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ABSTRACT: OBJECTIVE: The aim of this study was to compare the early repair response of cartilage defects in trochlea (TR) and medial femoral condyle (MFC) at 2-3 weeks after bone marrow stimulation. DESIGN: Bilateral full-thickness cartilage defects were generated in central trochlear groove and medial femoral condyle of skeletally mature rabbits. Four subchondral perforations were made on each defect, either by microfracture to 2 mm deep, or by drilling to 2 mm or 6 mm deep. Rabbits were sacrificed either on Day 14 postoperatively or on Day 21. Defects were analyzed by histology, stereology, histomorphometry and micro-computed tomography. Intact femurs (N=4) served as controls. RESULTS: Stromal cell density recruitment was similar in all defects, irrespective of defect location and surgical techniques used. There was a robust appearance of chondrocytes at Day 21 in TR defects with significantly higher volume fraction of chondrocytes in TR compared to MFC (p=0.013). Chondrogenic foci were observed in marrow penetrating holes, with a significantly higher frequency and larger foci in TR versus MFC defects at Day 21 (p=0.043 and p=0.0014, respectively). Micro-CT analysis showed that deep drilling elicited significantly more mineralized bone fill compared to shallower perforations at 2 and 3 weeks repair (all at p≤0.0008). CONCLUSIONS: Bone marrow stimulation induced greater chondrogenesis in TR vs MFC defects in adult rabbits, with more chondrocytes and larger chondrogenic foci appearing in TR versus MFC on Day 21 post-operation.
Osteoarthritis and Cartilage 04/2013; · 3.90 Impact Factor
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ABSTRACT: Background/Aim: Multidrug resistance poses a serious challenge in cancer therapy. To address this problem, we designed and synthesized Adva-27a, a novel non-ester GEM-difluorinated C-glycoside derivative of podophyllotoxin.
Adva-27a activity was evaluated in a variety of assays including inhibition of topoisomerase IIα, cytotoxic activity in drug-sensitive and drug-resistant cancer cell lines, metabolic stability in human liver microsomes and pharmacokinetic properties in rats.
Adva-27a exhibited dose-dependent human topoisomerase IIα inhibitory activity and dose-dependent growth inhibitory activity in several drug-sensitive and two multidrug-resistant cancer cell lines. In the multidrug-resistant cell lines, MCF-7/MDR (breast cancer) and H69AR (small-cell lung cancer), Adva-27a was significantly more potent than etoposide. The metabolic stability of Adva-27a in human liver microsomes and its pharmacokinetic properties in rats were better than those of etoposide.
Our studies have identified Adva-27a as a novel topoisomerase II inhibitor with superior cytotoxic activity against multidrug-resistant human cancer cells and more desirable pharmacokinetic properties than etoposide.
Anticancer research 10/2012; 32(10):4423-32. · 1.73 Impact Factor
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ABSTRACT: The mechanical properties of mammalian cells are largely determined by their cytoskeletons (CSKs), which comprise several distinct but interacting cytoplasmic molecular networks. To examine the influence of the CSK on cell mechanical properties, we deformed several mammalian cell-types (L929, CHO, HEK293, and U937) in suspension using time-varying non-uniform electric fields. Confocal fluorescent microscopy was also used to visualize and semi-quantitatively analyze CSK dimensions. We found mechanical properties of individually deformed cells to depend on cortical actin (CA) thickness. U937 and HEK293 cells with thin CA were more easily deformed than CHO and L929 cells, which bore thicker CA. In additional experiments, we treated U937 cells with latrunculin-A (Lat-A) and acrylamide (ACR), drugs that disrupt microfilaments (MF) and intermediate-filaments (IF), respectively, in order to assess their effects on the CSK and on the cell mechanical properties. We fit strain data using either a power-law or a viscoelasticity model of compliance. Our results demonstrated that maximal strain values observed under identical loading conditions were determined by the structural integrity and thickness of CA in suspended cells. Young's modulus values of individually deformed cells that were estimated using a power-law model showed a linear dependence on cortical actin thickness.
Journal of biomechanics 09/2012; · 2.66 Impact Factor
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ABSTRACT: An overall decline in the availability of osteogenic precursor cells and growth factors in the bone marrow microenvironment have been associated with impaired bone formation and osteopenia in humans. The objective of the current study was to determine if transplantation of mesenchymal stromal cells (MSC) from a healthy, young donor mouse into an osteopenic recipient mouse could enhance osseointegration of a femoral implant. MSC harvested from normal young adult mice differentiated into bone forming osteoblasts when cultured on implant grade titanium surfaces ex vivo and promoted bone formation around titanium-coated rods implanted in the femoral canal of osteopenic recipient mice. Micro computed tomographic imaging and histological analyses showed more, better quality, bone in the femur that received the MSC transplant compared with the contra-lateral control femur that received carrier alone. These results provide pre-clinical evidence that MSC transplantation promotes peri-implant bone regeneration and suggest the approach could be used in a clinical setting to enhance bone regeneration and healing in patients with poor quality bone.
Journal of Orthopaedic Research 01/2012; 30(8):1183-9. · 2.81 Impact Factor
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ABSTRACT: Chitosan, a natural polymer, is a promising system for the therapeutic delivery of both plasmid DNA and synthetic small interfering RNA. Reports attempting to identify the optimal parameters of chitosan for synthetic small interfering RNA delivery were inconclusive with high molecular weight at high amine-to-phosphate (N:P) ratios apparently required for efficient transfection. Here we show, for the first time, that low molecular weight chitosan (LMW-CS) formulations at low N:P ratios are suitable for the in vitro delivery of small interfering RNA. LMW-CS nanoparticles at low N:P ratios were positively charged (ζ-potential ~20 mV) with an average size below 100 nm as demonstrated by dynamic light scattering and environmental scanning electron microscopy, respectively. Nanoparticles were spherical, a shape promoting decreased cytotoxicity and enhanced cellular uptake. Nanoparticle stability was effective for at least 20 hours at N:P ratios above two in a slightly acidic pH of 6.5. At a higher basic pH of 8, these nanoparticles were unravelled due to chitosan neutralization, exposing their polynucleotide cargo. Cellular uptake ranged from 50% to 95% in six different cell lines as measured by cytometry. Increasing chitosan molecular weight improved nanoparticle stability as well as the ability of nanoparticles to protect the oligonucleotide cargo from nucleases at supraphysiological concentrations. The highest knockdown efficiency was obtained with the specific formulation 92-10-5 that combines sufficient nuclease protection with effective intracellular release. This system attained >70% knockdown of the messenger RNA, similar to commercially available lipoplexes, without apparent cytotoxicity. Contrary to previous reports, our data demonstrate that LMW-CS at low N:P ratios are efficient and nontoxic polynucleotide delivery systems capable of transfecting a plethora of cell lines.
International Journal of Nanomedicine 01/2012; 7:1399-414. · 3.13 Impact Factor
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ABSTRACT: Glucagon like peptide 1 (GLP-1), a blood glucose homeostasis modulating incretin, has been proposed for the treatment of type 2 diabetes mellitus (T2DM). However, native GLP-1 pharmacokinetics reveals low bioavailability due to degradation by the ubiquitous dipeptydil peptidase IV (DPP-IV) endoprotease. In this study, the glucosamine-based polymer chitosan was used as a cationic polymer-based in vitro delivery system for GLP-1, DPP-IV resistant GLP-1 analogues and siRNA targeting DPP-IV mRNA. We found chitosans to form spherical nanocomplexes with these nucleic acids, generating two distinct non-overlapping size ranges of 141-283 nm and 68-129 nm for plasmid and siRNA, respectively. The low molecular weight high DDA chitosan 92-10-5 (degree of deacetylation, molecular weight and N:P ratio (DDA-Mn-N:P)) showed the highest plasmid DNA transfection efficiency in HepG2 and Caco-2 cell lines when compared to 80-10-10 and 80-80-5 chitosans. Recombinant native GLP-1 protein levels in media of transfected cells reached 23 ng/L while our DPP-IV resistant analogues resulted in a fivefold increase of GLP-1 protein levels (115 ng/L) relative to native GLP-1, and equivalent to the Lipofectamine positive control. We also found that all chitosan-DPP-IV siRNA nanocomplexes were capable of DPP-IV silencing, with 92-10-5 being significantly more effective in abrogating enzymatic activity of DPP-IV in media of silenced cells, and with no apparent cytotoxicity. These results indicate that specific chitosan formulations may be effectively used for the delivery of plasmid DNA and siRNA in a combination therapy of type 2 diabetes.
European journal of pharmaceutical sciences: official journal of the European Federation for Pharmaceutical Sciences 11/2011; 45(1-2):138-49. · 2.61 Impact Factor
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ABSTRACT: Transforming growth factor-β (TGF-β) is a multifunctional cytokine that regulates a wide variety of cellular processes including proliferation, differentiation, and extracellular matrix deposition. Dysregulation of TGF-β signaling is associated with several diseases such as cancer and tissue fibrosis. TGF-β signals through two transmembrane proteins known as the type I (TGFBR1) and type II (TGFBR2) receptors. The levels of these receptors at the cell surface are tightly regulated by several mechanisms, including degradation following recruitment of the E3 ubiquitin ligase SMAD ubiquitination regulatory factor (Smurf) 2 by SMAD7. In addition, TGF-β co-receptors can modulate TGF-β signaling receptor activity in a cell-specific manner. We have previously identified a novel TGF-β co-receptor, CD109, a glycosyl phosphatidylinositol (GPI)-anchored protein that negatively regulates TGF-β signaling. Despite CD109's potential relevance as a regulator of TGF-β action in vivo, the mechanisms by which CD109 regulates TGF-β signaling are still incompletely understood. Previously, we have shown that CD109 downregulates TGF-β signaling by promoting TGF-β receptor localization into the lipid raft/caveolae compartment and by enhancing TGF-β receptor degradation. Here, we demonstrate that CD109 enhances SMAD7/Smurf2-mediated degradation of TGFBR1 in a ligand-dependent manner. Moreover, we show that CD109 regulates the localization and the association of SMAD7/Smurf2 with TGFBR1. Finally, we demonstrate that CD109's inhibitory effect on TGF-β signaling and responses require SMAD7 expression and Smurf2 ubiquitin ligase activity. Taken together, these results suggest that CD109 is an important regulator of SMAD7/Smurf2-mediated degradation of TGFBR1.
Journal of Cellular Biochemistry 09/2011; 113(1):238-46. · 2.87 Impact Factor
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ABSTRACT: Subchondral drilling and microfracture are bone marrow stimulation techniques commonly used for the treatment of cartilage defects. Few studies to date have examined the technical variants which may influence the success of the cartilage repair procedures. This study compared the effect of hole depth (6 mm vs. 2 mm) and hole type (drill vs. microfracture) on chondral defect repair using a mature rabbit model. Results from quantitative histomorphometry and histological scoring showed that deeper versus shallower drilling elicited a greater fill of the cartilage defect with a more hyaline character in the repair matrix indicated by significant improvement (p = 0.021) in the aggregate measure of increased cartilage defect fill, increased glycosaminoglycan and type II collagen content and reduced type I collagen content of total soft repair tissue. Compared to microfracture at the same 2 mm depth, drilling to 2 mm produced a similar quantity and quality of cartilage repair (p = 0.120) according to the aggregate indicator described above. We conclude that the depth of bone marrow stimulation can exert important influences on cartilage repair outcomes.
Journal of Orthopaedic Research 08/2011; 29(8):1178-84. · 2.81 Impact Factor
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ABSTRACT: Microfracture and drilling are bone marrow-stimulation techniques that initiate cartilage repair by providing access to cell populations in subchondral bone marrow. This study examined the effect of hole depth and of microfracture versus drilling on subchondral bone repair and cartilage repair in full-thickness chondral defects.
Repaired subchondral bone does not reconstitute its native structure and exhibits atypical morphologic features. Drilling deeper induces greater bone remodeling and is related to improved cartilage repair.
Controlled laboratory study.
Trochlear cartilage defects debrided of the calcified layer were prepared bilaterally in 16 skeletally mature rabbits. Drill holes were made to a depth of 2 mm or 6 mm and microfracture holes to 2 mm. Animals were sacrificed 3 months postoperatively, and joints were scanned by micro-computed tomography before histoprocessing. Bone repair was assessed with a novel scoring system and by 3-dimentional micro-computed tomography and compared with intact controls. Correlation of subchondral bone features to cartilage repair outcome was performed.
Although surgical holes were partly repaired with mineralized tissue, atypical features such as residual holes, cysts, and bony overgrowth were frequently observed. For all treatment groups, repair led to an average bone volume density similar to that of the controls but the repair bone was more porous and branched as shown by significantly higher bone surface area density and connectivity density. Deeper versus shallower drilling induced a larger region of repairing and remodeling subchondral bone that positively correlated with improved cartilage repair.
Incomplete reconstitution of normal bone structure and continued remodeling occurred in chondral defects 3 months after bone marrow stimulation. Deep drilling induced a larger volume of repairing and remodeling bone, which appeared beneficial for chondral repair.
Bone marrow stimulation does not reconstitute normal bone structure. Strategies that increase subchondral bone involvement in marrow stimulation could further benefit cartilage repair.
The American journal of sports medicine 05/2011; 39(8):1731-40. · 3.61 Impact Factor
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ABSTRACT: The optimal ratio of the polycation's amine to DNA phosphate group (N:P) for efficient polymer-based transfection always employs excess polycation versus DNA. Most of the excess polycation remains free in solution, unassociated with the polyplexes, but is essential for efficient transfection. The mechanism by which excess polycation increases transfection efficiency is not identified. We hypothesised that excess chitosan facilitates intracellular lysosomal escape of the polyplexes. We highlight here the essential role of excess chitosan by rescuing poorly transfecting low N:P ratio polyplexes, by adding free chitosan before or after polyplex addition to cells. We examined polyplex uptake, the kinetics of rescue, intracellular trafficking, and the effects of lysosomotropic agents. We found the facilitating role of excess chitosan to be downstream of cellular uptake. Live-cell confocal quantification of intracellular trafficking revealed prolonged colocalisation of low N:P polyplexes within lysosomes, compared to shorter residence times for both rescued or N:P 5 samples, followed by observation of free pDNA in the cytosol. These data demonstrate that excess polycation mediates enhanced transfection efficiency by promoting the release of polyplexes from the endo-lysosomal vesicles, revealing a critical intracellular barrier overcome by excess polycation and suggesting possible avenues for further optimisation of polymer-based gene delivery.
Biomaterials 03/2011; 32(20):4639-46. · 7.40 Impact Factor
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ABSTRACT: Transforming growth factor-β (TGF-β) is implicated in numerous pathological disorders, including cancer and mediates a broad range of biological responses by signaling through the type I and II TGF-β receptors. Internalization of these receptors via the clathrin-coated pits pathway facilitates SMAD-mediated signaling, whereas internalization via the caveolae pathway is associated with receptor degradation. Thus, molecules that modulate receptor endocytosis are likely to play a critical role in regulating TGF-β action. We previously identified CD109, a GPI-anchored protein, as a TGF-β co-receptor and a negative regulator of TGF-β signaling. Here, we demonstrate that CD109 associates with caveolin-1, a major component of the caveolae. Moreover, CD109 increases binding of TGF-β to its receptors and enhances their internalization via the caveolae. In addition, CD109 promotes localization of the TGF-β receptors into the caveolar compartment in the presence of ligand and facilitates TGF-β-receptor degradation. Thus, CD109 regulates TGF-β receptor endocytosis and degradation to inhibit TGF-β signaling. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.
Biochimica et Biophysica Acta 02/2011; 1813(5):742-53. · 4.66 Impact Factor
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ABSTRACT: Micro-computed tomography can be used to analyze subchondral bone features below treated cartilage defects in animal models. However, standardized methods for generating precise three-dimensional (3D) volumes of interest (VOI) below curved articular surfaces are lacking. The aims of this study were to develop standardized 3D VOI models adapted to the curved articular surface, and to characterize the subchondral bone specifically below a cartilage defect zone in intact and defect femoral trochlea. Skeletally mature rabbit distal femurs (N = 8 intact; N = 6 with acute debrided and microdrilled trochlear defects) were scanned by micro-computed tomography. Bone below the defect zone (3.5 mm width, 3.6 mm length, 1 mm deep) was quantified using simple geometric rectangular VOIs, and an optimized 3D VOI model with an adapted surface curvature, the Rectangle with Adapted Surface (RAS) model. In addition, a 250-μm-thick Curved-RAS model analyzed bone at three discrete subchondral levels. Simple geometric VOIs failed to analyze ~17% of the tissue volume, mainly near the top of the curved trochlear ridges. The RAS models revealed that after debridement and drilling, only 31% of the original bone remained within the VOI and bone loss was mainly accounted for by surgical debridement. Adapted surface VOIs are better than simple geometric VOI shapes for quantifying structural features of subchondral bone below a curved articular surface. Structural differences between the bone plate and cancellous bone were best captured using the smaller, depth-dependent Curved-RAS model.
Tissue Engineering Part C Methods 01/2011; 17(4):475-84. · 4.64 Impact Factor
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ABSTRACT: Asymmetrical flow field-flow fractionation (AF4) coupled with UV-vis spectrophotometry, multiangle light scattering (MALS), and dynamic light scattering (DLS) detection was used to analyze dispersions of DNA/rhodamine B labeled chitosan (Ch-rho) complexes frequently used as gene delivery vectors. The method yielded, in a single experiment, important characteristics of the complexes, such as their hydrodynamic radius, size distribution, conformation, composition, and the amount of free Ch-rho in the dispersions. Samples for analysis were obtained by varying experimental parameters known to influence the transfection efficiency of DNA/chitosan complexes, including the DNA concentration at mixing (82-164 μg/mL), the ratio of chitosan amino groups to DNA phosphate groups (3 ≤ N/P ratio ≤ 15), the chitosan molecular weight (10-76 kDa), and its degree of deacetylation. In all preparations, DNA/Ch-rho complexes had hydrodynamic radii ranging from 15 to 160 nm. Both the DNA concentration and the Ch-rho molecular weight influence the size distribution of the complexes: a greater fraction of large particles was detected in dispersions prepared with the most concentrated DNA solution or the Ch-rho of highest molar mass. All dispersions contained free Ch-rho in solution. The free Ch-rho content ranged from 53 to 92% of the total Ch-rho concentration in dispersions prepared with N/P ratios from 3 to 15, respectively, implying that the N/P ratio of the complexes ranged from 1.3 to 1.6 in all samples. The accuracy of the free Ch-rho determination by AF4/UV-vis/MALS+DLS was confirmed by an independent method involving (1) ultracentrifugation of the dispersions prepared with unlabeled chitosan and (2) analysis of the supernatant by the Orange II dye depletion method. This study demonstrates the ability of AF4/UV-vis/MALS+DLS to provide a complete physicochemical characterization of DNA/polycation complexes used in nonviral gene delivery.
Analytical Chemistry 11/2010; 82(23):9636-43. · 5.86 Impact Factor
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ABSTRACT: The transfection efficiency (TE) of chitosan-plasmid DNA (pDNA) polyplexes can be critically modulated by the polymer degree of deacetylation (DDA) and molecular weight (MW). This study was performed to test the hypothesis that the TE dependence on chitosan MW and DDA is related to the polyplex stability, hence their intracellular decondensation/unpacking kinetics. Major barriers to nonviral gene transfer were studied by image-based quantification. Although uptake increased with increased DDA, it did not appear to be a structure-dependent process affecting TE, nor was nuclear entry. Colocalization analysis showed that all chitosans trafficked through lysosomes with similar kinetics. Fluorescent resonant energy transfer (FRET) analysis revealed a distinct relationship between TE and polyplex dissociation rate. The most efficient chitosans showed an intermediate stability and a kinetics of dissociation, which occurred in synchrony with lysosomal escape. In contrast, a rapid dissociation before lysosomal escape was found for the inefficient low DDA chitosan whereas the highly stable and inefficient complex formed by a high MW and high DDA chitosan did not dissociate even after 24 hours. This study identified that the kinetics of decondensation in relation to lysosomal escape was a most critical structure-dependent process affecting the TE of chitosan polyplexes.
Molecular Therapy 10/2010; 18(10):1787-95. · 6.87 Impact Factor
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ABSTRACT: The development of new strategies for protein immobilization to control cell adhesion, growth and differentiation is of prime interest in the field of tissue engineering. Here we propose a versatile approach based on the interaction between two de novo designed peptides, Ecoil and Kcoil, for oriented immobilization of epidermal growth factor (EGF) on polyethylene terephthalate (PET) films. After amination of PET surfaces by ammonia plasma treatment, Kcoil peptides were covalently grafted in an oriented fashion using succinimidyl 6-[30-(2-pyridyldithio)-propionamido] hexanoate (LC-SPDP) linker, and the Kcoil-functionalized films were characterized by X-ray photoelectron spectroscopy (XPS). Bioactivity of Ecoil-EGF captured on Kcoil-functionalized PET via coiled-coil interactions was confirmed by EGF receptor phosphorylation analysis following A-431 cell attachment. We also demonstrated cell biological effects where tethered EGF enhanced adhesion, spreading and proliferation of human corneal epithelial cells compared to EGF that was either physically adsorbed or present in solution. Tethered EGF effects were most likely linked to the prolonged activation of both mitogen-activated protein kinase and phosphoinositidine 3-kinase pathways. Taken together, our results indicate that coiled-coil-based oriented immobilization is a powerful method to specifically tailor biomaterial surfaces for tissue engineering applications.
Biomaterials 09/2010; 31(27):7021-31. · 7.40 Impact Factor
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ABSTRACT: In vitro electromechanical and biomechanical testing of articular cartilage provide critical information about the structure and function of this tissue. Difficulties obtaining fresh tissue and lengthy experimental testing procedures often necessitate a storage protocol, which may adversely affect the functional properties of cartilage. The effects of storage at either 4°C for periods of 6 days and 12 days, or during a single freeze-thaw cycle at -20°C were examined in young bovine cartilage. Non-destructive electromechanical measurements and unconfined compression testing on 3 mm diameter disks were used to assess cartilage properties, including the streaming potential integral (SPI), fibril modulus (Ef), matrix modulus (Em), and permeability (k). Cartilage disks were also examined histologically. Compared with controls, significant decreases in SPI (to 32.3±5.5% of control values, p<0.001), Ef (to 31.3±41.3% [corrected] of control values, p=0.046), Em (to 6.4±8.5% of control values, p<0.0001), and an increase in k (to 2676.7±2562.0% of control values, p=0.004) were observed at day 12 of refrigeration at 4°C, but no significant changes were detected at day 6. A trend toward detecting a decrease in SPI (to 94.2±6.2% of control values, p=0.083) was identified following a single freeze-thaw cycle, but no detectable changes were observed for any biomechanical parameters. All numbers are mean±95% confidence interval. These results indicate that fresh cartilage can be stored in a humid chamber at 4°C for a maximum of 6 days with no detrimental effects to cartilage electromechanical and biomechanical properties, while one freeze-thaw cycle produces minimal deterioration of biomechanical and electromechanical properties. A comparison to literature suggested that particular attention should be paid to the manner in which specimens are thawed after freezing, specifically by minimizing thawing time at higher temperatures.
Journal of Biomechanical Engineering 06/2010; 132(6):064502. · 1.90 Impact Factor
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ABSTRACT: Primary chondrocytes cultured in agarose can escape the gel, accumulate at the interface between agarose and the culture medium, and form an outgrowing tissue. These outgrowths can appear as voluminous cartilage-like nodules that have never been previously investigated. In the present study, bovine articular chondrocytes from three age groups (fetal, young adult, aged) were seeded and cultured in agarose to test the hypothesis that hyaline-like cartilage outgrowths develop at the interface by appositional growth, in an age-dependant manner. Macroscopic appearance, cell content, cell division, cytoskeletal morphology, and extracellular matrix (ECM) composition were analyzed. Fetal chondrocytes produced a fibrous interfacial tissue while aged chondrocytes produced ECM-poor cell clusters. In contrast young adult chondrocytes produced large cartilaginous outgrowths, rich in proteoglycan and collagen II, where cells in the central region displayed a chondrocyte morphology. Cell proliferation was confined to the peripheral edge of these outgrowths, where elongated cell morphology, cell-cell contacts, and cell extensions toward the culture medium were seen. Thus these voluminous cartilaginous outgrowths formed in an appositional growth process and only for donor chondrocytes from young adult animals. This system offers an interesting ability to proliferate chondrocytes in a manner that results in a chondrocyte morphology and a cartilaginous ECM in central regions of the outgrowing tissue. It also provides an in vitro model system to study neocartilage appositional growth.
Connective tissue research 06/2010; 51(3):216-23. · 1.55 Impact Factor
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ABSTRACT: We describe a planar, micro-fabricated device for generating fringing non-uniform electric fields. We used it to measure the mechanical properties of individual mammalian cells in suspension by deforming them in time-varying, non-uniform electric fields. Electrical stresses generated by the planar microelectrodes were used to trap and stretch cells, while cell deformation was observed using optical microscopy. Two distinct cell types were compared after fitting strain data with a three-parameter 'standard linear solid' model of visco-elasticity, and with a two-parameter power-law method. Chinese hamster ovary (CHO) cells were approximately twice as stiff as U937 human promonocytes, and CHO cells displayed an elastic behaviour with recovery of initial shape, while U937 strain data bore witness to plastic deformation. Our results demonstrate that electrical stresses generated by micro-fabricated electrodes permit mechanical characterization of distinct mammalian cell types.
Journal of Micromechanics and Microengineering 05/2010; 20(6):065007. · 2.11 Impact Factor
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ABSTRACT: This study was designed to systematically evaluate the influence of pH and serum on the transfection process of chitosan-DNA complexes, with the objective of maximizing their efficiency. The hydrodynamic diameter of the complexes, measured by dynamic light scattering (DLS), was found to increase with salt and pH from 243 nm in water to 1244 nm in PBS at pH 7.4 and aggregation in presence of 10% serum. The cellular uptake of complexes into HEK 293 cells assessed by flow cytometry and confocal fluorescent imaging was found to increase at lower pH and serum. Based on these data, new methodology were tested and high levels of transfection (>40%) were achieved when transfection was initiated at pH 6.5 with 10% serum for 8-24 h to maximize uptake and then the media was changed to pH 7.4 with 10% serum for an additional 24-40 h period. Cytotoxicity of chitosan/DNA complexes was also considerably lower than Lipofectamine. Our study demonstrates that the evaluation of the influence of important parameters in the methodology of transfection enables the understanding of crucial physicochemical and biological mechanisms which allows for the design of methodologies maximising transgene expression.
Molecular Biotechnology 05/2010; 46(2):182-96. · 2.17 Impact Factor
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ABSTRACT: The composition of samples obtained upon complexation of DNA with chitosan was analyzed by asymmetrical flow field flow fractionation (AF4) with online UV-visible, multiangle light scattering (MALS), and dynamic light scattering (DLS) detectors. A chitosan labeled with rhodamine B to facilitate UV-vis detection of the polycation was complexed with DNA under conditions commonly used for transfection (chitosan glucosamine to DNA phosphate molar ratio of 5). AF4 analysis revealed that 73% of the chitosan-rhodamine remained free in the dispersion and that the DNA/chitosan complexes had a broad size distribution ranging from 20 to 160 nm in hydrodynamic radius. The accuracy of the data was assessed by comparison with data from batch-mode DLS and scanning electron microscopy. The AF4 combined with DLS allowed the characterization of small particles that were not detected by conventional batch-mode DLS. The AF4 analysis will prove to be an important tool in the field of gene therapy because it readily provides, in a single measurement, three important physicochemical parameters of the complexes: the amount of unbound polycation, the hydrodynamic size of the complexes, and their size distribution.
Biomacromolecules 02/2010; 11(3):549-54. · 5.48 Impact Factor
