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%).     

Novel cell patterning using microheater-controlled thermoresponsive plasma films

A novel approach is reported for cell patterning based on addressable microheaters and a poly(N-isopropyl acrylamide) (pNIPAM) themoresponsive coating. This thermoresponsive coating is created by a radio frequency NIPAM plasma and is denoted as plasma polymerized NIPAM (ppNIPAM). Films of ppNIPAM with a good retention of monomer side-chain functionality are produced using low-power continuous plasma deposition. Cell adhesion and cell detachment tests indicate that the surface switches between adhesive and nonadhesive behaviors as a function of temperature. The use of a photolithographically fabricated microheater array allows the ppNIPAM transition to occur spatially under the control of individual heaters. This localized change in the surface adhesive behavior is used to direct site-specific cell attachment. Patterned adhesion of two types of cells has been visualized on the array through fluorescent markers. Applications for diagnostic devices, cell-based sensors, tissue engineering, and cell transfection are envisioned.     

Venous shear stress enhances platelet mediated staphylococcal adhesion to artificial and damaged biological surfaces

We investigated the role of blood components in the adhesion of staphylococci to biological and artificial surfaces under well-defined flow conditions by using the Cone and Plate(let) Analyzer. An enzyme-linked immunosorbent assay-like binding assay with biotinylated bacteria determined the extent of bacterial adhesion to subendothelial extracellular matrix (ECM), polystyrene (PS) and adult bovine aortic endothelial (ABAE) cell monolayer. The presence of adsorbed plasma proteins on PS and ECM did not increase and in some cases reduced staphylococcal adhesion under flow conditions (200s(-1)). However, their presence on ABAE cells increased bacterial adhesion but to a level still lower than the adhesion to PS and ECM. In contrast, adhered platelets significantly increased staphylococcal adhesion to both PS and ECM, but did not affect the adhesion to ABAE cells. Furthermore, bacterial adhesion to the platelets coated ECM and PS under flow conditions (200s(-1)) was increased by 1.4 to 2.6-fold compare to static conditions. The platelet-enhanced bacterial adhesion was markedly inhibited by blockade of the platelet GPIb receptor. In conclusion, staphylococcal extensive adhesion to ECM and PS surfaces is increased by venous flow and mediated by surface adhered activated platelets via a GPIb dependent mechanism. On the other hand, ABAE cells demonstrated limited bacterial adhesion that is mediated by adsorbed plasma proteins. Our results suggest that under physiological venous flow conditions the intact vessel wall is less prone for bacterial adhesion than damaged vessel wall.     

Covalent functionalization of NiTi surfaces with bioactive peptide amphiphile nanofibers

Surface modification enables the creation of bioactive implants using traditional material substrates without altering the mechanical properties of the bulk material. For applications such as bone plates and stents, it is desirable to modify the surface of metal alloy substrates to facilitate cellular attachment, proliferation, and possibly differentiation. In this work we present a general strategy for altering the surface chemistry of nickel-titanium (NiTi) shape memory alloy in order to covalently attach self-assembled peptide amphiphile (PA) nanofibers with bioactive functions. Bioactivity in the systems studied here includes biological adhesion and proliferation of osteoblast and endothelial cell types. The optimized surface treatment creates a uniform TiO(2) layer with low levels of Ni on the NiTi surface, which is subsequently covered with an aminopropylsilane coating using a novel, lower temperature vapor deposition method. This method produces an aminated surface suitable for covalent attachment of PA molecules containing terminal carboxylic acid groups. The functionalized NiTi surfaces have been characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectroscopy (ToF-SIMS), and atomic force microscopy (AFM). These techniques offer evidence that the treated metal surfaces consist primarily of TiO(2) with very little Ni, and also confirm the presence of the aminopropylsilane overlayer. Self-assembled PA nanofibers presenting the biological peptide adhesion sequence Arg-Gly-Asp-Ser are capable of covalently anchoring to the treated substrate, as demonstrated by spectrofluorimetry and AFM techniques. Cell culture and scanning electron microscopy (SEM) demonstrate cellular adhesion, spreading, and proliferation on these functionalized metal surfaces. Furthermore, these experiments demonstrate that covalent attachment is crucial for creating robust PA nanofiber coatings, leading to confluent cell monolayers.     

基质刚度调节细胞-细胞外基质间黏附对肿瘤细胞迁移影响的模型研究

目的 研究细胞外基质(extracellular matrix,ECM)刚度对细胞和ECM间黏附及肿瘤迁移的影响.方法 建立基于网状波茨模型(cellular Potts model,CPM),模拟肿瘤细胞生长与细胞间免疫反馈过程,观察细胞力学行为改变对细胞-ECM黏附的影响状况,分析不同ECM下肿瘤细胞迁移的变化.结...     

两种方法分离子宫内膜干细胞的比较

背景：目前国内外尚无统一方法分离培养子宫内膜干细胞,实验重复性较差。 目的：寻找一种方便、高效、科学的体外分离子宫内膜干细胞的方法。 方法：采用混合法(机械和酶消化相结合)及胶原酶Ⅰ法分离子宫内膜干细胞,比较两种方法分离所得活细胞数及细胞生长周期的差异;并采用免疫细胞化学法和RT-PCR法检测CD90和CD117的表达情况。 结果与结论：经过15 d的原代培养后,子宫内膜干细胞形态呈圆梭形,细胞贴壁平铺生长,具有成纤维细胞形态,细胞排列无极性;细胞化学染色及RT-PCR法均检测到干细胞标志物CD90和CD117表达,证明培养的细胞具有干细胞特性。混合法获得的活细胞数多于胶原酶Ⅰ法,其差异有显著性意义 (P < 0.01),两种方法分离所得的细胞具有相同的生长周期。结果说明混合法是一种方便、高效、科学的体外分离子宫内膜干细胞的方法。     

Subpopulations of thyroid autoantibody secreting lymphocytes in Graves'' and Hashimoto thyroid glands.

Lymphocytes isolated from Graves' and Hashimoto thyroid tissue by enzymatic (dispase) digestion or mechanical disaggregation were markedly different in terms of their ability to synthesize thyroid autoantibodies in culture. Dispase digestion, followed by removal of thyroid follicular cells, gave a lymphocyte population with a high T:B cell ratio (6:1). However, the ability of these cell suspensions to synthesize microsomal (Mic) and thyroglobulin (Tg) antibodies spontaneously was significantly increased compared with lymphoid suspensions isolated by mechanical means. Spontaneous synthesis of thyroid autoantibodies was not markedly enhanced in cell suspensions prepared from patients' lymph node tissue by digestion compared with mechanical disaggregation. Further, Mic and Tg antibody production by thyroid lymphocytes prepared using dispase was inhibited by pokeweed mitogen (PWM) whereas in most cases suspensions prepared from the same tissues by mechanical dispersion synthesized low or undetectable levels of autoantibodies whether PWM was present or absent. Digestion of tissue debris remaining after mechanical removal of lymphocytes gave suspensions which had an increased proportion of suppressor/cytotoxic T cells compared with suspensions produced mechanically or by digestion alone; however, in terms of spontaneous autoantibody synthesis and PWM induced inhibition, these suspensions were similar to these obtained by digestion alone. It would therefore seem that enzymatic digestion of thyroid tissue resulted in the isolation of a lymphoid population which was different from that extracted by mechanical disaggregation. The digestion process appears to permit the recovery of lymphocytes closely associated with thyroid follicular cells and our studies suggest that it is this population which makes the major contribution to autoantibody synthesis.     

β1-integrin-mediated dynamic adhesion of colon carcinoma cells to extracellular matrix under laminar flow

To resist substantial wall shear stress exerted by blood flow metastasizing colon carcinoma cells have to form adhesive contacts with endothelial cells and subendothelial extracellular matrix (ECM). At secondary sites tumor cells have to stabilize these initial adhesive interactions to prevent detachment and recirculation. Previously we found that adhesion of colon carcinoma cells to ECM components under static conditions is mediated, in part, by various ₁-integrins. Since other malignant cells possess adhesive properties that are different under static and dynamic conditions, we analyzed human colon carcinoma cell adhesion under flow by decreasing the flow (wall shear stress, WSS) of cell suspensions and allowing cells to interact with collagen-coated surfaces in a laminar flow chamber. HT-29 colon carcinoma cells were used to study wall shear adhesion threshold (WSAT), dynamic adhesion rate (DAR) and adhesion stabilization rate (ASR). DAR was determined after a low flow period using a WSS set at 50% of WSAT. ASR was calculated 60 sec after reestablishment of high WSS. Glass slides were coated with collagen I (C I) or bovine serum albumin (BSA, negative control). In some experiments cells were pretreated with function-blocking anti-₁ or nonspecific IgG. Rolling of cells occurred on C I- and BSA-coated surfaces at high WSS. By decreasing WSS cell sticking without definite adhesion was found, and cells stuck to BSA at WSS lower than that found for C I. Further decreasing WSS below WSAT enabled stable cell adhesion to C I, but only a few cells adhered to BSA. ASR was found to be 73% of primarily adherent cells (to C I). Pretreatment with anti-₁ did not affect cell rolling but did inhibit cell sticking and adhesion completely, whereas nonspecific IgG was without effect. Activation of PKC using phorbol ester resulted in an increase of adhesive interactions under dynamic and static conditions, whereas its inhibition reduced adhesion. Adhesive interactions of HT-29 colon carcinoma cells with ECM-coated surfaces under laminar flow conditions occurred in various steps: (1) rolling, (2) sticking or initial adhesion, and (3) stabilization of adhesion. Under shear flow rolling of tumor cells on ECM-coated surfaces appeared to be mediated mainly by physical/mechanical and nonspecific surface-cell membrane interactions, whereas stabilized adhesion to ECM was specifically mediated by ₁-integrin binding to ECM components. PKC seems to be involved in the regulation of adhesion stabilization under static and flow conditions.     

Substratum roughness alters the growth, area, and focal adhesions of epithelial cells, and their proximity to titanium surfaces

Epithelial (E) cells were cultured on smooth tissue culture plastic (TCP), TCP-Ti, polished Ti (P), and rough grit-blasted Ti (B), acid-etched Ti (AE), and grit-blasted and acid-etchedTi (SLA) surfaces and their growth, area, adhesion, and membrane-Ti proximity assessed. Rough surfaces decreased the growth of E cells compared to smooth surfaces in cultures up to 28 days. In general rough surfaces decreased the spreading of E cells as assessed by their area with the most pronounced affect for the SLA surface. On the other hand, the strength of E cells adhesion as inferred by immunofluorescence staining of vinculin in focal adhesions indicated that E cells formed more and larger focal adhesions on the smooth P surface compared to the rougher AE surface. As this finding indicates a stronger adhesion to smooth surfaces, it is likely that E cells on rough surfaces are more susceptible to mechanical removal. An immunogold labeling method was developed to visualize focal adhesions using back-scattered electron imaging with a scanning electron microscope (SEM). On rough surfaces focal adhesions were primarily localized on to the ridges rather than the valleys and the cells tended to bridge over the valleys. Transmission electron microscopy (TEM) measurements of membrane proximity to the Ti surface indicated that average distance of cell to the Ti increased as the Ti surface roughness increased. Therefore, the size and shape of surface features are important determinants of epithelial adhesive behavior and epithelial coverage of rough surfaces would be difficult to attain if such surfaces become exposed.     

Extracellular matrix production by human osteoblasts cultured on biodegradable polymers applicable for tissue engineering

The nature of the extracellular matrix (ECM) is crucial in regulating cell functions via cell-matrix interactions, cytoskeletal organization, and integrin-mediated signaling. In bone, the ECM is composed of proteins such as collagen (CO), fibronectin (FN), laminin (LM), vitronectin (VN), osteopontin (OP) and osteonectin (ON). For bone tissue engineering, the ECM should also be considered in terms of its function in mediating cell adhesion to biomaterials. This study examined ECM production, cytoskeletal organization, and adhesion of primary human osteoblastic cells on biodegradable matrices applicable for tissue engineering, namely polylactic-co-glycolic acid 50:50 (PLAGA) and polylactic acid (PLA). We hypothesized that the osteocompatible, biodegradable polymer surfaces promote the production of bone-specific ECM proteins in a manner dependent on polymer composition.We first examined whether the PLAGA and PLA matrices could support human osteoblastic cell growth by measuring cell adhesion at 3, 6 and 12h post-plating. Adhesion on PLAGA was consistently higher than on PLA throughout the duration of the experiment, and comparable to tissue culture polystyrene (TCPS). ECM components, including CO, FN, LM, ON, OP and VN, produced on the surface of the polymers were quantified by ELISA and localized by immunofluorescence staining. All of these proteins were present at significantly higher levels on PLAGA compared to PLA or TCPS surfaces. On PLAGA, OP and ON were the most abundant ECM components, followed by CO, FN, VN and LN. Immunofluorescence revealed an extracellular distribution for CO and FN, whereas OP and ON were found both intracellularly as well as extracellularly on the polymer. In addition, the actin cytoskeletal network was more extensive in osteoblasts cultured on PLAGA than on PLA or TCPS.In summary, we found that osteoblasts plated on PLAGA adhered better to the substrate, produced higher levels of ECM molecules, and showed greater cytoskeletal organization than on PLA and TCPS. We propose that this difference in ECM composition is functionally related to the enhanced cell adhesion observed on PLAGA. There is initial evidence that specific composition of the PLAGA polymer favors the ECM. Future studies will seek to optimize ECM production on these matrices for bone tissue engineering applications.     

Limits of detection for time of flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS): detection of low amounts of adsorbed protein

Characterization of biomaterial surfaces requires analytical techniques that are capable of detecting a wide concentration range of adsorbed protein. This range includes detection of low amounts of adsorbed protein (<10 ng/cm2) that may be present on non-fouling biomaterials. X-ray Photoelectron Spectroscopy (XPS) and Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) are surface sensitive techniques capable of detecting adsorbed proteins. We have investigated the lower limits of detection of both XPS and ToF-SIMS on four model substrates each presenting unique challenges for analysis by XPS and ToF-SIMS: mica, poly(tetrafluoroethylene), allyl amine plasma polymer and heptyl amine plasma polymer. The detection limit for XPS ranged from 10 ng/cm2 of fibrinogen (on mica) to 200 ng/cm2 (on allyl amine plasma polymers). The detection limit for ToF-SIMS ranged from 0.1 ng/cm2 of fibrinogen to 100 ng/cm2, depending on the substrate and data analysis. Optimal conditions provided detection limits between 0.1 ng/cm2 and 15 ng/cm2 on all of the substrates used in this study. While both techniques were shown to be effective in detecting protein, the sensitivity of both XPS and ToF-SIMS was shown to be dependent on substrate surface chemistry and the organization of the adsorbed protein film. This study specifically highlights the applicability of ToF-SIMS in the characterization of low level protein adsorption.     

海马神经干细胞不同传代方法的比较

背景：体外培养神经干细胞,在悬浮培养时由于自身增殖特性会形成球,传代时将会面临如何将细胞球分离成单细胞的问题。 目的：寻求理想的大鼠海马神经干细胞传代方法,以获得大量可增生的神经干细胞以供研究。 方法：分离新生1 d SD大鼠海马神经干细胞,原代培养至五六天时,分别用机械吹打法、胰蛋白酶、TrypLE和Accutase消化法分离神经干细胞球。之后每7 d传代1次,连续传代3次。分别于每次传代后第1天和传代后第4天计数活细胞比例和细胞球数目,实验重复3次。 结果与结论：神经干细胞球经3种酶消化后获得的均是单细胞;经机械吹打后既有单个细胞,也有小细胞球分布于培养液中。在酶消化法中,Accutase消化法传代后神经干细胞的活细胞比例明显高于胰蛋白酶消化(P < 0.01)和TrypLE消化法   (P < 0.05)。同时,Accutase消化法传代后新形成的细胞球数目也较其余各组多(P < 0.01)。提示在实验条件下,Accutase消化法能够较好地将神经干细胞球分离成存活率较高、能快速形成新的克隆球的单个细胞,是较为理想的神经干细胞分离传代方法。     

Theoretical considerations on mechanisms of harvesting cells cultured on thermoresponsive polymer brushes

Poly (N-isopropylacrylamide) (PNIPAM) brushes and hydrogels serve as temperature-responsive cell culture substrates. The cells adhere at 37 °C and are detached by cooling to below the lower critical solution temperature T(LCST) ≈ 32 °C, an effect hitherto attributed to change in PNIPAM hydration. The article proposes a mechanism coupling the change of hydration to integrin mediated environmental sensing for cell culture on brushes and hydrogels in serum containing medium. Hydration is associated with swelling and higher osmotic pressure leading to two effects: (i) The lower osmotic pressure in the collapsed brush/hydrogel favors the adsorption of serum borne extracellular matrix (ECM) proteins enabling cell adhesion; (ii) Brush/hydrogel swelling at T < T(LCST) gives rise to a disjoining force f(cell) due to confinement by the ventral membrane of a cell adhering via integrin-ECM bonds. f(cell) places the integrin-ECM bonds under tension thus accelerating their dissociation and promoting desorption of ECM proteins. Self consistent field theory of PNIPAM brushes quantifies the effect of the polymerization degree N, the area per chain Σ, and the temperature, T on ECM adsorption, f(cell) and the dissociation rate of integrin-ECM bonds. It suggests guidelines for tuning Σ and N to optimize adhesion at 37 °C and detachment at T < T(LCST). The mechanism rationalizes existing experimental results on the influence of the dry thickness and the RGD fraction on adhesion and detachment.     

The effect of silica nanoparticle-modified surfaces on cell morphology, cytoskeletal organization and function

Chemical and morphological characteristics of a biomaterial surface are thought to play an important role in determining cellular differentiation and apoptosis. In this report, we investigate the effect of nanoparticle (NP) assemblies arranged on a flat substrate on cytoskeletal organization, proliferation and metabolic activity on two cell types, Bovine aortic endothelial cells (BAECs) and mouse calvarial preosteoblasts (MC3T3-E1). To vary roughness without altering chemistry, glass substrates were coated with monodispersed silica nanoparticles of 50, 100 and 300 nm in diameter. The impact of surface roughness at the nanoscale on cell morphology was studied by quantifying cell spreading, shape, cytoskeletal F-actin alignment, and recruitment of focal adhesion complexes (FAC) using image analysis. Metabolic activity was followed using a thiazolyl blue tetrazolium bromide assay. In the two cell types tested, surface roughness introduced by nanoparticles had cell type specific effects on cell morphology and metabolism. While BAEC on NP-modified substrates exhibited smaller cell areas and fewer focal adhesion complexes compared to BAEC grown on glass, MC3T3-E1 cells in contrast exhibited larger cell areas on NP-modified surfaces and an increased number of FACs, in comparison to unmodified glass. However, both cell types on 50 nm NP had the highest proliferation rates (comparable to glass control) whereas cells grown on 300 nm NP exhibited inhibited proliferation. Interestingly, for both cell types surface roughness promoted the formation of long, thick F-actin fibers, which aligned with the long axis of each cell. These findings are consistent with our earlier result that osteogenic differentiation of human mesenchymal progenitor cells is enhanced on NP-modified surfaces. Our finding that nanoroughness, as imparted by nanoparticle assemblies, effects cellular processes in a cell specific manner, can have far reaching consequences on the development of "smart" biomaterials especially for directing stem cell differentiation.     

细胞力学2022年度研究进展

细胞的力学微环境在调控其生理功能方面起关键作用。体内细胞经常受到剪切、拉伸、压缩等多种力学载荷,并且可以通过黏附分子(如整合素-配体素的结合)连接到细胞外基质上,进而可以感知外基质的硬度。细胞力学主要研究活细胞在力学载荷下的力学特性和行为,以及这些特征和行为与细胞功能的关系。本文综述2022年度细胞力学领域的研究进展,主要关注整合素-配体素间的相互作用,以及外基质硬度和力学载荷对细胞生理行为和形态发生的影响。     

Limits of detection for time of flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS): detection of low amounts of adsorbed protein

Characterization of biomaterial surfaces requires analytical techniques that are capable of detecting a wide concentration range of adsorbed protein. This range includes detection of low amounts of adsorbed protein (<10 ng/cm²) that may be present on non-fouling biomaterials. X-ray Photoelectron Spectroscopy (XPS) and Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) are surface sensitive techniques capable of detecting adsorbed proteins. We have investigated the lower limits of detection of both XPS and ToF-SIMS on four model substrates each presenting unique challenges for analysis by XPS and ToF-SIMS: mica, poly(tetrafluoroethylene), allyl amine plasma polymer and heptyl amine plasma polymer. The detection limit for XPS ranged from 10 ng/cm² of fibrinogen (on mica) to 200 ng/cm² (on allyl amine plasma polymers). The detection limit for ToF-SIMS ranged from 0.1 ng/cm² of fibrinogen to 100 ng/cm², depending on the substrate and data analysis. Optimal conditions provided detection limits between 0.1 ng/cm² and 15 ng/cm² on all of the substrates used in this study. While both techniques were shown to be effective in detecting protein, the sensitivity of both XPS and ToF-SIMS was shown to be dependent on substrate surface chemistry and the organization of the adsorbed protein film. This study specifically highlights the applicability of ToF-SIMS in the characterization of low level protein adsorption.     

Mediation of the migration of endothelial cells and fibroblasts on polyurethane nanocomposites by the activation of integrin-focal adhesion kinase signaling

Model surfaces of polyurethane-gold nanocomposites (PU-Au) were used to examine cell behavior on nanophase-segregated materials. Previously we showed that endothelial cell (EC) migration on these materials was modulated by the PI3K/Akt/eNOS pathway. The present study, investigated the expressions of alpha5/beta3 (α5β3) integrin, focal adhesion kinase (FAK), and other downstream signal molecules such as the Rho family and matrix metalloproteinases 2 (MMP-2) induced by the materials in two different cells, that is bovine arterial endothelial cells (BAEC) and human skin fibroblasts (HSF). Both cells proliferated better on the more phase-separated PU-Au 43.5 ppm than on the less phase-separated controls (PU and PU-Au 174 ppm). On PU-Au 43.5 ppm, BAEC compared to HSF had denser actin fibers and were more extended. BAEC became rounded with Y-27632 treatment and shrunk with LY294002 treatment. Treatment by inhibitors only caused slight changes in HSF. The migration distance of BAEC on PU-Au 43.5 ppm was greater than that of HSF, and was significantly reduced by LY294002 or Y-27632 but not SU-1498. The expressions of p-FAK, p-RhoA, p-Rac/Cdc42, MMP2, and α5β3 integrin induced by PU-Au 43.5 ppm were more pronounced in BAEC versus HSF. Further enhancement in MMP2 and α5β3 integrin expressions by FAK-GFP transfection was more remarkable for cells on PU-Au 43.5 ppm. Our findings suggested that the integrin α5β3/FAK pathway may be induced by nanophase-separated materials in both ECs and fibroblasts to promote their proliferation/migration, while the crosstalk between the PI3K/Akt/eNOS pathway and FAK/Rho-GTPase activation may account for the greater effect in ECs than in fibroblasts.     

Retention of endothelial cell adherence to porcine-derived extracellular matrix after disinfection and sterilization

Extracellular matrices (ECM) derived from porcine tissue are associated with rapid and extensive repopulation with host cells when used as scaffolds for in vivo tissue repair. Cell adhesion to substrates used for tissue engineering has been studied extensively but the factors that mediate this phenomenon in ECM scaffolds following treatment with oxidants and sterilants have not been examined. Cell adhesion assays were used to examine human microvascular endothelial cell (HMEC) attachment to ECM graft materials harvested from small intestinal submucosa (SIS) and urinary bladder matrix (UBM) following decellularization and sterilization procedures designed to render the ECM safe for clinical use. HMECs were able to attach directly to these ECM scaffolds via several attachment proteins present within the ECM, including type I collagen, type IV collagen, and fibronectin. The ability of the SIS ECM and UBM ECM to support the growth and proliferation of HMEC was also examined. HMEC were able to grow to single-layer confluence on both surfaces of SIS and UBM sheets. The endothelial cells were also able to penetrate the SIS and UBM at later time points if they were seeded on the abluminal side of the ECM sheets. The ability of the processed ECM to support HMEC attachment and proliferation is similar to that reported for unprocessed ECM and may therefore play a role in the rapid remodeling response observed when these matrices are implanted in vivo as scaffolds for wound repair.     

Function of linear and cyclic RGD-containing peptides in osteoprogenitor cells adhesion process.

Cell adhesion directly influences cell growth, differentiation and migration as well as morphogenesis, integrity and repair. The extracellular matrix (ECM) elaborated by osteoblast cells constitutes a regulator of the cell adhesion process and then of the related phenomenon. These regulatory effects of ECM are mediated through integrins and some of them are able to bind RGD sequences. The aim of this study was to determine the role of the sequence and the structure of RGD-containing peptides (linear and cyclic) as well as their role in the cell adhesion process. Cell adhesion assays onto ECM proteins coated surfaces were performed using a range of linear and cyclic RGD-containing peptides. We showed a different human osteoprogenitor cell adhesion according to the coating for ECM proteins and for RGD-peptides. Inhibition assays using peptides showed different responses depending on the coated protein. Depending on the amino-acid sequence and the structure of the peptides (cyclic linear), we observed 100% inhibition of cell adhesion onto vitronectin. These results suggest the importance of sequence, structure and conformation of the peptide, which may play a crucial function in the ligand/receptor interaction and/or in the stability of the interaction.     

Cell adhesion on an artificial extracellular matrix using aptamer-functionalized PEG hydrogels

The development of an artificial extracellular matrix (ECM) is important to regenerative medicine because the ECM plays complex and dynamic roles in the regulation of cell behavior. In this study, nucleic acid aptamers were applied to functionalize hydrogels for mimicking the adhesion sites of the ECM. The results showed that nucleic acid aptamers could be incorporated into polyethylene glycol (PEG) hydrogels via free radical polymerization. The incorporation of the aptamers produced only a moderate effect on the mechanical properties of the PEG hydrogels. Importantly, the results also showed that the aptamers effectively induced cell type-specific adhesion to the PEG hydrogels without affecting cell viability. The cell adhesion was a function of the aptamer concentration, the spacer length and the cell seeding time. In addition, cell adhesion to the aptamer-functionalized hydrogel could be attenuated by means of aptamer inactivation in a physiological condition. Thus, aptamer-functionalized hydrogels are promising biomaterials for the development of artificial ECMs.