynamic processes not only increase the accuracy of simulations, but also improve our understanding of the function and regulation of molecules within cells. In this study we used chitosan as a multifunctional biomaterial for selective micropatterning of cells, peptide delivery and covalent bonding with quantum dots (QD) to decrease the cytotoxicity of QD. Our results demonstrate the efficacy of chitosan–QD–peptide–Alexa Fluor 488 in controlling the spread and spatial organization of cells. Cationic chitosan also provided an efficient delivery mechanism to live cells. We used the shift from green to red fluorescence of the chitosan dual color QD peptide to detect biological activity. This methodology has potential applications in high throughput screening of inhibitors and activators of biological mechanisms and pathways and for use in the pharmaceutical industry.

The effect of topology of chitosan biomaterials on the differentiation and proliferation of neural stem cells

Gan Wang, Qiang Ao, Kai Gong, Aijun Wang, Lu Zheng, Yandao Gong, Xiufang Zhang — 2010-04-05

Abstract: Neural stem cells (NSCs) are capable of self-renewal and differentiation into three principle central nervous system cell types under specific local microenvironments. Chitosan films (Chi-F), chitosan porous scaffolds (Chi-PS) and chitosan multimicrotubule conduits (Chi-MC) were used to investigate their effects on the differentiation and proliferation of NSCs isolated from the cortices of fetal rats. In the presence of 10% fetal bovine serum most NSCs cultured on Chi-F differentiated into astrocytes, NSCs cultured on Chi-MC showed a significant increase in neuronal differentiation, while Chi-PS somewhat promoted NSCs to differentiate into neurons. However, in serum-free medium with 20ngml−1 basic fibroblast growth factor NSCs cultured on Chi-F showed the greatest proliferation, NSCs cultured on Chi-MC showed moderate cell proliferation, but NSCs cultured on Chi-PS exhibited the least cell proliferation. These observations indicate that chitosan topology can play an important role in regulating differentiation and proliferation of NSCs and raise the possibility of the utilization of chitosan in various structural biomaterials in neural tissue engineering.

Fabrication of porous polysaccharide-based scaffolds using a combined freeze-drying/cross-linking process

2010-03-08

Abstract: Biocompatible three-dimensional (3-D) porous scaffolds are of great interest for tissue engineering applications. We here present a novel combined freeze-drying/cross-linking process to prepare porous polysaccharide-based scaffolds. This process does not require an organic solvent or porogen agent. We unexpectedly found that cross-linking of biomacromolecules such as pullulan and dextran with sodium trimetaphosphate could be performed during freeze-drying. We have demonstrated that the freeze-drying pressure modulates the degree of porosity. High freeze-drying pressure scaffolds presented pores with a mean diameter of 55±4μm and a porosity of 33±12%, whereas low freeze-drying pressure scaffolds contained larger pores with a mean diameter of 243±14μm and a porosity of 68±3%. Porous scaffolds of the desired shape could be easily obtained and were stable in culture medium for weeks. In vitro viable mesenchymal stem cells were found associated with porous scaffolds in higher proportions than with non-porous scaffolds. Moreover, cells penetrated deeper into scaffolds with larger pores. This novel combined freeze-drying/cross-linking processing of polysaccharides enabled the fabrication of biocompatible scaffolds with controlled porosity and architectures suitable for 3-D in vitro culture and biomedical applications.

Encapsulation of fibroblasts causes accelerated alginate hydrogel degradation

N.C. Hunt, A.M. Smith, U. Gbureck, R.M. Shelton, L.M. Grover — 2010-03-22

Abstract: Calcium-alginate hydrogel has been widely studied as a material for cell encapsulation for tissue engineering. At present, the effect that cells have on the degradation of alginate hydrogel is largely unknown. We have shown that fibroblasts encapsulated at a density of 7.5×105cellsml−1 in both 2% and 5% w/v alginate remain viable for at least 60days. Rheological analysis was used to study how the mechanical properties exhibited by alginate hydrogel changed during 28days in vitro culture. Alginate degradation was shown to occur throughout the study but was greatest within the first 7days of culture for all samples, which correlated with a sharp release of calcium ions from the construct. Fibroblasts were shown to increase the rate of degradation during the first 7days when compared with acellular samples in both 2% and 5% w/v gels, but after 28days both acellular and cell-encapsulating samples retained disc-shaped morphologies and gel-like spectra. The results demonstrate that although at an early stage cells influence the mechanical properties of encapsulating alginate, over a longer period of culture, the hydrogels retain sufficient mechanical integrity to exhibit gel-like properties. This allows sustained immobilization of the cells at the desired location in vivo where they can produce extracellular matrix and growth factors to expedite the healing process.

Novel thermosensitive calcium alginate microspheres: Physico-chemical characterization and delivery properties

2010-03-12

Abstract: The system described in this paper was obtained by soaking calcium alginate (CaAlg) microspheres in a water solution of poly-[(3-acrylamidopropyl)-trimethylammonium chloride-b-N-isopropylacrylamide] [poly(AMPTMA-b-NIPAAM)], a new block co-polymer recently synthesized by atom transfer radical polymerization (ATRP). The block co-polymer is characterized by a lower critical solution temperature (LCST) of 41°C in aqueous 0.1M NaCl solution, and can be anchored on the CaAlg microspheres by means of polyion interactions. Polycations (permanently positively charged blocks) and polyanions (free alginate carboxylic groups) interact, leading to microspheres with thermosensitive properties. As an effect of interaction with the microspheres the LCST of the co-polymer is lowered to 36–38°C. In this temperature range a colloidal water suspension of the microspheres collapses, forming macroscopic aggregates. The new system shows, at human body temperature, an improved ability to carry and deliver both hydrophobic and hydrophilic molecules in comparison with unmodified CaAlg microspheres. The release properties of the microspheres loaded with different model drugs can be appropriately modulated by the amount of the poly(AMPTMA-b-NIPAAM). Furthermore, the microspheres show the interesting capability of retaining the activity of a loaded enzyme (horseradish peroxidase), used as a model protein. The results obtained indicate that the proposed drug delivery system may be suitable for drug depot applications.

Alginate-controlled formation of nanoscale calcium carbonate and hydroxyapatite mineral phase within hydrogel networks

2010-03-31

Abstract: A one-step method was used to make nanostructured composites from alginate and calcium carbonate or calcium phosphate. Nanometer-scale mineral phase was successfully formed within the gel network of alginate gel beads, and the composites were characterized. It was found that calcite was the dominating polymorph in the calcium carbonate mineralized beads, while stoichiometric hydroxyapatite was formed in the calcium phosphate mineralized beads. A combination of electron microscopy, Fourier-transform infrared spectroscopy, thermogravimetric analysis and powder X-ray diffraction showed that alginate played an active role in controlling mineral size, morphology and polymorphy. For the calcium phosphate mineralized beads, alginate was shown to modulate stoichiometric hydroxyapatite with low crystallinity at room temperature, which may have important applications in tissue engineering. The results presented in this work demonstrate important aspects of alginate-controlled crystallization, which contributes to the understanding of composite material design.

Development of bone-like composites via the polymer-induced liquid-precursor (PILP) process. Part 1: Influence of polymer molecular weight

Sang-Soo Jee, Taili T. Thula, Laurie B. Gower — 2010-03-31

Abstract: Bone is an organic–inorganic composite consisting primarily of collagen fibrils and hydroxyapatite crystals intricately interlocked to provide skeletal and metabolic functions. Non-collagenous proteins (NCPs) are also present, and although only a minor component, the NCPs are thought to play an important role in modulating the mineralization process. During secondary bone formation, an interpenetrating structure is created by intrafibrillar mineralization of the collagen matrix. Many researchers have tried to develop bone-like collagen–hydroxyapatite (HA) composites via the conventional crystallization process of nucleation and growth. While those methods have been successful in inducing heterogeneous nucleation of HA on the surface of collagen scaffolds, they have failed to produce a composite with the interpenetrating nanostructured architecture of bone. Our group has shown that intrafibrillar mineralization of type I collagen can be achieved using a polymer-induced liquid-precursor (PILP) process. In this process, acidic polypeptides are included in the mineralization solution to mimic the function of the acidic NCPs, and in vitro studies have found that acidic peptides such as polyaspartate induce a liquid-phase amorphous mineral precursor. Using this PILP process, we have been able to prepare collagen–HA composites with the fundamental nanostructure of bone, wherein HA nanocrystals are embedded within the collagen fibrils. This study shows that through further optimization a very high degree of mineralization can be achieved, with compositions matching that of bone. Synthetic collagen sponges were mineralized with calcium phosphate while analyzing various parameters of the reaction, with the focus of this report on the molecular weight of the polymeric process-directing agent. In order to determine whether intrafibrillar mineralization was achieved, an in-depth characterization of the mineralized composites was performed, including wide-angle X-ray diffraction, electron microscopy and thermogravimetric analyses. The results of this work lead us closer to the development of bone-like collagen–HA composites that could become the next generation of synthetic bone grafts.

Structure–property relationships of a biopolymer network: The eggshell membrane

Fernando G. Torres, Omar P. Troncoso, Franco Piaggio, Alfredo Hijar — 2010-03-15

Abstract: The eggshell membrane (ESM) is a biopolymer network that may have potential applications in biomedicine, but it also may reveal important details regarding the behaviour of biopolymer networks. In this paper, we have studied the mechanical and morphological properties of the ESM in order to reveal important structure–property relationships. Light optical microscopy and atomic force microscopy were used to assess the morphology of the ESM. The mechanical properties of membranes and individual fibres were studied by means of tensile tests and nanoindentation tests, respectively. The mechanical behaviour of ESM networks in different environmental conditions showed a non-linear and a linear regime. As for elastomers and other biopolymer systems, the non-linear regime was modelled by the Mooney–Rivlin relation. The Young’s modulus in the linear regime of the network was related to the Young’s modulus of the individual fibres using Gibson and Ashby analysis for cellular solids. The results of morphological characterization were used to relate the properties of individual fibres to the properties of the whole networks. This enabled us to predict the macroscopical properties of the network based on the properties of the individual fibres. It was found that the ESM networks behaved as both Mooney–Rivlin and Hookean materials in different environmental conditions. This study helps elucidate the properties of the biopolymer networks found in nature and describes important mechanical properties for the use of the ESM as a biomaterial.

Effects of a surface topography composite with puerariae radix on human STRO-1-positive stem cells

2010-03-18

Abstract: Human skeletal stem cells (STRO-1 positive/STRO-1+) respond to different topographical features in various ways. On a flat surface these cells spread and tend to develop a fibroblast-like morphology. On a microgrooved surface enriched skeletal stem cell populations prefer to stretch along the grooves, which affects their cellular structure and differentiation, a phenomenon known as contact guidance. Growth factors, hormones and chemicals can also stimulate cell differentiation. A traditional Chinese medicine, puerariae radix, has previously been observed to stimulate bone formation. The active ingredients have been identified as isoflavones with estrogen-like bioactivity. This study combined the effects of microgrooved topology and hormone-like isoflavones in the biodegradable polymer polycaprolactone (PCL). Human osteogenic cells (STRO-1+) were cultured on flat PCL, grooved PCL and puerariae powder-impregnated grooved PCL for 5weeks. Coomassie staining indicated that cell growth and survival was similar on flat PCL, grooved PCL and grooved PCL impregnated with 1 wt.% or 2 wt.% puerariae powder. Grooved PCL impregnated with 2 wt.% puerariae powder was observed to have an influence on protein expression, as observed by positive osteocalcin staining. Protein expression profiles were analyzed by difference gel electrophoresis to identify proteins that showed modulation of expression in response to these different environments. Overall, our results suggest that puerariae powder has an additive effect, along with microgrooved topographical stimulation, to promote changes in the STRO-1+ proteome that affect cell phenotype.

Plasma-induced polymerization as a tool for surface functionalization of polymer scaffolds for bone tissue engineering: An in vitro study

2010-03-11

Abstract: A commonly applied strategy in the field of tissue engineering (TE) is the use of temporary three-dimensional scaffolds for supporting and guiding tissue formation in various in vitro strategies and in vivo regeneration approaches. The interactions of these scaffolds with highly sensitive bioentities such as living cells and tissues primarily occur through the material surface. Hence, surface chemistry and topological features have principal roles in coordinating biological events at the molecular, cellular and tissue levels on timescales ranging from seconds to weeks. However, tailoring the surface properties of scaffolds with a complex shape and architecture remains a challenge in materials science. Commonly applied wet chemical treatments often involve the use of toxic solvents whose oddments in the construct could be fatal in the subsequent application. Aiming to shorten the culture time in vitro (i.e. prior the implantation of the construct), in this work we propose a modification of previously described bone TE scaffolds made from a blend of starch with polycaprolactone (SPCL). The modification method involves surface grafting of sulfonic or phosphonic groups via plasma-induced polymerization of vinyl sulfonic and vinyl phosphonic acid, respectively. We demonstrate herein that the presence of these anionic functional groups can modulate cell adhesion mediated through the adsorbed proteins (from the culture medium). Under the conditions studied, both vitronectin adsorption and osteoblast proliferation and viability increased in the order SPCL≪sulfonic-grafted SPCL

Synthesis and characterization of mesoporous Gd2O3 nanotube and its use as a drug-carrying vehicle

Yen-Po Chang, Kun-Ho Liu, Chih-Shin Chao, San-Yuan Chen, Dean-Mo Liu — 2010-03-11

Abstract: Gd2O3 nanotubes were constructed for the first time by assembling highly crystalline Gd2O3 nanoparticles through the use of combined soft template and sol–gel methods. Amphiphilic block copolymer was used as structure-directing agent and gadolinium isopropoxide as inorganic precursor in non-aqueous solution. The amphiphilic copolymer molecules are known to undergo self-organization above a critical micelle concentration, forming micellular architecture that further provides a structurally ordered active site for the nucleation and growth of Gd monomers. The resulting self-assembly of the Gd2O3 nanocrystals led to the formation of Gd2O3 tubular nanostructure after pyrolytic removal of the template. Transmission electron microscopy analysis indicated a mesoporous channel array along the [110] direction of the nanotubes where the wall of nanotube is well organized by the assembly of a highly crystalline framework of Gd2O3 nanocrystals. This Gd2O3 nanotube exhibited weak superparamagnetic property and was found to be able to carry and elute a model molecule, i.e. ibuprofen (IBU), in a controllable manner via an external magnetic field. The mechanism of IBU release from the nanotubes with and without the use of magnetic stimulus was proposed.

Bioengineering of the silica-polymerizing enzyme silicatein-α for a targeted application to hydroxyapatite

2010-03-11

Abstract: Since its discovery, numerous biotechnological approaches have aimed to explore the silica-polymerizing catalytic activity of the enzyme silicatein. In vivo, silicatein catalyzes polymerization of amorphous silica nanospheres from soluble precursors. In vitro, it directs the formation of nanostructured biosilica. This is of interest for various applications that strive to benefit from both the advantages of the biological system (i.e., silica synthesis under physiological conditions) and the cell mineralization-stimulating effect of biosilica. However, so far immobilization of silicatein has been hampered by the complex multistep procedure required. In addition, the chemical surface modifications involved not only restrict the choice of carrier materials but also render application of silicatein to hydroxyapatite (HA) of mineralized tissue impossible. Here we describe the bioengineering of silicatein, adapted for application in the fields of bone regeneration, tissue engineering, and dental care. Inspired by Glu-rich sequences of mammalian proteins that confer binding affinity to HA, a novel protein-tag was developed, the Glu-tag. Following expression of Glu-tagged silicatein, the HA-binding capacity of the enzyme is demonstrated in combination with synthetic and dental HA. Furthermore, immobilized Glu-tagged silicatein catalyzes synthesis of biosilica coatings on both synthetic HA nanofibrils and dental HA. Hence, Glu-tagged silicatein reveals a considerable biomedical potential with regenerative and prophylactic implementations.

Biomimetic remineralization as a progressive dehydration mechanism of collagen matrices – Implications in the aging of resin–dentin bonds

2010-03-22

Abstract: Biomineralization is a dehydration process in which water from the intrafibrillar compartments of collagen fibrils are progressively replaced by apatites. As water is an important element that induces a lack of durability of resin–dentin bonds, this study has examined the use of a biomimetic remineralization strategy as a progressive dehydration mechanism to preserve joint integrity and maintain adhesive strength after ageing. Human dentin surfaces were bonded with dentin adhesives, restored with resin composites and sectioned into sticks containing the adhesive joint. Experimental specimens were aged in a biomimetic analog-containing remineralizing medium and control specimens in simulated body fluid for up to 12 months. Specimens retrieved after the designated periods were examined by transmission electron microscopy for the presence of water-rich regions using a silver tracer and for collagen degradation within the adhesive joints. Tensile testing was performed to determine the potential loss of bond integrity after ageing. Control specimens exhibited severe collagen degradation within the adhesive joint after ageing. Remineralized specimens exhibited progressive dehydration, as manifested by silver tracer reduction and partial remineralization of water-filled microchannels within the adhesive joint, as well as intrafibrillar remineralization of collagen fibrils that were demineralized initially as part of the bonding procedure. Biomimetic remineralization as a progressive dehydration mechanism of water-rich, resin-sparse collagen matrices enables these adhesive joints to resist degradation over a 12-month ageing period, as verified by the conservation of their tensile bond strength. The ability of the proof of concept biomimetic remineralization strategy to prevent bond degradation warrants further development of clinically relevant delivery systems.

Synthesis of nanobioglass and formation of apatite rods to occlude exposed dentine tubules and eliminate hypersensitivity

A.R. Curtis, N.X. West, B. Su — 2010-03-04

Abstract: The occlusion of patent dentine tubules may reduce or eliminate hypersensitivity by restricting dentinal fluid movement. The efficacy of a novel sol–gel nanobioglass and a melt-derived bioglass to occlude tubules and promote apatite formation was tested by mechanically brushing a slurry of bioglass powder and human saliva onto dentine possessing exposed tubules. Scanning electron microscopy, focused ion beam and energy-dispersive X-ray spectroscopy were used to characterize the powders and assess tubule occlusion. Melt-derived bioglass possessed an irregular particle morphology and had a mean size of 3.30±0.42μm. The sol–gel bioglass particles were spherical, with a mean size of 0.65±0.19μm. Dentine treated with melt-derived bioglass exhibited a tightly adherent continuous apatite layer. Treatment with nanobioglass resulted in particle deposition within tubules and formation of apatite rods which were tightly adherent to tubule walls and continuous to a measured depth of 270μm.

Teeth restored using fiber-reinforced posts: In vitro fracture tests and finite element analysis

M. Schmitter, P. Rammelsberg, J. Lenz, S. Scheuber, K. Schweizerhof, S. Rues — 2010-03-15

Abstract: In dentistry the restoration of decayed teeth is challenging and makes great demands on both the dentist and the materials. Hence, fiber-reinforced posts have been introduced. The effects of different variables on the ultimate load on teeth restored using fiber-reinforced posts is controversial, maybe because the results are mostly based on non-standardized in vitro tests and, therefore, give inhomogeneous results. This study combines the advantages of in vitro tests and finite element analysis (FEA) to clarify the effects of ferrule height, post length and cementation technique used for restoration. Sixty-four single rooted premolars were decoronated (ferrule height 1 or 2mm), endodontically treated and restored using fiber posts (length 2 or 7mm), composite fillings and metal crowns (resin bonded or cemented). After thermocycling and chewing simulation the samples were loaded until fracture, recording first damage events. Using UNIANOVA to analyze recorded fracture loads, ferrule height and cementation technique were found to be significant, i.e. increased ferrule height and resin bonding of the crown resulted in higher fracture loads. Post length had no significant effect. All conventionally cemented crowns with a 1-mm ferrule height failed during artificial ageing, in contrast to resin-bonded crowns (75% survival rate). FEA confirmed these results and provided information about stress and force distribution within the restoration. Based on the findings of in vitro tests and computations we concluded that crowns, especially those with a small ferrule height, should be resin bonded. Finally, centrally positioned fiber-reinforced posts did not contribute to load transfer as long as the bond between the tooth and composite core was intact.

Augmentation of bone defect healing using a new biocomposite scaffold: An in vivo study in sheep

2010-03-25

Abstract: Previous studies support resorbable biocomposites made of poly(l-lactic acid) (PLA) and β-tricalcium phosphate (TCP) produced by supercritical gas foaming as a suitable scaffold for tissue engineering. The present study was undertaken to demonstrate the biocompatibility and osteoconductive properties of such a scaffold in a large animal cancellous bone model. The biocomposite (PLA/TCP) was compared with a currently used β-TCP bone substitute (ChronOS™, Dr. Robert Mathys Foundation), representing a positive control, and empty defects, representing a negative control. Ten defects were created in sheep cancellous bone, three in the distal femur and two in the proximal tibia of each hind limb, with diameters of 5mm and depths of 15mm. New bone in-growth (osteoconductivity) and biocompatibility were evaluated using microcomputed tomography and histology at 2, 4 and 12months after surgery. The in vivo study was validated by the positive control (good bone formation with ChronOS™) and the negative control (no healing with the empty defect). A major finding of this study was incorporation of the biocomposite in bone after 12months. Bone in-growth was observed in the biocomposite scaffold, including its central part. Despite initial fibrous tissue formation observed at 2 and 4months, but not at 12months, this initial fibrous tissue does not preclude long-term application of the biocomposite, as demonstrated by its osteointegration after 12months, as well as the absence of chronic or long-term inflammation at this time point.

Mechanical properties, electronic structure and bonding of α- and β-tricalcium phosphates with surface characterization

L. Liang, P. Rulis, W.Y. Ching — 2010-03-31

Abstract: The mechanical properties and electronic structure of α- and β-tricalcium phosphate (TCP) crystals are studied by using two ab initio density functional methods, the Vienna Ab initio Simulation Package (VASP) and the orthogonalized linear combination of atomic orbitals method. Based on the VASP optimized crystal structures, the elastic constants of α- and β-TCP are obtained using an effective stress–strain computational scheme. From the calculated elastic constants, the bulk modulus, shear modulus, Young’s modulus and Poisson’s ratios are obtained. The results show that the mechanical properties of the two crystals are comparable and that α-TCP is somewhat softer than β-TCP. Comparison with experimental extrapolations of the elastic constants shows significant differences, which attest to the difficulty of obtaining single crystal samples. The calculated electronic structure results show that both crystals are large gap insulators with a direct band gap of 4.89eV for α-TCP and 5.25eV for β-TCP. Effective charge calculations show that, on average, β-TCP has slightly less charge transfer per Ca than α-TCP. The (010) ((001)) surface model for α-TCP (β-TCP) is studied using a supercell slab geometry and fully relaxed to obtain the optimized structures. The estimated surface formation energies are 0.777 and 0.842Jm−2 for α-TCP and β-TCP, respectively. The electronic structures of the two surface models are compared with the bulk models. Charge density analysis shows that the surfaces of both TCP crystals are positively charged overall owing to the presence of Ca ions near the surfaces.

Synthesis of high surface area mesostructured calcium phosphate particles

SuXiu Ng, Jun Guo, Jan Ma, Say Chye Joachim Loo — 2010-03-15

Abstract: High surface area mesostructured calcium phosphate (MCP) particles were synthesized using surfactants (F127 and P123) and the textural properties were optimized in this study. The synthesized MCP samples subsequently underwent surfactant washing for surfactant removal, which achieved surface areas of >200m2g–1. It was found that both F127 and P123 gave similar surface areas (SBET), but differing pore diameters (Dpore). Other synthesis parameters, such as synthesis temperature, surfactant concentration and pH, were also studied to further optimize the textural properties of MCP. It was found that the highest surface area MCP was synthesized at 25°C and pH 12, with 80wt.% surfactant concentration. Further, in vitro loading and delivery tests on optimized MCP showed an enhanced loading efficiency of bovine serum albumin and lysozyme as compared with non-mesostructured calcium phosphate.

Microwave-processed nanocrystalline hydroxyapatite: Simultaneous enhancement of mechanical and biological properties

Susmita Bose, Sudip Dasgupta, Solaiman Tarafder, Amit Bandyopadhyay — 2010-03-15

Abstract: Despite the excellent bioactivity of hydroxyapatite (HA) ceramics, poor mechanical strength has limited the applications of these materials primarily to coatings and other non-load-bearing areas as bone grafts. Using synthesized HA nanopowder, dense compacts with grain sizes in the nanometer to micrometer range were processed via microwave sintering between 1000 and 1150°C for 20min. Here we demonstrate that the mechanical properties, such as compressive strength, hardness and indentation fracture toughness, of HA compacts increased with a decrease in grain size. HA with 168±86nm grain size showed the highest compressive strength of 395±42MPa, hardness of 8.4±0.4GPa and indentation fracture toughness of 1.9±0.2MPam1/2. To study the in vitro biological properties, HA compacts with grain size between 168nm and 1.16μm were assessed for in vitro bone cell–material interactions with human osteoblast cell line. Vinculin protein expression for cell attachment and bone cell proliferation using MTT assay showed that surfaces with finer grains provided better bone cell–material interactions than coarse-grained samples. Our results indicate simultaneous improvements in mechanical and biological properties in microwave sintered HA compacts with nanoscale grain size.

Biofilm formation on bone grafts and bone graft substitutes: Comparison of different materials by a standard in vitro test and microcalorimetry

Martin Clauss, Andrej Trampuz, Olivier Borens, Marc Bohner, Thomas Ilchmann — 2010-03-12

Abstract: We analyzed the initial adhesion and biofilm formation of Staphylococcus aureus (ATCC 29213) and S. epidermidis RP62A (ATCC 35984) on various bone grafts and bone graft substitutes under standardized in vitro conditions. In parallel, microcalorimetry was evaluated as a real-time microbiological assay in the investigation of biofilm formation and material science research. The materials β-tricalcium phosphate (β-TCP), processed human spongiosa (Tutoplast™) and poly(methyl methacrylate) (PMMA) were investigated and compared with polyethylene (PE). Bacterial counts (log10 cfu per sample) were highest on β-TCP (S. aureus 7.67±0.17; S. epidermidis 8.14±0.05) while bacterial density (log10 cfu per surface) was highest on PMMA (S. aureus 6.12±0.2, S. epidermidis 7.65±0.13). Detection time for S. aureus biofilms was shorter for the porous materials (β-TCP and processed human spongiosa, p<0.001) compared to the smooth materials (PMMA and PE), with no differences between β-TCP and processed human spongiosa (p>0.05) or PMMA and PE (p>0.05). In contrast, for S. epidermidis biofilms the detection time was different (p<0.001) between all materials except between processed human spongiosa and PE (p>0.05). The quantitative analysis by quantitative culture after washing and sonication of the material demonstrated the importance of monitoring factors like specific surface or porosity of the test materials. Isothermal microcalorimetry proved to be a suitable tool for an accurate, non-invasive and real-time microbiological assay, allowing the detection of bacterial biomass without removing the biofilm from the surface.

A novel modular device for 3-D bone cell culture and non-destructive cell analysis

2010-03-15

Abstract: Synthetic materials have emerged as bone substitutes for filling bone defects of critical sizes. Because bone healing requires a mechanically resistant matrix (scaffold) attractive to osteogenic cells and must allow revascularization for nutrient and oxygen supply, scaffold-based strategies focus on the further development of chemical and physical qualities of the material. Cellular ingrowth towards the scaffold center is critical; therefore selective information from inner regions, in particular from the central part, is essential.In this paper we introduce a novel modular in vitro system for three-dimensional (3-D) in vitro bone cell cultures. This 3-D system is developed exclusively for in vitro research purposes, with special emphasis on the geometrical scaffold design (pore size, pore design). The system is composed of a stack of titanium slices which are mounted on a clamp and which enable the separate monitoring of cell growth patterns on every single slice of the slide stack. In this way we are able to gain selective information about the regulation of the cell physiology in the inner part of the 3-D construct which can be used for the development of an optimized scaffold design for orthopedic implants.

Evaluation of a new press-fit in situ setting composite porous scaffold for cancellous bone repair: Towards a “surgeon-friendly” bone filler?

M. Peroglio, L. Gremillard, D. Eglin, P. Lezuo, M. Alini, J. Chevalier — 2010-03-15

Abstract: In this study, a composite porous material obtained by coating a poly(ester urethane) foam with a calcium phosphate cement is proposed as novel cancellous bone filler with easy handling, in situ hardening and press-fitting properties. The coating can be applied to the foam in the surgical theater, allowing refinement of scaffold shape to the needs of the ongoing surgery. An innovative experiment was developed in order to determine the setting curve of the composite scaffold as well as the time of manipulation available to the surgeon without risk of material damage. This composite material is soft and can be press-fit in a cavity without damaging the scaffold in the first 5min after coating application. The composite scaffold hardens quickly (22min) and, once the cement has set, its compressive strength and fracture energy are increased by over an order of magnitude as compared to the initial poly(ester urethane) foam. This set of interesting properties makes calcium phosphate cement-coated elastomeric scaffolds a new promising strategy for cancellous bone filling.