Peptide-grafted poly(ethylene glycol) hydrogels support dynamic adhesion of endothelial progenitor cells

This study investigated the dynamic adhesion of endothelial progenitor cells (EPCs) to peptide-grafted poly(ethylene glycol) diacrylate (PEGDA) hydrogels and determined the relative ability of RGDS, REDV and YIGSRG peptides to reduce the velocity of EPC rolling. Circulating EPCs are key mediators of endothelium repair and have been shown to accelerate re-endothelialization, which is important in reducing the incidence of restenosis following stent placement and occlusion of small diameter vascular grafts. However, to exploit these capabilities for tissue engineering applications, more knowledge is needed about EPC binding to the vascular wall under shear and, in particular, whether the incorporation of peptide ligands into biomaterials can support the process of EPC rolling or maintain EPC adhesion. This study specifically examined one type of EPCs endothelial colony forming cells (ECFCs), based on their ability to be expanded in culture and differentiate into mature endothelial cells. The amount of grafted PEG–peptide was shown to be dependent on the concentration of PEG–peptide grafting solution photopolymerized onto the hydrogel surface. The ECFC strength of adhesion on PEG–RDGS grafted hydrogels exceeded 350 dyn cm^?2 for 85% of adherent cells. PEG–RGDS grafted hydrogels supported ECFC rolling, whereas ECFC velocity on the negative control PEG–RGES grafted hydrogels and on the “blank slate” PEGDA hydrogels was substantially higher than the cutoff velocity for cell rolling. The ECFC rolling velocity on PEG–RDGS grafted hydrogels depended on the shear rate; as shear rate was increased from 20 s^?1 to 120 s^?1, ECFC rolling velocity increased from 103 ± 3 μm s^?1 to 741 ± 28 μm s^?1. REDV and YIGSRG, which are known to preferentially support endothelial cell adhesion, also supported ECFC rolling. Interestingly, the rolling velocity of ECFCs on PEG–REDV grafted hydrogels was significantly lower than on PEG–YIGSRG or on PEG–RGDS grafted hydrogels. Understanding the dynamic adhesion of ECFCs to peptide-grafted hydrogels is the first step towards understanding the similarities and differences of EPCs from mature endothelial cells and improving the ability to sequester EPCs to biomaterial surfaces in order to promote intravascular re-endothelialization.     

Covalently immobilized RGD gradient on PEG hydrogel scaffold influences cell migration parameters

Understanding the influence of a controlled spatial distribution of biological cues on cell activities can be useful to design “cell instructive” materials, able to control and guide the formation of engineered tissues in vivo and in vitro. To this purpose, biochemical and mechanical properties of the resulting biomaterial must be carefully designed and controlled. In this work, the effect of covalently immobilized RGD peptide gradients on poly(ethylene glycol) diacrylate hydrogels on cell behaviour was studied. We set up a mechanical device generating gradients based on a fluidic chamber. Cell response to RGD gradients with different slope (0.7, 1 and 2 mM cm^?1) was qualitatively and quantitatively assessed by evaluating cell adhesion and, in particular, cell migration, compared to cells seeded on hydrogels with uniform distribution of RGD peptides. To evaluate the influence of RGD gradient and to exclude any concentration effect on cell response, all analyses were carried out in a specific region of the gradients which displayed the same average concentration of RGD (1.5 mM). Results suggest that cells recognize the RGD gradient and adhere onto it assuming a stretched shape. Moreover, cells tend to migrate in the direction of the gradient, as their speed is higher than that of cells migrating on hydrogels with a uniform distribution of RGD and increases by increasing RGD gradient steepness. This increment is due to an augmentation of bias speed component of the mean squared speed, that is, the drift of the cell population migrating on the anisotropic surface provided by the RGD gradient.     

Influence de la densité de peptides RGD greffés en surface de polyéthylène téréphtalate sur l’attachement des MC3T3

The aim of this study was to evaluate the impact of different densities on MC3T3 cells attachment onto polyethylene terephthalate (PET) film surfaces. Biomimetic modifications were performed by means of a three-step reaction procedure: creation of COOH functions onto PET surface, coupling agent grafting and finally immobilization of peptides. The originality of this work consist, in one hand on quantifying RGD peptides densities grafted onto PET, and on the other hand on studying MC3T3 cells responses after seeding on such biomimetic surfaces. After each functionnalization step, modifications were validated by several physicochemical techniques: X-Ray Photoelectron Spectroscopy permitted to prove the grafting and high-resolution β-imager coupled with use of radiolabelled amino acids served in evaluation of peptides densities. Moreover, this last technique permit us to ensure stability of binding between peptides and polymer. The efficiency of this new route for biomimetic modification of PET surface was demonstrated by measuring the adhesion at 15 hours of osteoblast like cells. Study of cellular comportment was realized by means of focal contact proteins (vinculin, actin) immunostaining.     

Dynamic cell-adhesive microenvironments and their effect on myogenic differentiation

Integrin-mediated cell adhesion plays a central role in cell behavior on biomaterial surfaces and influences various cell functions. Photoactivatable RGD adhesive peptides were used to investigate the effect of the density and time point of bioadhesive ligand presentation on cell adhesion, proliferation and differentiation. PEGylated self-assembled monolayers were functionalized with RGD and caged RGD ligands and seeded with C2C12 myoblasts. The cultures were irradiated at various time points between 1 and 48 h after cell seeding in order to increase RGD surface concentration at defined time points. Attachment, spreading and myogenic differentiation of C2C12 myoblasts strongly varied with the density of RGD at the surface. Proliferation and myogenesis were further regulated by the time point at which RGD was presented to the cell, reaching highest levels when RGD exposure occurred ≤6 h after cell seeding. These results provide fundamental insights in cell–biomaterial interactions of C2C12 myoblasts in terms of temporal integrin-mediated cell responses.     

Engineering high-density endothelial cell monolayers on soft substrates

This study demonstrates that a confluent monolayer of endothelial cells (ECs) can be tissue engineered on a soft substrate with a cell density and morphology that approximates in vivo conditions. We achieved formation of a confluent EC monolayer on polydimethylsiloxane (PDMS) elastomer by microcontact printing of fibronectin (FN) in a square lattice array of 3 μm diameter circular islands at a 6 μm pitch. Uniform coatings of FN or serum proteins on PDMS or on tissue-culture-treated polystyrene failed to support the equivalent EC density and/or confluence. The ECs on the FN micropatterned PDMS achieved a density of 1,536 ± 247 cells mm^?2, close to the 3,215 ± 336 cells mm^?2 observed in vivo from porcine pulmonary artery and significantly higher (2- to 5-fold) than EC density on other materials. The probable mechanism for enhanced EC adhesion, growth and density is increased focal adhesion (FA) formation between the ECs and the substrate. After 14 days culture, the micropatterned FN surface increased the average number of FAs per cell to 35 ± 10, compared to 7 ± 6 for ECs on PDMS uniformly coated with FN. Thus, microscale patterning of FN into FA-sized, circular islands on PDMS elastomer promotes the formation of EC monolayers with in vivo-like cell density and morphology.     

Mechanisms underlying the attachment and spreading of human osteoblasts: From transient interactions to focal adhesions on vitronectin-grafted bioactive surfaces

The features of implant devices and the reactions of bone-derived cells to foreign surfaces determine implant success during osseointegration. In an attempt to better understand the mechanisms underlying osteoblasts attachment and spreading, in this study adhesive peptides containing the fibronectin sequence motif for integrin binding (Arg-Gly-Asp, RGD) or mapping the human vitronectin protein (HVP) were grafted on glass and titanium surfaces with or without chemically induced controlled immobilization. As shown by total internal reflection fluorescence microscopy, human osteoblasts develop adhesion patches only on specifically immobilized peptides. Indeed, cells quickly develop focal adhesions on RGD-grafted surfaces, while HVP peptide promotes filopodia, structures involved in cellular spreading. As indicated by immunocytochemistry and quantitative polymerase chain reaction, focal adhesions kinase activation is delayed on HVP peptides with respect to RGD while an osteogenic phenotypic response appears within 24 h on osteoblasts cultured on both peptides. Cellular pathways underlying osteoblasts attachment are, however, different. As demonstrated by adhesion blocking assays, integrins are mainly involved in osteoblast adhesion to RGD peptide, while HVP selects osteoblasts for attachment through proteoglycan-mediated interactions. Thus an interfacial layer of an endosseous device grafted with specifically immobilized HVP peptide not only selects the attachment and supports differentiation of osteoblasts but also promotes cellular migration.     

Study of cellular dynamics on polarized CoCrMo alloy using time-lapse live-cell imaging

The physico-chemical processes and phenomena occurring at the interface of metallic biomedical implants and the body dictate their successful integration in vivo. Changes in the surface potential and the associated redox reactions at metallic implants can significantly influence several aspects of biomaterial/cell interactions such as cell adhesion and survival in vitro. Accordingly, there is a voltage viability range (voltages which do not compromise cellular viability of the cells cultured on the polarized metal) for metallic implants. We report on cellular dynamics (size, polarity, movement) and temporal changes in the number and total area of focal adhesion complexes in transiently transfected MC3T3-E1 pre-osteoblasts cultured on CoCrMo alloy surfaces polarized at the cathodic and anodic edges of its voltage viability range (?400 and +500 mV (Ag/AgCl), respectively). Nucleus dynamics (size, circularity, movement) and the release of reactive oxygen species (ROS) were also studied on the polarized metal at ?1000, ?400 and +500 mV (Ag/AgCl). Our results show that at ?400 mV, where reduction reactions dominate, a gradual loss of adhesion occurs over 24 h while cells shrink in size during this time. At +500 mV, where oxidation reactions dominate (i.e. metal ions form, including Cr⁶⁺), cells become non-viable after 5 h without showing any significant changes in adhesion behavior right before cell death. Nucleus size of cells at ?1000 mV decreased sharply within 15 min after polarization, which rendered the cells completely non-viable. No significant amount of ROS release by cells was detected on the polarized CoCrMo at any of these voltages.     

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

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

The promotion of chondrogenesis in adipose-derived adult stem cells by an RGD-chimeric protein in 3D alginate culture

The dynamic regulation of integrin-binding peptides is crucial for chondrogenic differentiation. Here, we revealed the feasibility for flexible modification of RGD by embedding a large molecular weight and slightly charged (isoelectric point, 6–6.25) RGD-chimeric protein (CBD–RGD) with cellulose-binding domain (CBD) in three dimensional (3D) alginate beads to evaluate the chondrogenesis of adipose-derived adult stem cells (ADAS). The binding of CBD–RGD with cells and its diffusion from alginate beads were studied on fluorescein isothiocyanate (FITC)-conjugated CBD–RGD. The increases in gene expression (Sox9, Aggrecan, fibronectin and collagen II), accumulation of chondrogenic matrices and decrease of collagen X gene expression during TGF-β3 induction were only observed for those beads containing 10 mg/g CBD–RGD initially, with 20.18 ± 0.73% of that released in a week. The contrary was observed for beads with CBD–RGD 20 mg/g initially and having higher persistence (only 8.6 ± 2.17% released in a week). The 10 mg/g CBD–RGD-mediated enhancement was demonstrated via the activation of integrin α5 and β1-dependent pathway, and especially related to the upregulation of Sox9 gene and the temporary block of fibronectin expression as well as sustained inhibition of RhoA activity in the early differentiation stage. Thus, we speculated that the dynamic mobility of CBD–RGD may account for the enhanced chondrogenesis. It was concluded that the CBD–RGD–alginate culture system promoted the chondrogenesis of mesenchymal stem cells coordinated with TGF-β3 induction in an RGD dose-dependent manner.     

Cyclo-(DfKRG) peptide grafting onto Ti-6Al-4V: physical characterization and interest towards human osteoprogenitor cells adhesion

In the present paper, specific interest has been devoted to the design of new hybrid materials associating Ti-6Al-4V alloy and osteoprogenitor cells through the grafting of two RGD containing peptides displaying a different conformation (linear RGD and cyclo-DfKRG) onto titanium surface. Biomimetic modification was performed by means of a three-step reaction procedure: silanization with APTES, cross-linking with SMP and finally immobilization of peptides thanks to thiol bonding. The whole process was performed in anhydrous conditions to ensure homogeneous biomolecules layout as well as to guarantee a sufficient amount of biomolecules grafted onto surfaces. The efficiency of this new route for biomimetic modification of titanium surface was demonstrated by measuring the adhesion between 1 and 24 h of osteoprogenitor cells isolated from HBMSC. Benefits of the as-proposed method were related to the high concentration of peptides grafted onto the surface (around 20 pmol/mm(2)) as well as to the capacity of cyclo-DfKRG peptide to interact with integrin receptors. Moreover, High Resolution beta-imager (using [(35)S]-Cys) has exhibited the stability of peptides grafted onto the surface when treated in harsh conditions.     

Increased expression of the intercellular adhesion molecule-1 (ICAM-1) on peripheral blood neutrophils in acute pancreatitis

PurposeConsidering the important role of neutrophils’ activation in the pathogenesis of acute pancreatitis (AP), the aim of our study was to evaluate the expression of leukocytes’ adhesion molecules in patients with AP.Patients/methodsThirty-five patients (16 women and 19 men; age 32–77 years, median 56 years) with AP were prospectively included into our study. The absolute number of leukocytes was estimated by haematologic analyser. Surface neutrophils antigens (CD) were assayed by the direct fluorescence method for whole blood, using a flow cytometer.ResultsAt the day 1, significant increase of ICAM-1 expression was found in patients with severe AP (S-AP) (7280 mm⁻³ vs 2850 mm⁻³ in healthy control; p < 0.05). In the days 2, 3 and 5 it sharply decreased and peaked again to 4860 mm⁻³ at the day 10. In patients with mild AP (M-AP), not significant elevation of ICAM-1 quickly returned to normal level. In both forms of AP, neutrophil CD62L (L-selectin) expression reached the highest level at the day 1 (8800 mm⁻³ and 9020 mm⁻³, respectively in M-AP and S-AP, in comparison to 3400 mm⁻³ in control; p < 0.05). Expression of CD69 (neutrophils’ marker of early activation) significantly increased in both M-AP and S-AP.ConclusionsWe have found an early and significant increase of peripheral blood neutrophil CD54/ICAM-1 expression, specific for S-AP but not for M-AP. It may provide a good marker predicting severe course of pancreatitis.     

The influence of poly(ethylene oxide) grafting via siloxane tethers on protein adsorption

Amphiphilic PEO–silanes (a–c) having siloxane tethers of varying lengths with the general formula α-(EtO)₃Si–(CH₂)₂–oligodimethylsiloxane_n-block-poly(ethylene oxide)₈–OCH₃ [n = 0 (a), n = 4 (b), and n = 13 (c)] were grafted onto silicon wafers and resistance to adsorption of plasma proteins was measured. Distancing the PEO segment from the hydrolyzable triethoxysilane [(EtO)₃Si] grafting group by a oligodimethylsiloxane tether represents a new method of grafting PEO chains to surfaces. Properties of surfaces grafted with a–c were compared to surfaces grafted with a traditional PEO–silane containing a propyl spacer [(EtO)₃Si–(CH₂)₃–poly(ethylene oxide)₈–OCH₃, PEO control]. As the siloxane tether length increased, chain density of PEO–silanes grafted onto oxidized silicon wafers decreased and hydrophobicity of the PEO–silane increased which led to a decrease in surface hydrophilicity. Despite decreased surface hydrophilicity, resistance to the adsorption of bovine serum albumin (BSA) increased in the order: PEO control < a < b ≈ c and to human fibrinogen (HF) increased in the order: PEO control < a < b < c.     

ACL reconstruction: Effect of bone dowel on tibial tunnel enlargement

PurposeTo evaluate prospectively if the impaction of a bone dowel in the tibial tunnel prevents the tunnels from enlarging beyond their original diameter.MethodsSeventeen patients underwent arthroscopically assisted ACL reconstruction with hamstring autologous graft. All patients underwent CT of the knee on the day of surgery, at 3 months and 12 months post-op.ResultsOn the day of surgery, the median cross-sectional areas of the tunnels were 77.0 and 79.0 mm², respectively at 15 mm and 20 mm from the tip of the posterior wall of the tunnel. At 3 months, the median cross-sectional areas of the tunnels were 70.0 and 65.0 mm², at 15 mm and 20 mm. At 12 months post-op, the median cross-sectional areas of the tunnels were 69.0 and 69.0 mm². The median enlargement of the tunnels between 3 months and 12 months post-op was 0.0 mm² at 15 mm and ? 2.0 mm² at 20 mm.ConclusionsThe impaction of an autologous bone dowel in the tibial tunnel during hamstring ACL reconstruction keeps the tunnels from enlarging beyond their original diameter, and there is no further enlargement of the tunnels after 3 months post-op.     

Morphometric characteristics of neutrophils stimulated by adhesion and hypochlorite

The aim of this work was to compare cell form, size and volume as well as the locomotor activity of polymorphonuclear leukocytes (PMNLs) stimulated by adhesion to glass and exposed to hypochlorous acid at non-toxic dose. After 20 min of adhesion to a glass surface, volume, cell surface area and projection area of PMNLs were equaled to 143.1 ± 21.4 μm³, 288.8 ± 28.8 μm² and 248.3 ± 32.3 μm², respectively. Projection area of PMNLs exposed to NaOCl was noticeably enlarged as compared with control samples. The cell volume of 20 min adherent cells exposed to NaOCl was enlarged in comparison with both control cells and 5 min adhered exposed to NaOCl cells. NaOCl exposure induced a degranulation of PMNLs as measured by lysozyme release. Granules could be found both above the cell surface and on the substratum near the cell. The S/V ratio for PMNLs increased (from 1.52 to 2.02 μm⁻¹) with the increasing of cell activation time. But at NaOCl addition the reverse tendency was observed (from 2.10 to 1.87 μm⁻¹). In cells exposed to NaOCl the redistribution and decrease of concentration of F-actin took place. This observation supports the hypothesis that the priming of PMNLs with hypochlorous acid modifies cell motility and morphology and reflects also on other functions.     

Ability of polyurethane foams to support cell proliferation and the differentiation of MSCs into osteoblasts

In bone tissue reconstruction, the use of engineered constructs created by mesenchymal stem cells (MSCs) that differentiate and proliferate into three-dimensional porous scaffolds is an appealing alternative to autologous and heterologous bone grafts. Scaffolds considered in this work are represented by polyurethane (PU) foams. Two PU foams (EC-1 and EC-2) were synthesized and characterized for morphology, mechanical properties and in vitro interaction with the osteoblast-like cell line MG63 and MSCs from human bone marrow. EC-1 and EC-2 showed similar densities (0.20 g cm^?3) with different morphologies: EC-1 showed a more homogeneous pore size (average Φ = 691 μm) and distribution, with a 35% open porosity, whereas EC-2 evidenced a wide range of pore dimension, with an average pore size of 955 μm and a 74% open porosity. The compressive properties of the two foams were similar in the dry condition and both showed a strong decrease in the wet condition. In vitro tests showed good MG63 cell proliferation, as confirmed by the results of the MTT assay and scanning electron microscopy (SEM) observations, with a higher cell viability on EC-2 foam 7 days post-seeding. In the experiments with MSCs, SEM observations showed the presence of an inorganic phase deposition starting day 7 onto EC-1, day 14 onto EC-2. The inorganic particles (CaP) deposition was much more evident onto the pore surface of both foams at day 30, indicating good differentiation of MSCs into osteoblasts. Both PU foams therefore appeared to stimulate cell adhesion and proliferation in vitro, sustaining the MSCs’ growth and differentiation into osteoblasts.     

Adhesion and Proliferation of Human Adipo-Stromal Cells for Two- or Three-Dimensional Poly(ethylene terephthalate) Substrates with or without RGD Immobilization

The adhesion and proliferation of human adipo-stromal cells was investigated for poly(ethylene terephthalete) (PET) films of two-dimensional (2D) substrate and non-woven fabrics of three-dimensional (3D) substrate after seeding at the same cell density and culturing at the same medium volume to surface area of the substrates ratio. When seeded by a static seeding method, more cells adhered on the film than on the non-woven fabric. However, the number ratio of cells proliferated to those initially adhered was similar. For the non-woven fabric, cell proliferation was enhanced by an agitated culture method. NaOH treatment introduced carboxyl groups into the surface of substrates, irrespective of the substrate type. Cell adhesion of a peptide (Arg-Gly-Asp, RGD) was chemically immobilized through the carboxyl groups on the non-woven fabric surface at a density of 10 pmol/cm². RGD immobilization significantly increased the number of cells adhered after the agitated seeding method, but did not affect the cell proliferation. Phosphorylation of focal adhesion kinase (FAK) was also enhanced by the RGD immobilization on the PET non-woven fabrics.     

Bioactive polymer grafting onto titanium alloy surfaces

Bioactive polymers bearing sulfonate (styrene sodium sulfonate, NaSS) and carboxylate (methylacrylic acid, MA) groups were grafted onto Ti6Al4V alloy surfaces by a two-step procedure. The Ti alloy surfaces were first chemically oxidized in a piranha solution and then directly subjected to radical polymerization at 70 °C in the absence of oxygen. The grafted surfaces were characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS) and the toluidine blue colorimetric method. Toluidine blue results showed 1–5 μg cm^?2 of polymer was grafted onto the oxidized Ti surfaces. Grafting resulted in a decrease in the XPS Ti and O signals from the underlying Ti substrate and a corresponding increase in the XPS C and S signals from the polymer layer. The ToF-SIMS intensities of the S^? and SO^? ions correlated linearly with the XPS atomic percent S concentrations and the ToF-SIMS intensity of the TiO₃H₂^? ion correlated linearly with the XPS atomic per cent Ti concentration. Thus, the ToF-SIMS S^?, SO^? and TiO₃H₂^? intensities can be used to quantify the composition and amount of grafted polymer. ToF-SIMS also detected ions that were more characteristic of the polymer molecular structure (C₆H₄SO₃^? and C₈H₇SO₃^? from NaSS, C₄H₅O₂^? from MA), but the intensity of these peaks depended on the polymer thickness and composition. An in vitro cell culture test was carried out with human osteoblast-like cells to assess the influence of the grafted polymers on cell response. Cell adhesion after 30 min of incubation showed significant differences between the grafted and ungrafted surfaces. The NaSS grafted surfaces showed the highest degree of cell adhesion while the MA-NaSS grafted surfaces showed the lowest degree of cell adhesion. After 4 weeks in vivo in rabbit femoral bones, bone was observed to be in direct contact with all implants. The percentage of mineralized tissue around the implants was similar for NaSS grafted and non-grafted implants (59% and 57%). The MA-NaSS grafted implant exhibited a lower amount of mineralized tissue (47%).     

Shape-dependent cell migration and focal adhesion organization on suspended and aligned nanofiber scaffolds

In the body, cells dynamically respond to chemical and mechanical cues from the extracellular matrix (ECM), yet precise mechanisms by which biophysical parameters (stiffness, topography and alignment) affect cell behavior remain unclear. Here, highly aligned and suspended multilayer polystyrene (PS) nanofiber scaffolds are used to study biophysical influences on focal adhesion complex (FAC) arrangement and associated migration behavior of mouse C2C12 cells arranged in specific shapes: spindle, parallel and polygonal. Furthermore, the role of cytoskeletal-altering drugs including blebbistatin, nocodazole and cytochalasin-D on FAC formation and migratory behavior is investigated. For the first time, this work reports that cells on suspended fiber networks, including cells with administered drugs, elongated along the fiber axes and developed longer (～ 4×) and more concentrated FAC clusters compared to cells on flat PS control substrates. Additionally, substrate designs which topographically restrict sites of cell attachment and align adhesions were found to promote higher migration speeds (spindle: 52 μm h^?1, parallel: 39 μm h^?1, polygonal: 25 μm h^?1, flat: 32 μm h^?1). This work demonstrates that suspended fiber topography-induced concentration of FACs along fiber axes generates increased migration potential as opposed to flat surfaces, which diffuse and randomly orient adhesions.     

Poly(ethylene glycol) analogs grafted with low molecular weight poly(ethylene imine) as non-viral gene vectors

A novel class of non-viral gene vectors consisting of low molecular weight poly(ethylene imine) (PEI) (molecular weight 800 Da) grafted onto degradable linear poly(ethylene glycol) (PEG) analogs was synthesized. First, a Michael addition reaction between poly(ethylene glycol) diacrylates (PEGDA) (molecular weight 258 Da) and d,l-dithiothreitol (DTT) was carried out to generate a linear polymer (PEG–DTT) having a terminal thiol, methacrylate and pendant hydroxyl functional groups. Five PEG–DTT analogs were synthesized by varying the molar ratio of diacrylates to thiols from 1.2:1 to 1:1.2. Then PEI (800 Da) was grafted onto the main chain of the PEG–DTTs using 1,1′-carbonyldiimidazole as the linker. The above reaction gave rise to a new class of non-viral gene vectors, (PEG–DTT)–g-PEI copolymers, which can effectively complex DNA to form nanoparticles. The molecular weights and structures of the copolymers were characterized by gel permeation chromatography, ¹H nuclear magnetic resonance and Fourier transform infrared spectroscopy. The size of the nanoparticles was <200 nm and the surface charge of the nanoparticles, expressed as the zeta potential, was between +20 and +40 mV. Cytotoxicity assays showed that the copolymers exhibited much lower cytotoxicities than high molecular weight PEI (25 kDa). Transfection was performed in cultured HeLa, HepG2, MCF-7 and COS-7 cells. The copolymers showed higher transfection efficiencies than PEI (25 kDa) tested in four cell lines. The presence of serum (up to 30%) had no inhibitory effect on the transfection efficiency. These results indicate that this new class of non-viral gene vectors may be a promising gene carrier that is worth further investigation.     

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

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