Acta Biomaterialia - Articles in Press

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

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.

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

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

Abstract: 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, namely the intrinsic parameters governing the vibrational behavior of the orthorhombic structure of polyethylene. According to 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.

Anticoagulant activity of enzymatically synthesized amylose derivatives containing carboxy or sulfonate groups - Accepted Manuscript

2010-03-08

Abstract: Heparin is an extracellular matrix polysaccharide. It is widely employed as an anticoagulant and can be used to form an anticoagulant surface on various medical devices such as renal dialysis devices to prevent thrombosis. However, heparin may cause hemorrhage and thrombocytopenia. Moreover, commercially available heparin may be contaminated with viruses and allergens of animal origin, as it is derived mainly from porcine or bovine tissue. To avoid these problems, we prepared succinated and sulfonated enzymatically synthesized amylose (SucESA and SulfESA, respectively) and assessed their anticoagulant activity. SucESA and SulfESA inhibited factor Xa activity in normal human plasma to an equal extent. However, SucESA strongly inhibited thrombin activity, whereas SulfESA only inhibited it slightly. These results suggest that SucESA inhibits the activities of both factor Xa (or its upstream coagulation factors) and thrombin and that SulfESA inhibits only factor Xa activity (or that of its upstream coagulation factors). SucESA and SulfESA with a high degree of substitution strongly inhibited factor Xa and thrombin activity compared with those of the derivatives with a low degree of substitution, even when present in high concentrations. This suggests that the density of the anion group determines the degree of inhibition of factor Xa and thrombin activity. SucESA, which has a high molecular weight, inhibited thrombin activity to greater degree than low molecular weight SucESA. Because SucESA and SulfESA inhibited both purified factor Xa and thrombin with no relation to AT, it is suggested that SucESA and SulfESA inhibit through direct action to both enzymes.

Hydroxyl radical release from dental resins: EPR evidence - Accepted Manuscript

2010-03-08

Abstract: It is well known that polymeric free radicals remain trapped inside dental resins for a long time after photopolymerization. Moreover, although these high molecular mass compounds have very limited mobility, there is evidence to suggest that they disappear progressively over time. The purpose of this study was to provide new experimental data to help understand this phenomenon. To determine whether low molecular mass free radicals are released by dental composites stored in hydrophilic media, we used electron paramagnetic resonance (EPR) spectroscopy to perform spin-trapping experiments on experimental and commercial samples stored in ethanol. Under these conditions, ethoxy radicals were produced. Further experiments demonstrated that hydroxyl radicals were firstly released from the methacrylated resin and secondly that they reacted with ethanol molecules to produce “secondary” ethoxy free radicals. In addition to the well-known monomer toxicity of methacrylated resins, we may have identified a new source of concern for these biomaterials

Modulation of polycaprolactone composite properties through incorporation of mixed phosphate glass formulations - Accepted Manuscript

2010-03-08

Abstract: Phosphate-based glasses (PGs) and their composites are of interest as bone repair and tissue engineering scaffolds due to the totally degradable nature of the materials. This study investigated the effect of Si and Fe on the properties of PG particulate-filled polycaprolactone (PCL) matrix composites. Two glass compositions were investigated (in mol%): 50 P2O5, 40 CaO and 10 SiO2 or Fe2O3 (Si10 and Fe10 respectively). All composites contained 40 vol% particulate filler, either of Si10, Fe10, or a blend (40Si10/0Fe10, 30Si10/10Fe10, 20Si10/20Fe10, 10Si10/30Fe10, 0Si10/40Fe10). Ion release, weight loss and composite mechanical properties were characterized as a function of time in deionised water (DW) and phosphate buffered saline (PBS), respectively. The potential for calcium phosphate deposition was assessed in simulated body fluid (SBF).Calcium and phosphate ions released in DW increased in tandem with the rate of composite weight loss, which increased with Si10 content. A Si10 content dependent rate of pH reduction was observed in DW. At day 56, PG in 40Si10/0Fe10 composite completely dissolved, whereas 67% of the 0Si10/40Fe10 remained. The initial flexural strength of 40Si10/0Fe10 composites was significantly lower when compared to the other materials. An increase in Si10 content led to an increase in Young’s modulus and a concomitant decrease in flexural strain. It was found that the PCL molecular weight (Mw) was dramatically decreased with an increase in Si10 content. FTIR analysis showed that Si incorporation into PG led to their reaction with the PCL ester bonds resulting in a reduction in PCL Mw when processed at elevated temperatures. Changes in mechanical properties with time in PBS were glass blend dependent and a more rapid rate of reduction was observed in Si10 dominant composites. At day 28 in SBF, surface deposited brushite was formed in 20Si10/20Fe10 PG containing composites. Therefore the properties of PCL-PG composites could be tailored by controlling the phosphate glass blend composition.

The resorption of nanocrystalline calcium phosphates by osteoclast-like cells - Accepted Manuscript

2010-03-08

Abstract: Nanocrystalline calcium phosphates containing carbonate have a high similarity to bone mineral. The reactions of bone cells (primary osteoblasts and osteoclast-like cells) on these materials as well as on sintered β-tricalcium phosphate (β-TCP) and HA (HA) confirmed a good biocompatibility of the nanocrystalline samples. However, osteoclastic differentiation was constrained on the carbonate-rich samples, leading to a small number of osteoclast-like cells on the materials and few resorption pits. The grain size of the calcium phosphate ceramics (nano vs. micro) was less important than expected due to physico-chemical considerations. When comparing the nanocrystalline samples, the highest resorption rate was found for nHA with low carbonate content which strongly stimulated the differentiation of osteoclast-like cells on its surface.

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

2010-03-08

Abstract: Biocompatible three-dimensional porous scaffolds are of great interest for tissue engineering applications. We present here a novel combined freeze-drying/cross-linking process to prepare porous polysaccharide-based scaffolds. This process does not require organic solvent nor porogen agent. We unexpectedly found that cross-linking of biomacromolecules such as pullulan and dextran with sodium trimetaphosphate could be performed during a freeze-drying process. We evidenced that the freeze-drying pressures modulate the degree of porosity. High freeze-drying pressure scaffolds presented pores with a mean diameter of 55±4 microns and a porosity of 33%±12%, whereas low freeze-drying pressure scaffolds contained larger pores with a mean diameter of 243±14 microns and a porosity of 68%±3%. The porous scaffolds could be easily obtained at the desired shape 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 process of polysaccharides enabled the fabrication of biocompatible scaffolds with controlled porosity and architecture suitable for 3D in vitro culture and biomedical applications.

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

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

Abstract: Cell-based assays in the pharmaceutical and biotechnology industries have become an important tool. 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 (QDs) to decrease the cytotoxicity of QDs. Our results demonstrate the efficacy of chitosan-QD-peptide-Alexa Fluor 488 regions 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.

Synthesis of nano-bioglass and formation of apatite-rods to occlude exposed dentine tubules and eliminate hypersensitivity - Accepted Manuscript

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 nano-bioglass 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 (SEM), focused ion beam (FIB) and energy disperse x-ray spectroscopy (EDXS) was used to characterize the powders and assess tubule occlusion. Melt-derived bioglass possessed an irregular particle morphology and 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 nano-bioglass 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.

Photopolymerizable and injectable polyurethanes for biomedical applications: synthesis and biocompatibility - Accepted Manuscript

2010-03-02

Abstract: Two types of photopolymerizable and injectable polyurethane acrylates (PUAs), which were based on poly(propylene glycol) (PPG) or poly(caprolactone diol) (PCL) and hydroxyethyl methacrylate (HEMA), were synthesized and characterized in order to provide information regarding their use as an injectable material for biomedical applications. Structural characteristics of the biomaterials, including the degree of phase separation, were evaluated by Fourier transform infrared spectroscopy (FTIR). The viscosities of the obtained biomaterials make them suitable to be injected, molded and photopolymerized by using visible light, as demonstrated by the injection test. The cured polymers had mechanical properties comparable to some soft tissues, such as skin. An in vitro cell-polyurethane cytotoxicity study was carried out with mesenchymal stem cells (MSCs) from rat tibias and femurs. The proliferation/viability of the cells in the presence of the synthesized material was assessed by the MTT assay, collagen synthesis analysis, and the expression of alkaline phosphatase. The results that were obtained through the in vitro tests indicated that PUAs are cytocompatible. The in vivo experiments were correlated with the in vitro tests and showed low levels of toxicity for the obtained biomaterials. Histology cross-sections showed that the biomaterials induced no significant inflammatory reaction. Our study demonstrates the potential of the synthesized photocurable polyurethanes to be used in biomedical applications. Furthermore, the obtained injectable polymer systems employ minimally invasive procedures and can be molded in situ before photopolymerization with visible light.

Injectable oxidized hyaluronic acid/adipic acid dihydrazide hydrogel for nucleus pulposus regeneration - Uncorrected Proof

Yu-Chun Chen, Wen-Yu Su, Feng-Huei Lin — 2010-03-02

Abstract: Injectable hydrogel allows irregular surgical defects to be completely filled, lessens the risk of implant migration, and minimizes surgical defects due to the solution–gel state transformation. Here, we first propose a method for preparing oxidized hyaluronic acid/adipic acid dihydrazide (oxi-HA/ADH) injectable hydrogel by chemical cross-linking under physiological conditions. Fourier transform infrared spectrometry and trinitrobenzene sulfonate assay were used to confirm the oxidation of hyaluronic acid. Rheological properties were measured to evaluate the working ability of the hydrogel for further clinical application. The oxi-HA/ADH in situ forming hydrogel can transform from liquid form into a gel-like matrix within 3–8min, depending on the operational temperature. Furthermore, hydrogel degradation and cell assessment is also a concern for clinical application. Injectable oxi-HA/ADH8 hydrogel can maintain its gel-like state for at least 5weeks with a degradation percentage of 40%. Importantly, oxi-HA/ADH8 hydrogel can assist in nucleus pulposus cell synthesis of type II collagen and aggrecan mRNA gene expression according to the results of real-time PCR analysis, and shows good biocompatibility based on cell viability and cytotoxicity assays. Based on the results of the current study, oxi-HA/ADH hydrogel may possess several advantages for future application in nucleus pulposus regeneration.

Synergistic effects of electrospun PLLA fiber dimension and pattern on neonatal mouse cerebellum C17.2 stem cells - Accepted Manuscript

Liumin He, Susan Liao, Daping Quan, Kun Ma, Casey Chan, S. Ramakrishna — 2010-03-02

Abstract: Topographical features, including fiber dimension and pattern, are important aspect in developing a fibrous scaffold for tissue engineering. In this study, poly(L-lactide) (PLLA) aligned fibers with the diameter of 307 ± 47 nm, 500 ± 53 nm, 679 ± 72, 917 ± 84 nm and random fibers with the diameter of 327 ± 40 nm, 545 ± 54 nm, 746 ± 82 nm, 1150 ± 109 nm were obtained by optimizing electrospinning parameters. We cultured neonatal mouse cerebellum C17.2 cells on the PLLA fibers and the neural stem cells (NSCs) exhibited significantly different growth and differentiation with the fiber dimension and pattern. On aligned fibers, cell viability and proliferation was best on 500 nm fibers, and reduced on smaller or larger fibers. However, on random fibers, cell viability and proliferation was best with the smallest (350 nm) and largest (1150 nm) diameter fibers. Polarized and elongated cells were orientated along the fiber direction on the aligned fibers with focal contacts bridging the cell body and aligned fibers. Cells of spindle and polygonal morphologies were randomly distributed on the random fibers with no focal contacts observed. Moreover, longer neurites were obtained on the aligned fibers than random fibers within the same diameter range. Therefore, the surface topographic morphologies of fibrous scaffolds, including fiber pattern, dimension and mesh size played roles in regulating the viability, proliferation and neurite outgrowth of neural stem cells. Nevertheless, our results indicated that 500-nm aligned fiber is the most promising for fine-tuning the design for a nerve scaffold.

Microtopographical effects of natural scaffolding on cardiomyocyte function and arrhythmogenesis - Uncorrected Proof

U. Shah, H. Bien, E. Entcheva — 2010-03-02

Abstract: A natural myocardial patch for heart regeneration derived from porcine urinary bladder matrix (UBM) was previously reported to outperform synthetic materials (Dacron and expanded polytetrafluoroethylene (ePTFE)) used in current surgical treatments. UBM, an extracellular matrix prepared from urinary bladder, has intricate three-dimensional architecture with two distinct sides: the luminal side with a smoother surface relief; and the abluminal side with a fine mesh of nano- and microfibers. This study tested the ability of this natural scaffold to support functional cardiomyocyte networks, and probed how the local microtopography and composition of the two sides affects cell function. Cardiomyocytes isolated from neonatal rats were seeded in vitro to form cardiac tissue onto luminal (L) or abluminal (Ab) UBM. Immunocytochemistry of contractile cardiac proteins demonstrated growth of cardiomyocyte networks with mature morphology on either side of UBM, but greater cell compactness was seen in L. Fluorescence-based imaging techniques were used to measure dynamic changes in intracellular calcium concentration upon electrical stimulation of L and Ab-grown cells. Functional differences in cardiac tissue grown on the two sides manifested themselves in faster calcium recovery (p<0.04) and greater hysteresis (difference in response to increasing and decreasing pacing rates) for L vs Ab side (p<0.03). These results suggest that surface differences may be leveraged to engineer the desired cardiomyocyte responses and highlight the potential of natural scaffolds for fostering heart repair.

Mesoporous Silica Nanotubes Coated with Multilayered Polyelectrolytes for pH-Controlled Drug Release - Accepted Manuscript

Yun-Jie Yang, Xia Tao, Qian Hou, Yi Ma, Xuan-Li Chen, Jian-Feng Chen — 2010-03-02

Abstract: Two kinds of inorganic/organic hybrid composites based on mesoporous silica nanotubes (MSNTs) and pH-responsive polyelectrolytes have been developed as pH-controlled drug delivery systems via the layer-by-layer self-assembly technique. One system was based on alternatively loading poly(allylamine hydrochloride) and sodium poly(styrene sulfonate) onto as-prepared MSNTs to load and release of positively charged doxorubicin drug. The other system was synthesized by alternatively coating sodium alginate and chitosan onto the amine-functionalized MSNTs and applied as vehicles for the loading and release of negatively charged model drug, sodium fluorescein. The controlled release of drug molecules from these delivery systems was achieved by changing the pH value of the release media. Results of in vitro cell cytotoxicity assays further indicated that the cell-killing efficacy of the loaded doxorubicin drug against human fibrosarcoma (HT-1080) and human breast adenocarcinoma (MCF-7) cells was pH-dependent. Therefore, these hybrid composites could be potentially applicable for pH-controlled drug delivery systems.

Surface modification with an antithrombin-heparin complex for anticoagulation: studies on a model surface with gold as substrate - Accepted Manuscript

2010-03-02

Abstract: Gold was used as a substrate for immobilization of an antithrombin-heparin (ATH) covalent complex to investigate ATH as a surface modifier to prevent blood coagulation. Three different surface modification methods were used to attach ATH to gold: (a) by direct chemisorption, (b) using dithiobis (succinimidyl propionate) (DSP) as a linker molecule, and (c) using polyethylene oxide (PEO) as a linker/spacer. The ATH-modified surfaces were compared to analogous heparinized surfaces. Water contact angles and XPS confirmed the modifications and provided data on surface properties and possible orientation. Ellipsometry measurements showed that surface coverage of DSP and PEO was high. ATH and heparin densities were quantified using radioiodination and QCM, respectively. The surface density of ATH was greatest on the DSP surface (0.17 μg/cm2) and lowest on the PEO (0.05 μg/cm2). Low uptake on the PEO surface was likely due to the protein resistance of the PEO component. Using radioiodinated antithrombin (AT) it was shown that ATH-immobilized surfaces bound significantly greater amounts from both buffer and plasma than the analogous heparinized surfaces. Immunoblot analysis of proteins adsorbed from plasma demonstrated that surfaces chemisorbed with PEO, whether or not subsequently modified with ATH, inhibited non-specific adsorption. The immunoblot response for AT was stronger on the DSP-ATH than on the heparin surfaces, thus confirming the results from radiolabelling. The ATH surfaces again showed higher selectivity for AT binding than analogous heparin modified surfaces, indicating the enhanced anticoagulant potential of ATH for biomaterial surface modification.

The Effects of TGF-β3 and Preculture Period of Osteogenic Cells on the Chondrogenic Differentiation of Rabbit Marrow Mesenchymal Stem Cells Encapsulated in a Bilayered Hydrogel Composite - Accepted Manuscript

2010-03-02

Abstract: In this work, injectable, biodegradable hydrogel composites of crosslinked oligo(poly(ethylene glycol) fumarate) (OPF) and gelatin microparticles (MPs) were utilized to fabricate a bilayered osteochondral construct. Rabbit marrow mesenchymal stem cells (MSCs) were encapsulated with transforming growth factor-β3 (TGF-β3)-loaded MPs in the chondrogenic layer and cocultured with cells of different periods of osteogenic preculture (0, 3, 6 and 12 days) in the osteogenic layer to investigate the effects of TGF-β3 delivery and coculture on the proliferation and differentiation of cells in both layers. The results showed that, in the chondrogenic layer, TGF-β3 significantly stimulated chondrogenic differentiation of MSCs. Additionally, cells of various osteogenic preculture periods in the osteogenic layer, along with TGF-β3, enhanced gene expression for MSC chondrogenic markers to different extents. In the osteogenic layer, cells maintained their alkaline phosphatase activity during the coculture; however, mineralization was delayed by the presence of TGF-β3. Overall, this study demonstrated the fabrication of bilayered hydrogel composites that mimic the structure and function of osteochondral tissue, along with the application of these composites as cell and growth factor carriers, while illustrating that encapsulated cells of different degrees of osteogenic differentiation can significantly influence the chondrogenic differentiation of cocultured progenitor cells in both the presence and absence of chondrogenic growth factors.

PMMA-based bone cements containing magnetite particles for the hyperthermia of cancer - Accepted Manuscript

M. Kawashita, K. Kawamura, Z. Li — 2010-03-02

Abstract: Polymethylmethacrylate (PMM A)-based cements containing magnetite (Fe3O4) particles were prepared and their structure and properties were investigated. The Fe3O4 particles were uniformly dispersed in the cement matrix and constituted a maximum of 60 wt% of the total weight of cement. The setting time of the cement increased and the maximum temperature during the setting reaction decreased with increasing Fe3O4 content. The compressive strength of cement increased with increasing Fe3O4 content. Cement with 50 wt% Fe3O4 particles generated heat in alternating magnetic fields of 300 and 120 Oe at a frequency of 100 kHz.

Interactions of human bone cells with diamond-like carbon polymer hybrid coatings - Accepted Manuscript

2010-03-02

Abstract: Diamond-like carbon (DLC) coatings produced with plasma accelerating filtered pulsed arc discharge (FPAD) method display an excellent adherence to the substrate and improve its corrosion resistance. This article reports the interactions of human osteoblastic cells with DLC and two DLC polymer hybrid (DLC-p-h) coatings deposited on smooth, matt and rough silicon wafers with FPAD method. The DLC-p-h materials were DLC polytetrafluoroethylene hybrid (DLC-PTFE-h) and DLC polydimethylsiloxane hybrid (DLC-PDMS-h) coatings. Biocompatibility of the coatings was assayed by using mesenchymal stem cells, primary osteoblasts and Saos-2 cells. Human mesenchymal stem cells proliferated when cultured on DLC and DLC-PTFE-h, but their numbers diminished on DLC-PDMS-h. In all three cell types studied, phalloidin-TRITC staining disclosed cell-type typical organization of actin cytoskeleton on DLC and DLC-PTFE-h, but minimal and disorganized stress fibers on cells cultured on DLC-PDMS-h. Microtubular cytoskeleton was similarly disorganized on DLC-PDMS-h. Cells on DLC-PDMS-h developed a peculiar form of membrane damage with nuclear staining by propidium iodide associated with granular calcein staining of the cytoplasm. Active caspase-3 labeling was only seen in cells cultured on DLC-PDMS-h, indicating that these cells undergo apoptosis induced by defective cell adhesion. Results suggest that DLC-PDMS-h coatings might be useful in orthopedic applications where an implant or implant-facet should be protected against bone overgrowth while DLC and DLC-PTFE-h coatings might improve osseointegration.

Bismuth-doped injectable calcium phosphate cement with improved radiopacity and potent antimicrobial activity for root canal filling - Accepted Manuscript

Fangping Chen, Changsheng Liu, Yuhao Mao — 2010-03-02

Abstract: A bismuth-doped injectable calcium phosphate cement (BD-ICPC) with improved radiopacity, potent antimicrobial activity and sealability was developed in this work by introducing bismuth salicylite basic (BSB) into the powder phase of calcium phosphate cement (CPC). The results showed that the radiopacity and sealability of BD-ICPC were improved compared with pure ICPC. Although BSB has a retarding effect on the setting rate of the cement, the addition of BSB reduced the viscosity and yield stress of BD-ICPC, thus enhanced its injectability. It was noteworthy that BD-ICPC had a potent antimicrobial activity with improved sealability. In addition, BD-ICPC afforded a uniform and tight adaptation to the root canal wall. These results indicated that the BD-ICPC possessed the combination of in vitro good radiopacity, high injectability, potent antimicrobial activity, improved sealability and tight adaptation to the root canals. It is expected to be used as a novel root canal filling material.

The mechanical stress–strain properties of single electrospun collagen type I nanofibers - Uncorrected Proof

C.R. Carlisle, C. Coulais, M. Guthold — 2010-03-02

Abstract: Knowledge of the mechanical properties of electrospun fibers is important for their successful application in tissue engineering, material composites, filtration and drug delivery. In particular, electrospun collagen has great potential for biomedical applications due to its biocompatibility and promotion of cell growth and adhesion. Using a combined atomic force microscopy (AFM)/optical microscopy technique, the single fiber mechanical properties of dry, electrospun collagen type I were determined. The fibers were electrospun from a 80mgml−1 collagen solution in 1,1,1,3,3,3-hexafluro-2-propanol and collected on a striated surface suitable for lateral force manipulation by AFM. The small strain modulus, calculated from three-point bending analysis, was 2.82GPa. The modulus showed significant softening as the strain increased. The average extensibility of the fibers was 33% of their initial length, and the average maximum stress (rupture stress) was 25MPa. The fibers displayed significant energy loss and permanent deformations above 2% strain.

The effects of pore architecture in silk fibroin scaffolds on the growth and differentiation of mesenchymal stem cells expressing BMP7 - Uncorrected Proof

Yufeng Zhang, Wei Fan, Zhaocheng Ma, Chengtie Wu, Wei Fang, Gang Liu, Yin Xiao — 2010-02-26

Abstract: The pore architecture of scaffolds is known to play a critical role in tissue engineering as it provides the vital framework for seeded cells to organize into a functioning tissue. In this report we have investigated the effects of different concentrations of silk fibroin protein on three-dimensional (3D) scaffold pore microstructure. Four pore size ranges of silk fibroin scaffolds were made by the freeze drying technique, with the pore sizes ranging from 50 to 300μm. The pore sizes of the scaffolds decreased as the concentration of fibroin protein increased. Human bone marrow mesenchymal stromal cells (BMSC) transfected with the BMP7 gene were cultured in these scaffolds. A cell viability colorimetric assay, alkaline phosphatase assay and reverse transcription-polymerase chain reaction were performed to analyze the effect of pore size on cell growth, the secretion of extracellular matrix (ECM) and osteogenic differentiation. Cell migration in 3D scaffolds was confirmed by confocal microscopy. Calvarial defects in SCID mice were used to determine the bone forming ability of the silk fibroin scaffolds incorporating BMSC expressing BMP7. The results showed that BMSC expressing BMP7 preferred a pore size between 100 and 300μm in silk fibroin protein fabricated scaffolds, with better cell proliferation and ECM production. Furthermore, in vivo transplantation of the silk fibroin scaffolds combined with BMSC expressing BMP7 induced new bone formation. This study has shown that an optimized pore architecture of silk fibroin scaffolds can modulate the bioactivity of BMP7-transfected BMSC in bone formation.

In vitro dissolution and mechanical behavior of c-axis preferentially oriented hydroxyapatite thin films fabricated by pulsed laser deposition - Uncorrected Proof

Hyunbin Kim, Renato P. Camata, Shafiul Chowdhury, Yogesh K. Vohra — 2010-02-26

Abstract: Owing to its resemblance to the major inorganic constituent of bone and tooth, hydroxyapatite is recognized as one of the most biocompatible materials and is widely used in systems for bone replacement and regeneration. In this study the pulsed laser deposition technique was chosen to produce hydroxyapatite with different crystallographic orientations in order to investigate some of the material properties, including its in vitro dissolution behavior, as well as mechanical properties. The crystallographic orientations of hydroxyapatite coatings can be carefully controlled, mainly by varying the energy density of the KrF excimer laser (248nm) used for deposition. Nanoindentation results showed that highly c-axis oriented hydroxyapatite coatings have higher hardness and Young’s modulus values compared with the values of randomly oriented coatings. After 24h immersion in simulated physiological solution the overall surface morphology of the highly oriented coatings was dramatically altered. The porosity was drastically increased and sub-micron pores were formed throughout the coatings, whereas the average size of the grains in the coatings was not significantly changed. The composition of the textured hydroxyapatite coatings remained essentially unchanged. Their c-axis texture, on the other hand, was rather enhanced with an increase in immersion time. The c-axis oriented hydroxyapatite surfaces are likely to promote preferentially oriented growth through a cyclic process of dissolution and reprecipitation, followed by homoepitaxial growth. The remarkable morphological and microstructural changes after dissolution suggest a capability of highly textured hydroxyapatite as a tissue engineering scaffold with an interconnecting porous network that may be beneficial for cellular activity.

Stability of antibacterial self-assembled monolayers on hydroxyapatite - Uncorrected Proof

2010-02-26

Abstract: Open fractures are common in battlefields, motor vehicle accidents, gunshot wounds, sports injuries, and high-energy falls. Such fractures are treated using hydroxyapatite (HA)-based bone graft substitutes. However, open fracture wounds are highly susceptible to bacterial infections. Hence, this study was focused on incorporating antibacterial properties to HA using silver (Ag) carrying self-assembled monolayers (SAMs). Also, the stability of Ag carrying SAMs on HA was investigated under sterilization and physiological conditions. Initially, the –COOH terminated phosphonic acid SAMs of two different chain lengths (11 carbon atoms – shorter chain and 16 carbon atoms – longer chain) were deposited on HA. Antibacterial SAMs (ASAMs) were prepared by chemically attaching Ag to shorter and longer chain SAMs coated HA. X-ray photoelectron spectroscopy, atomic force microscopy, and contact angle goniometry collectively confirmed the attachment of Ag onto SAMs coated HA. The bacterial adhesion study showed that the adherence of Staphylococcus aureus was significantly reduced on ASAMs coated HA when compared to control-HA. The stability studies showed that gas plasma, dry heat and autoclave degraded most of the ASAMs on HA. UV irradiation did not damage the shorter chain ASAMs as vigorously as other treatments, while it degraded the longer chain ASAMs completely. Ethylene oxide treatment did not degrade the longer chain ASAMs unlike all other treatments but it severely damaged the shorter chain ASAMs. Both shorter and longer chain ASAMs significantly desorbed from the HA surfaces under physiological conditions although longer chain ASAMs exhibited better stability than shorter chain ASAMs. This study demonstrated the potential for using ASAMs to provide antibacterial properties to HA and the need for developing techniques to improve stability of SAMs under sterilization and physiological conditions.

Calcification of cartilage formed in vitro on calcium polyphosphate bone substitutes is regulated by inorganic polyphosphate - Uncorrected Proof

Jean-Philippe St-Pierre, Robert M. Pilliar, Marc D. Grynpas, Rita A. Kandel — 2010-02-26

Abstract: A major challenge to the successful clinical application of bioengineered cartilage remains its integration to surrounding tissues upon implantation. One way to address this consists of generating biphasic constructs composed of articular cartilage formed in vitro on the top surface and integrated with the porous sub-surface of a bone substitute material – in the case of this study, calcium polyphosphate (CPP). To improve the mechanical integrity of the cartilage–bone substitute interface, attempts have been made to generate a zone of calcified cartilage (ZCC) within the CPP–cartilage interface, thereby mimicking the native joint architecture. The purpose of this work was to establish the effects of the degradation products of CPP on cartilage calcification in order to explain the observed positioning of a ZCC away from the interface junction. It was determined that polyphosphate released from the CPP accumulates within in vitro-grown cartilage and inhibits cartilage calcification in a concentration and chain length (i.e. molecular weight) dependent manner. It was found that this effect is transient as chondrocytes express exopolyphosphatases which hydrolyze polyphosphate to release orthophosphate. Hence, the generation of biphasic constructs with a properly located ZCC will require tailoring of CPP substrates with lower degradation rates or the upregulation of exopolyphosphatases by chondrocytes.

Thorough analysis of silicon substitution in biphasic calcium phosphate bioceramics: A multi-technique study - Uncorrected Proof

2010-02-26

Abstract: Four samples of composition Ca10(PO4)6−x(SiO4)x(OH)2−x, with x=0.0, 0.1, 0.2 and 0.5, were prepared and characterized using powder X-ray and neutron powder diffraction, and 1H, 31P and 29Si nuclear magnetic resonance (NMR) spectroscopy. The composition of the Si-substituted HAp phases was determined by joint Rietveld refinements from powder X-ray and powder neutron diffraction data. Taking into account electroneutrality, a chemical formula for the Si-substituted HAp phases with indication of the incorporated silicate amount is proposed. Solid-state 29Si NMR confirms the presence of only Q0 species, in good agreement with the presence of substituted HAp and β-TCP phases only. Thanks to NMR spectroscopy, two types of protons in the Si-substituted HAp phase were identified, the new site corresponding to species engaged in hydrogen bonding with silicate anions. This allowed further refinement of the formulae for these phases with very good quantitative agreement for populations derived from the refinement and integration of NMR data.

A novel platform for in situ investigation of cells and tissues under mechanical strain - Uncorrected Proof

W.W. Ahmed, M.H. Kural, T.A. Saif — 2010-02-26

Abstract: The mechanical micro-environment influences cellular responses such as migration, proliferation, differentiation and apoptosis. Cells are subjected to mechanical stretching in vivo, e.g., epithelial cells during embryogenesis. Current methodologies do not allow high-resolution in situ observation of cells and tissues under applied strain, which may reveal intracellular dynamics and the origin of cell mechanosensitivity. A novel polydimethylsiloxane substrate was developed, capable of applying tensile and compressive strain (up to 45%) to cells and tissues while allowing in situ observation with high-resolution optics. The strain field of the substrate was characterized experimentally using digital image correlation, and the deformation was modeled by the finite element method, using a Mooney–Rivlin hyperelastic constitutive relation. The substrate strain was found to be uniform for >95% of the substrate area. As a demonstration of the system, mechanical strain was applied to single fibroblasts transfected with GFP-actin and whole transgenic Drosophila embryos expressing GFP in all neurons during live imaging. Three observations of biological responses due to applied strain are reported: (1) dynamic rotation of intact actin stress fibers in fibroblasts; (2) lamellipodia activity and actin polymerization in fibroblasts; (3) active axonal contraction in Drosophila embryo motor neurons. The novel platform may serve as an important tool in studying the mechanoresponse of cells and tissues, including whole embryos.

Poly(dimethylsiloxane) elastomers with tethered peptide ligands for cell adhesion studies - Uncorrected Proof

Yuanzi Wu, Sean R. Coyer, Hongwei Ma, Andrés J. García — 2010-02-22

Abstract: Poly(dimethylsiloxane) (PDMS) is the choice of material for a wide range of biological and non-biological applications because of its chemical inertness, non-toxicity, ease of handling and commercial availability. However, PDMS exhibits uncontrolled protein adsorption and cell adhesion and it has proved difficult to functionalize to present bioactive ligands. We present a facile strategy for functional surface modification of PDMS using commercial reagents to engineer polymer brushes of oligo(ethylene glycol) methacrylate that prevent cell adhesion and can be functionalized to display bioadhesive ligands. The polymer brushes resist biofouling and prevent cell adhesion and bioadhesive peptides can be tethered either uniformly or constrained to micropatterned domains using standard peptide chemistry approaches. This approach is relevant to various biomedical and biotechnological applications.

The effect of BMP-2 on micro- and macroscale osteointegration of biphasic calcium phosphate scaffolds with multiscale porosity - Uncorrected Proof

2010-02-22

Abstract: It is well established that scaffolds for applications in bone tissue engineering require interconnected pores of the order of 100μm for bone growth and nutrient and waste transport. As a result, most studies have focused on macroporosity (>100μm). More recently researchers have also investigated the role of microporosity in calcium phosphate (CaP)-based scaffolds. Osteointegration into macropores improves when scaffold rods or struts contain micropores, typically defined as pores less than ∼50μm. We recently demonstrated multiscale osteointegration, or growth into both macropores and micropores (<10μm), of rods in biphasic CaP (BCP) scaffolds. The combined effect of BMP-2, a potent osteoinductive growth factor, and multiscale porosity has yet to be investigated. In this study we implanted BCP scaffolds into porcine mandibles for 3, 6, 12 and 24weeks and evaluated the effect of BMP-2 on multiscale osteointegration. The results showed that in this in vivo model BMP-2 influences osteointegration at the microscale but not at the macroscale. Cell density was higher in the rod micropores for scaffolds containing BMP-2 compared with controls at all time points, but BMP-2 was not required for bone formation in micropores. In contrast, there was essentially no difference in the fraction of bone in macropores for scaffolds with BMP-2 compared with controls. Additionally, bone in macropores seemed to have reached steady-state by 3weeks. Multiscale osteointegration results in composites that are fully osteointegrated, with no ‘dead space’, and are likely to have not only a continuous cell network but also the potential for enhanced load transfer and improved mechanical properties.

Assessing the biocompatibility of degradable metallic materials: State-of-the-art and focus on the potential of genetic regulation - Uncorrected Proof

Agung Purnama, Hendra Hermawan, Jacques Couet, Diego Mantovani — 2010-02-22

Abstract: For decades, the design, development and use of metallic biomaterials has focused on the corrosion resistance of these materials once implanted in the human body. Recently, degradable metallic biomaterials (DMMs) have been proposed for some specific applications, including paediatric, orthopaedic and cardiovascular applications. DMMs are expected to disappear via corrosion after providing structural support for a certain period of time depending on the application site. Over the past decades, a wide-ranging and comprehensive set of in vitro, in vivo and for some cases also ex vivo tests have been proposed and exhaustively investigated for conventional corrosion-resistant metallic biomaterials. Standardization and regulatory bodies in the United States, Japan and Europe have therefore developed tests to license corrosion-resistant metals for use as “biomaterials”. This is not the case for DMMs. Once implanted, this new class of biomaterials is expected to support the healing process of a diseased tissue or organ while degrading at a potentially adjustable degradation rate. The tests developed for corrosion-resistant metals cannot simply be transposed to DMMs. These tests can in some cases be adapted, but the expected unique properties of DMMs should also inspire and lead to the design and the development of new specific tests. The current challenge is how to assess the tolerance of surrounding tissues and organs to the presence of degradation products. This work precisely focuses on this topic. The tests usually used to assess the biocompatibility of conventional corrosion-resistant metals are briefly reviewed. Then, genetic regulation is proposed as an original and novel approach to assess the biocompatibility of DMMs. This method appears to predict cell behaviour in the presence of degradation products that are closely related to DNA damage. Various genes have been related to the toxicity and inflammatory responses, indicating their role as biomarkers to assess the toxicity of degradation products. Finally, some gene families that have the potential to be applied as biomarkers of degradation product toxicity are summarized.

The history of biodegradable magnesium implants: A review - Uncorrected Proof

Frank Witte — 2010-02-22

Abstract: Today, more than 200years after the first production of metallic magnesium by Sir Humphry Davy in 1808, biodegradable magnesium-based metal implants are currently breaking the paradigm in biomaterial science to develop only highly corrosion resistant metals. This groundbreaking approach to temporary metallic implants is one of the latest developments in biomaterials science to be rediscovered. It is a challenging topic, and several secrets still remain that might revolutionize various biomedical implants currently in clinical use. Magnesium alloys were investigated as implant materials long ago. A very early clinical report was given in 1878 by the physician Edward C. Huse. He used magnesium wires as ligature for bleeding vessels. Magnesium alloys for clinical use were explored during the last two centuries mainly by surgeons with various clinical backgrounds, such as cardiovascular, musculoskeletal and general surgery. Nearly all patients benefited from the treatment with magnesium implants. Although most patients experienced subcutaneous gas cavities caused by rapid implant corrosion, most patients had no pain and almost no infections were observed during the postoperative follow-up. This review critically summarizes the in vitro and in vivo knowledge and experience that has been reported on the use of magnesium and its alloys to advance the field of biodegradable metals.

Editorial - Uncorrected Proof

Diego Mantovani, Frank Witte — 2010-02-22

More than 200years after the first production of elemental magnesium by Sir Humphrey Davy, the past decade has witnessed increased attention to magnesium-based alloys by biomaterial scientists and medical device developers. Surprisingly, the concept of using magnesium as an implantable material is relatively old. In the early part of the twentieth century magnesium was investigated extensively for several medical applications, but with the advent and adoption of stainless steels as metallic implants this interest waned in the post-World War II period to little, if any, research. The attraction of a lightweight metal with mechanical properties suitable for many applications brought a renewed focus on magnesium alloys in the automotive and aerospace industries. This interest spread to the current, rapidly growing interest in magnesium-based alloys for medical applications. In the words of the author of Ecclesiastes, “there is nothing new under the sun”.

Controllable inhibition of cellular uptake of oxidized low-density lipoprotein: Structure–function relationships for nanoscale amphiphilic polymers - Uncorrected Proof

2010-02-18

Abstract: A family of anionic nanoscale polymers based on amphiphilic macromolecules (AMs) was developed for controlled inhibition of highly oxidized low-density lipoprotein (hoxLDL) uptake by inflammatory macrophage cells, a process that triggers the escalation of a chronic arterial disease called atherosclerosis. The basic AM structure is composed of a hydrophobic portion formed from a mucic acid sugar backbone modified at the four hydroxyls with lauroyl groups conjugated to hydrophilic poly(ethylene glycol) (PEG). The AM structure–activity relationships were probed by synthesizing AMs with six key variables: length of the PEG chain, carboxylic acid location, type of anionic charge, number of anionic charges, rotational motion of the anionic group, and PEG architecture. All AM structures were confirmed by nuclear magnetic resonance spectroscopy and their ability to inhibit hoxLDL uptake in THP-1 human macrophage cells was compared in the absence and presence of serum. We report that AMs with one, rotationally restricted carboxylic acid within the hydrophobic portion of the polymer was sufficient to yield the most effective AM for inhibiting hoxLDL internalization by THP-1 human macrophage cells under serum-containing conditions. Further, increasing the number of charges and altering the PEG architecture in an effort to increase serum stabilization did not significantly impair the ability of AMs to inhibit hoxLDL internalization, suggesting that selected modifications to the AMs could potentially promote multifunctional characteristics of these nanoscale macromolecules.

Influence of chemical structures of benzodioxole-based coinitiators on the properties of the unfilled dental resin - Uncorrected Proof

Suqing Shi, Pu Xiao, Kemin Wang, Yongkuan Gong, Jun Nie — 2010-02-18

Abstract: To investigate the influence of chemical structures of benzodioxole-based coinitiator on the initiating reactivity and the mechanical properties of cured samples for the unfilled dental resin, a mixture of 2,2-bis[4-(2-hydroxy-3-methacryloxyprop-1-oxy)phenyl]propane (bis-GMA) and triethylene glycol dimethacrylate (TEGDMA) (70/30 wt.%) was photoinduced by combinations of camphorquinone (CQ) and benzodioxole derivatives. 2-(N,N-Dimethylamino)ethyl methacrylate (DMEM) was used as control. The kinetics was monitored by a real-time Fourier transformation infrared spectroscopy (FTIR) and the dynamic mechanical analysis was performed on a dynamic mechanical analyzer (DMA). The cytotoxicity property of the cured samples was evaluated by MTT assay in vitro using VERO as reference cell lines. The results indicated that the 4-position phenyl ring substituents of the benzodioxole-based coinitiator had great influence on the initiating reactivity. Incorporating substituents with π electron acceptors in the 4-position of phenyl ring led to the decrease of the rate of polymerization (Rp) of the CQ/benzodioxole derivatives. However, the electron-donating substituents were useful to increase the reactivity. When compared with CQ/amine initiating systems, the combination of CQ and benzodioxole compounds caused lower Rp but the comparable final double bond conversion. All the cured films initiated by CQ/benzodioxole derivatives had almost the same glass transition temperature (Tg) and storage modulus. Indirect cytotoxicity assessment indicated low cytotoxicity of benzodioxole derivatives. These results were very useful for the design of benzodioxole derivatives with satisfactory reactivity and biocompatibility, and are very important for clinical applications.

Influences of tensile load on in vitro degradation of an electrospun poly(l-lactide-co-glycolide) scaffold - Uncorrected Proof

Ping Li, Xiaoliang Feng, Xiaoling Jia, Yubo Fan — 2010-02-18

Abstract: Scaffolds for tissue engineering, regenerative medicine and implantation are usually subjected to different mechanical loads during in vitro and in vivo degradation. In this study, the in vitro degradation process of electrospun poly(l-lactide-co-glycolide) (PLGA) scaffolds was examined under continuous tensile load and compared with that under no load. As PLGA degraded in phosphate-buffered saline solution (pH 7.4) at 37°C over a 7-week period, the tensile elastic modulus and ultimate strength of the loaded specimen increased dramatically, followed by a decrease, which was much faster than that of the unloaded specimen, whereas break elongation of the loaded samples declined more quickly over the whole degradation period. Moreover, molecular weight, thermal properties and lactic acid release showed greater degradation under load. Also, a ruptured morphology was more obvious after degradation under tensile load. The results demonstrate that tensile load increased the degradation rate of electrospun PLGA and it may be necessary to consider the effects of mechanical load when designing or applying biodegradable scaffolds. Finally, some possible explanation for the faster degradation under load is given.

Synthesis and characterization of poly(methyl methacrylate)-based experimental bone cements reinforced with TiO2–SrO nanotubes - Uncorrected Proof

S.M.Z. Khaled, Paul A. Charpentier, Amin S. Rizkalla — 2010-02-18

Abstract: In an attempt to overcome existing limitations of experimental bone cements we here demonstrate a simple approach to synthesizing strontium-modified titania nanotubes (n-SrO–TiO2 tubes) and functionalize them using the bifunctional monomer methacrylic acid. Then, using ‘grafting from’ polymerization with methyl methacrylate, experimental bone cements were produced with excellent mechanical properties, radiopacity and biocompatibility. Transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy-dispersive spectroscopy mapping and backscattered SEM micrographs revealed a uniform distribution of SrO throughout the titanium matrix, with retention of the nanotubular morphology. Nanocomposites were then reinforced with 1, 2, 4 and 6 wt.% of the functionalized metal oxide nanotubes. Under the mixing and dispersion regime employed in this study, 2 wt.% appeared optimal, exhibiting a more uniform dispersion and stronger adhesion of the nanotubes in the poly(methyl methacrylate) matrix, as shown by TEM and SEM. Moreover, this optimum loading provided a significant increase in the fracture toughness (KIC) (20%) and flexural strength (40%) in comparison with the control matrix (unfilled) at P<0.05. Examination of the fracture surfaces by SEM showed that toughening was provided by the nanotubes interlocking with the acrylic matrix and crack bridging during fracture. On modifying the n-TiO2 tubes with strontium oxide the nanocomposites exhibited a similar radiopacity to a commercial bone cement (CMW® 1), while exhibiting a significant enhancement of osteoblast cell proliferation (242%) in vitro compared with the control at P<0.05.

Hydroxyapatite nanorods/poly(vinyl pyrolidone) composite nanofibers, arrays and three-dimensional fabrics: Electrospun preparation and transformation to hydroxyapatite nanostructures - Uncorrected Proof

2010-02-17

Abstract: Electrospinning has been recognized as an efficient technique for fabricating polymer nanofibrous biomaterials. However, the study of electrospun inorganic biomaterials with well-designed three-dimensional (3-D) structures is still limited and little reported. In this study hydroxyapatite (HAp) nanorods with an average diameter of ∼7nm and length of ∼27nm were synthesized through a simple precipitation method and used for the fabrication of inorganic/organic [poly(vinyl pyrolidone) (PVP)] composite nanofibers by electrospinning in ethanol solution. 3-D fabrics and aligned nanofiber arrays of the HAp nanorods/PVP composite were obtained as precursors. Thereafter, 3-D single phase HAp fabrics, tubular structures and aligned nanofiber arrays were obtained after thermal treatment of the corresponding composite precursors. Cytotoxicity experiments indicated that the HAp fabric scaffold had good biocompatibility. In vitro experiments showed that mesenchymal stem cells could attach to the HAp fabric scaffold after culture for 24h.

Femtosecond laser induced fixation of calcium alkali phosphate ceramics on titanium alloy bone implant material - Uncorrected Proof

Christian Symietz, Erhard Lehmann, Renate Gildenhaar, Jörg Krüger, Georg Berger — 2010-02-17

Abstract: Femtosecond lasers provide a novel method of attaching bioceramic material to a titanium alloy, thereby improving the quality of bone implants. The ultrashort 30fs laser pulses (790nm wavelength) penetrate a thin dip-coated layer of fine ceramic powder, while simultaneously melting a surface layer of the underlying metal. The specific adjustment of the laser parameters (pulse energy and number of pulses per spot) avoids unnecessary melting of the bioactive calcium phosphate, and permits a defined thin surface melting of the metal, which in turn is not heated throughout, and therefore maintains its mechanical stability. It is essential to choose laser energy densities that correspond to the interval between the ablation fluences of both materials involved: about 0.1–0.4Jcm−2. In this work, we present the first results of this unusual technique, including laser ablation studies, scanning electron microscopy and optical microscope images, combined with EDX data.

Amorphous calcium phosphates: Synthesis, properties and uses in biomaterials - Uncorrected Proof

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.

Patterned transgene expression in multiple-channel bridges after spinal cord injury - Uncorrected Proof

2010-02-17

Abstract: Patterning of gene delivery on sub-millimeter length scales within tissue engineering scaffolds is fundamental to recreating the complex architectures of tissues. Surface-mediated delivery of lipoplexes mixed with fibronectin was investigated to pattern vectors within 250μm channels on poly(lactide-co-glycolide) (PLG) bridges. Initial studies performed in vitro on PLG surfaces indicated that a DNA density of 0.07μgmm–2 inside each channel with a weight ratio of DNA to fibronectin of 1:20 maximized the number of transfected cells and the levels of transgene expression. Patterned vectors encoding for nerve growth factor (NGF) resulted in localized neurite extension within the channel. Translation to three-dimensional multiple-channel bridges enabled patterned transfection of different vectors throughout the channels for DNA:fibronectin ratios of 1:4 and multiple DNA depositions, with a large increase of neural cell bodies and neurite extension for delivery of DNA encoding for NGF. In vivo, the immobilization of non-viral vectors within the channels resulted in localized transfection within the pore structure of the bridge immediately around the channels of the bridge containing DNA. This surface immobilization strategy enables patterned gene delivery in vitro and in vivo on length scales of hundreds of microns and may find utility in strategies aimed at regenerating tissues with complex architectures.

Varying the diameter of aligned electrospun fibers alters neurite outgrowth and Schwann cell migration - Uncorrected Proof

2010-02-17

Abstract: Aligned, electrospun fibers have shown great promise in facilitating directed neurite outgrowth within cell and animal models. While electrospun fiber diameter does influence cellular behavior, it is not known how aligned, electrospun fiber scaffolds of differing diameter influence neurite outgrowth and Schwann cell (SC) migration. Thus, the goal of this study was to first create highly aligned, electrospun fiber scaffolds of varying diameter and then assess neurite and SC behavior from dorsal root ganglia (DRG) explants. Three groups of highly aligned, electrospun poly-l-lactic acid (PLLA) fibers were created (1325+383nm, large diameter fibers; 759+179nm, intermediate diameter fibers; and 293+65nm, small diameter fibers). Embryonic stage nine (E9) chick DRG were cultured on fiber substrates for 5days and then the explants were stained with neurofilament, S100 and DAPI. Neurite length and SC migration distance were determined. In general, the direction of neurite extension and SC migration were guided along the aligned fibers. On the small diameter fiber substrate, the neurite length was 42% and 36% shorter than those on the intermediate and large fiber substrates, respectively. Interestingly, SC migration did not correlate with that of neurite extension in all situations. SCs migrated equivalently with extending neurites in both the small and large diameter scaffolds, but lagged behind neurites on the intermediate diameter scaffolds. Thus, in some situations, topography alone is sufficient to guide neurites without the leading support of SCs. Scanning electron microscopy images show that neurites cover the fibers and do not reside exclusively between fibers. Further, at the interface between fibers and neurites, filopodial extensions grab and attach to nearby fibers as they extend down the fiber substrate. Overall, the results and observations suggest that fiber diameter is an important parameter to consider when constructing aligned, electrospun fibers for nerve regeneration applications.

Crystal templating dendritic pore networks and fibrillar microstructure into hydrogels - Uncorrected Proof

Scott A. Zawko, Christine E. Schmidt — 2010-02-17

Abstract: Native tissues contain space-filling dendritic pore networks, such as vasculature, for the efficient distribution of oxygen and nutrients; however, it is not yet possible to create tissue-engineered scaffolds with dendritic porosity. Fibers are also important structural features of native tissues because they provide sites for cell anchorage, promote cell guidance and contribute to mechanical stability. Here, we have developed a “crystal templating” technique, which is simple and inexpensive, for fabricating polymer scaffolds with space-filling dendritic pore networks and fibrillar microtopography. To do this, we grow dendritic urea crystals in solution cast films of hyaluronic acid (HA), photocrosslink the HA around the crystal network to lock in the dendritic configuration, and dissolve the crystals to obtain empty pores. During in situ crystal growth the HA biopolymer is phase separated from the long narrow urea crystals and shaped into a fibrillar microstructure. The porous fibrillar HA scaffolds created by crystal templating may be applicable as regenerative patches for skin and other tissues.

Hardness in arthropod exoskeletons in the absence of transition metals - Corrected Proof

B.W. Cribb, C.-L. Lin, L. Rintoul, R. Rasch, J. Hasenpusch, H. Huang — 2010-02-11

Abstract: The arthropod cuticle is a remarkable and versatile biological material commonly composed of chitin and proteins. Lessons can be learned from the way it is adapted to fit its functions. The larval jewel beetle, Pseudotaenia frenchi, demonstrates hardness in the cutting edge of the mandibles in excess of the mineralized carapace of stone crabs and compares favourably with some stainless steels. Yet this is a form of cuticle which is devoid of transition metals or mineralization. In seeming contradiction, the similarly dark coloured adult beetle mandibles contain the transition metal manganese, but are significantly softer. Energy dispersive X-ray analysis and infrared spectroscopy have been used to investigate the differences in composition of mandible cuticle of the adult and larval beetles.

Sol–gel silica-based biomaterials and bone tissue regeneration - Uncorrected Proof

Daniel Arcos, María Vallet-Regí — 2010-02-11

Abstract: The impact of bone diseases and trauma in developed and developing countries has increased significantly in the last decades. Bioactive glasses, especially silica-based materials, are called to play a fundamental role in this field due to their osteoconductive, osteoproductive and osteoinductive properties. In the last years, sol–gel processes and supramolecular chemistry of surfactants have been incorporated to the bioceramics field, allowing the porosity of bioglasses to be controlled at the nanometric scale. This advance has promoted a new generation of sol–gel bioactive glasses with applications such as drug delivery systems, as well as regenerative grafts with improved bioactive behaviour. Besides, the combination of silica-based glasses with organic components led to new organic–inorganic hybrid materials with improved mechanical properties. Finally, an effort has been made to organize at the macroscopic level the sol–gel glass preparation. This effort has resulted in new three-dimensional macroporous scaffolds, suitable to be used in tissue engineering techniques or as porous pieces to be implanted in situ. This review collects the most important advances in the field of silica glasses occurring in the last decade, which are called to play a lead role in the future of bone regenerative therapies.

Hydrogel/calcium phosphate composites require specific properties for three-dimensional culture of human bone mesenchymal cells - Corrected Proof

J. Sohier, P. Corre, P. Weiss, P. Layrolle — 2010-02-11

Abstract: To provide multipotent cells with a three-dimensional environment closer to bone matrix, an engineered construct mimicking bone components has been designed and evaluated. A biocompatible hydrogel (silated hydroxypropylmethyl cellulose) was used as an extra-cellular matrix while biphasic calcium phosphate ceramic particles were used to replace mineralized matrix. Finally, human bone mesenchymal cells were cultured in three dimensions in the resulting constructs to study their cell viability, proliferation, interactions within the composites, and maintenance of their osteogenic potential. This approach resulted in homogeneous structures in which cells were viable and retained their osteoblastic differentiation potential. However, the cells did not proliferate nor colonize the constructs, possibly because of a lack of suitable interactions with their micro-environment.

Light-sensitive intelligent drug delivery systems of coumarin-modified mesoporous bioactive glass - Uncorrected Proof

H.-M. Lin, W.-K. Wang, S.-G. Shyu — 2010-02-11

Abstract: Functionalized mesoporous bioactive glasses (MBG) with photoactive coumarin demonstrates photo-responsive dimerization resulting in reversible gate operation. Coumarin-modified MBG was used as a drug delivery carrier to investigate drug storage/release characteristics using phenanthrene as a model drug. Irradiation with UV light (>310nm) induced photo-dimerization of the coumarin-modified MBG, which led to the pores’ closing with cyclobutane dimers and trapping of the guest phenanthrene in the mesopores. However, irradiating the dimerized-coumarin-modified MBG with shorter wavelength UV light (∼250nm) regenerates the coumarin monomer derivative by the photo-cleavage of cyclobutane dimers, such that trapped guest molecules are released from the mesopores. The structural, morphological, textural and optical properties are well characterized by X-ray diffraction, transmission electron microscopy, N2 adsorption/desorption, and UV–visible spectroscopy. The results reveal that the MBG exhibits the typical ordered characteristics of the hexagonal mesostructure. The system demonstrates great potential in light-sensitive intelligent drug delivery systems and disease therapy fields.

Enhanced antiproliferative activity of carboplatin-loaded chitosan–alginate nanoparticles in a retinoblastoma cell line - Corrected Proof

Suphiya Parveen, Moutushy Mitra, S. Krishnakumar, Sanjeeb K. Sahoo — 2010-02-10

Abstract: In the present study the potential of carboplatin-loaded chitosan–alginate nanoparticles (CANPs) for the treatment of retinoblastoma was investigated. The carboplatin-loaded CANPs were ∼300nm in size, exhibited a high zeta potential of ∼36mV and drug encapsulation of ∼20wt.%. The CANPs were further characterized by Fourier transform infrared spectroscopy, differential scanning calorimetry and transmission electron microscopy. In vitro release studies revealed fast release of ∼25% of the drug during the first 24h, followed by sustained release. CANPs demonstrated greater and sustained antiproliferative activity of the drug in a dose- and time-dependent manner (carboplatin IC50=0.56μgml−1, carboplatin-loaded CANPs IC50=0.004μgml−1), as well as an enhanced apoptotic effect as compared with the drug in solution in a retinoblastoma cell line (Y79). The higher cytotoxic effect of CANPs may be due to their greater cellular uptake as compared with native carboplatin. It was also demonstrated that clathrin-mediated endocytosis plays a key role in the internalization of CANPs in the Y79 cell line. In conclusion, biodegradable chitosan nanoparticles could be used as an effective ocular drug delivery system for sustained intracellular delivery of carboplatin for the treatment of retinoblastoma.

Synthesis and characterization of hydroxyapatite whiskers by hydrothermal homogeneous precipitation using acetamide - Uncorrected Proof

Hongquan Zhang, Brian W. Darvell — 2010-02-10

Abstract: Long and uniform HA whiskers with high crystallinity, controlled morphology and high aspect ratio were successfully synthesized by hydrothermal homogeneous precipitation using acetamide. Compared with the additive urea, which is commonly used to raise the pH to drive nucleation and growth of HA crystals, acetamide has a low hydrolysis rate under the required hydrothermal conditions. This allows better and easier control, giving rise to rapid growth of whiskers at a low supersaturation. Whisker length and width were in turn determined by solution conditions, including the concentration of Ca and PO4. Whiskers had a mean length of 60–116μm and an aspect ratio of 68–103 for starting solutions containing 42–84mmoll−1 Ca and 25–50mmoll−1 PO4 with a fixed Ca/P ratio of 1.67. Such whiskers are favourable for their improved bone bonding and bioactivity, as well as their mechanical properties. Whiskers were slightly Ca-deficient with Ca/P=1.60–1.65, with the preferred direction of growth along the c-axis. Variation of acetamide concentration did not affect the constitution, the crystallinity or the crystal growth habit.

Preparation, characterization and in vitro analysis of novel structured nanofibrous scaffolds for bone tissue engineering - Uncorrected Proof

J. Wang, X. Yu — 2010-02-08

Abstract: In a previous study, a three-dimensional nanofibrous spiral scaffold for bone tissue engineering was developed, which showed enhanced human osteoblast cell attachment, proliferation and differentiation compared with traditional cylinder scaffolds, owing to the incorporation of spiral structures and nanofiber. However, the application of these scaffolds to bone tissue engineering was limited by their weak mechanical strength. This limitation triggered the design for novel structured scaffolds with reinforced physical characteristics. In this study, spiral polycaprolactone (PCL) nanofibrous scaffolds were inserted into poly(lactide-co-glycolide) (PLGA) microsphere sintered tubular scaffolds to form integrated scaffolds to provide mechanical properties and bioactivity appropriate for bone tissue engineering. Four experiment groups were designed: PLGA cylinder scaffold; PLGA tubular scaffold; PLGA tubular scaffold with PCL spiral structured inner core; PLGA tubular scaffold with PCL nanofiber containing spiral structured inner core. The morphology, porosity and mechanical properties of the scaffolds were characterized. Furthermore, human osteoblastic cells were seeded on these scaffolds, and the cell attachment, proliferation, differentiation and mineralized matrix deposition on the scaffolds were evaluated. The integrated scaffolds had Young’s modulus 250–300MPa, and compressive strength 8–11MPa under uniaxial compression. With the addition of an inner highly porous insert to the tubular shell, human osteoblast cells seeded on the integrated scaffolds showed slightly higher cell proliferation, 20–25% more alkaline phosphatase expression and twofold higher calcium deposition than those on the cylinder and tubular scaffolds. Furthermore, compared with sintered PLGA cylinder scaffolds, the integrated scaffolds allowed better cellular infiltration Therefore, this design demonstrates great potential for integrated scaffolds in bone tissue engineering applications.

Influence of polymer content in Ca-deficient hydroxyapatite–polycaprolactone nanocomposites on the formation of microvessel-like structures - Corrected Proof

2010-02-08

Abstract: Calcium phosphate (CaP) ceramics are widely used in bone tissue engineering due to their good osteoconductivity. The mechanical properties of CaP can be modified by the addition of small volume fractions of biodegradable polymers such as polycaprolactone (PCL). Nevertheless, it is also important to evaluate how the polymer content influences cell–material or cell–cell interactions because of potential consequences for bone regeneration and vascularization. In this study we assessed the general biocompatibilty of Ca-deficient hydroxyapatite (CDHA)–PCL disks containing nominally 11 and 24% polycaprolactone using human umbilical vein endothelial cells and human primary osteoblasts. Confocal microscopy showed that both CDHA–PCL variants supported the growth of both cell types. In terms of the endothelial cells grown on CDHA–PCL nanocomposites with 24% PCL, an increased expression of the endothelial marker vWF compared to CDHA–PCL with 11% PCL was observed in real-time polymerase chain reaction analysis. In addition to monocultures, co-cultures of outgrowth endothelial cells, derived from peripheral blood, and primary osteoblasts were assessed as an example of a more complex test system for bone regeneration and vascularization. Constructs based on CDHA with different PCL contents were investigated with regard to the formation of microvessel-like structures induced by the co-culture process using confocal microscopy and quantitative image analysis. Furthermore, the osteogenic differentiation of the co-culture was assessed. As a result, more pre-vascular structures were observed after 1week on the CDHA–PCL disks with 24% PCL, whereas after 4weeks of culture the extent of microvessel-like structure formation was slightly higher on the CDHA with 11% PCL. In contrast to this, variation of PCL content had no effect on the osteogenic differentiation in the co-culture.

Mechanical and microstructural properties of polycaprolactone scaffolds with one-dimensional, two-dimensional, and three-dimensional orthogonally oriented porous architectures produced by selective laser sintering - Corrected Proof

Shaun Eshraghi, Suman Das — 2010-02-08

Abstract: This article reports on the experimental determination and finite element modeling of tensile and compressive mechanical properties of solid polycaprolactone (PCL) and of porous PCL scaffolds with one-dimensional, two-dimensional and three-dimensional orthogonal, periodic porous architectures produced by selective laser sintering (SLS). PCL scaffolds were built using optimum processing parameters, ensuring scaffolds with nearly full density (>95%) in the designed solid regions and with excellent geometric and dimensional control (within 3–8% of design). The tensile strength of bulk PCL ranged from 10.5 to 16.1MPa, its modulus ranged from 343.9 to 364.3MPa, and the tensile yield strength ranged from 8.2 to 10.1MPa. These values are consistent with reported literature values for PCL processed through various manufacturing methods. Across porosity ranged from 56.87% to 83.3%, the tensile strength ranged from 4.5 to 1.1MPa, the tensile modulus ranged from 140.5 to 35.5MPa, and the yield strength ranged from 3.2 to 0.76MPa. The compressive strength of bulk PCL was 38.7MPa, the compressive modulus ranged from 297.8 to 317.1MPa, and the compressive yield strength ranged from 10.3 to 12.5MPa. Across porosity ranged from 51.1% to 80.9%, the compressive strength ranged from 10.0 to 0.6MPa, the compressive modulus ranged from 14.9 to 12.1MPa, and the compressive yield strength ranged from 4.25 to 0.42MPa. These values, while being in the lower range of reported values for trabecular bone, are the highest reported for PCL scaffolds produced by SLS and are among the highest reported for similar PCL scaffolds produced through other layered manufacturing techniques. Finite element analysis showed good agreement between experimental and computed effective tensile and compressive moduli. Thus, the construction of bone tissue engineering scaffolds endowed with oriented porous architectures and with predictable mechanical properties through SLS is demonstrated.