Synergistic effect of titanium alloy and collagen type I on cell adhesion, proliferation and differentiation of osteoblast-like cells

A number of studies have demonstrated the pivotal role of collagen in modulating cell growth and differentiation. In bone, where the extracellular matrix is composed of approximately 85% type I collagen, cellular interaction with matrix components has been shown to be important in the regulation of the osteoblast phenotype. Preservation or enhancement of normal osteoblast function and appositional bone formation after implant placement represents a strategy that can be useful for the purpose of improving osseointegration. In order to further improve biocompatibility, we combined two known favorable compounds, namely the titanium alloy, Ti6A14V, with type I collagen. We assessed the in vitro behavior of primary osteoblasts grown on both fibrillar collagen-coated and tropocollagen-coated Ti6A14V in comparison with uncoated titanium alloy, using an improved adsorption procedure. As parameters of biocompatibility, a variety of processes, including cell attachment, spreading, cytoskeletal organization, focal contact formation, proliferation and expression of a differentiated phenotype, were investigated. Our results demonstrated for the first time that in comparison to uncoated titanium alloy, collagen-coated alloy enhanced spreading and resulted in a more rapid formation of focal adhesions and their associated stress fibers. Growing on collagen-coated Ti6A14V, osteoblasts had a higher proliferative capacity and the intracellular expression of osteopontin was upregulated compared to uncoated titanium alloy. Type I collagen-coated titanium alloy exhibits favorable effects on the initial adhesion and growth activities of osteoblasts, which is encouraging for its potential use as bone graft material. Moreover, collagen type I may serve as an excellent biocompatible carrier for osteotropic factors such as cell adhesion molecules (e.g. fibronectin) or bone-specific growth factors.     

The attachment and growth behavior of osteoblast-like cells on microtextured surfaces

In previous studies, we showed that the application of microgrooves on a surface can direct cellular morphology and the deposition of mineralized matrix of osteoblast-like cells (Biomaterials 20 (1999) 1293; Clin. Oral Impl Res. 11 (2000) 325). In this study, we evaluated the attachment and growth behavior of these cells, using scanning- and transmission electron microscopy (SEM/TEM). Smooth and microgrooved polystyrene substrates were made (groove depth 0.5-1.5 microm, groove- and ridge width 1-10 microm). On these substrates, osteoblast-like cells were cultured for periods up to 16 days. SEM showed that the cells, and their extensions, closely followed the surface on smooth and wider grooved (>5 microm) substrates. In contrast, narrow grooves (<2 microm) were bridged. After 16 days of incubation, the matrix showed extensive deposition of collagen fibrils, and the formation of calcified nodules. With TEM it was shown that on the smooth and wider grooved substrates, focal adhesions were spread throughout the surface. However, on narrow grooves focal adhesions were always positioned on the edges of surface ridges only. Apparently, most extracellular matrix (ECM) was produced by the cells that directly adhered to the substrate. Deposition of ECM was seen in the surface grooves, as well as in between the cell layers. On basis of the current study and previous experiments, we conclude that microgrooves are able to influence bone cell behavior by (1) determining the alignment of cells and cellular extensions, (2) altering the formation and placement of cell focal adhesions, and (3) altering ECM production. Therefore, microgrooved surfaces seem interesting to be applied on bone-anchored implants.     

Cell-matrix adhesions on poly(vinyl alcohol) hydrogels

Cell-matrix adhesions regulate cell morphology, intracellular signaling, gene expression, and phenotype. Understanding how different methods of attaching matrix proteins to substrates affect the molecular arrangement of these adhesions offers the possibility of controlling cell function and architecture. The goal of this study was to visualize and quantify the cell-matrix adhesions formed by human fibroblasts on the matrix protein fibronectin covalently attached to poly(vinyl) alcohol (PVA) hydrogels. These adhesions were then compared with the cell adhesions formed in routine cell culture on fibronectin noncovalently coated onto glass coverslips or those formed on fibronectin covalently immobilized onto glass coverslips. Cell adhesions were characterized by immunofluorescence confocal microscopy utilizing paxillin as a marker for focal adhesions and alpha(5) integrin as a marker for fibrillar adhesions. As expected, distinct focal and fibrillar adhesions were observed in routine cell culture on coverslips coated noncovalently with fibronectin. Cells cultured on fibronectin covalently linked to PVA demonstrated diminished spatial separation of paxillin and alpha(5) integrin, accompanied by a reduction in fibrillar adhesions and fibronectin fibrillogenesis. Cells on fibronectin covalently immobilized on glass displayed the strongest marker colocalization and the most complete loss of fibrillar adhesions and lack of fibrillogenesis. These results indicate that fibronectin-conjugated PVA promotes the formation of cell adhesion structures intermediate in composition between those formed on noncovalently attached and covalently immobilized fibronectin. Furthermore, they imply that bioactive polymers can selectively induce specific cell-matrix adhesions, a characteristic that may have consequences in various tissue-engineering applications.     

Cell adhesion mechanisms on laterally mobile polymer films

In contrast with the majority of substrates used to study cell adhesion, the natural extracellular matrix (ECM) is dynamic and remodeled over time. Here we use amphiphilic block copolymers to create self-assembled supported films with tunable lateral mobility. These films are intended to serve as partial mimics of the ECM in order to better understand cell adhesion responses, specifically in the context of dynamic substrates. Block copolymers are end-labeled with RGD peptide ligands to allow for integrin-mediated cell adhesion, and the addition of a trace hydrophobic homopolymer is used to control the film lateral mobility. We find that NIH 3T3 fibroblasts cultured on these biomimetic films exhibit non-linear spreading behavior in response to substrate mobility. In the absence of RGD ligands, however, fibroblasts do not spread. Employing quantitative analysis of focal adhesions (FA) and integrin ligation, we discover the presence of FA-dependent and FA-independent mechanisms responsible for the biphasic cell spreading behavior. The use of designed biomimetic platforms therefore yields insight into ECM mechanosensing by revealing that cells can engage distinct mechanisms to promote adhesion onto substrates with different time-dependent properties.     

Adriamycin-induced inhibition of melanoma cell invasion is correlated with decreases in tumor cell motility and increases in focal contact formation

Tumor cell adhesion to the extracellular matrix (ECM) is closely linked with tumor cell invasion and metastasis. In this study, we demonstrate that low levels of adriamycin, a widely used anticancer drug, can inhibit the invasion of highly metastatic K1735-M2 mouse melanoma cellsin vitro through a reconstituted basement membrane extract. Adriamycin-induced inhibition of melanoma cell invasion occurred at levels of the drug (i.e. 1 ng/ml) that did not inhibit tumor cell growth, suggesting that the observed inhibition in tumor cell invasion was not due to the well-documented ability of adriamycin to interfere with DNA and/or RNA synthesis. Rather, these studies indicated that adriamycin-induced inhibition of melanoma cell invasion was accompanied by a corresponding decrease in the ability of adriamycin-treated tumor cells to migrate in response to several isolated ECM components including fibronectin, laminin and basement membrane (type IV) collagen. The decreased migration of adriamycin-treated tumor cells was not accompanied by a decrease in the adhesion or spreading of the adriamycin-treated cells on substrata coated with these ECM components. Instead, adriamycin-treated cells actually exhibited a slightly increased propensity (compared to untreated control cells) to adhere on fibronectin-, laminin-, and type IV collagen-coated substrata. Additionally, adriamycin treatment caused a dramatic increase in focal contact formation by these melanoma cells, as assessed by fluorescent microscopy of actin and vinculin. In addition to providing a useful model for which to study the molecular and cellular basis for focal contact formation, these results further emphasize the results of several other investigators that have suggested an important role for focal contacts in modulating tumor cell motility, invasion and metastasis.     

The effect of substrate microtopography on focal adhesion maturation and actin organization via the RhoA/ROCK pathway

Recently, a growing number of reports have reported that micro- or nanoscale topography enhances cellular functions such as cell adhesion and stem cell differentiation, but the mechanisms responsible for this topography-mediated cell behavior are not fully understood. In this study, we examine the underlying processes and mechanisms behind specific topography-mediated cellular functions. Formation of focal adhesions (FA) was studied by culturing cells on different kinds of topographies, including a flat surface and surfaces with a micropatterned topography (2 μm lattice pattern with 3 μm intervals). We found that the formation and maturation of focal adhesions were highly dependent on the topography of the substrate although the shape, morphology and spreading of cells on the different substrates were not significantly affected. Focal adhesion maturation and actin polymerization were also promoted in cells cultured on the micropatterned substrate. These differences in cell adhesion led us to focus on the Rho GTPases, RhoA and downstream pathways since a number of reports have demonstrated that RhoA-activated cells have highly enhanced focal adhesions and actin activation such as polymerization. By inhibiting the Rho-associated kinase (ROCK) and downstream myosin II, we found that the FA formation, actin organization, and FAK phosphorylation were dramatically decreased. The topographical dependency of FA formation was also highly decreased. These results show that the FA formation and actin cytoskeleton organization of cells on the microtopography is regulated by the RhoA/ROCK pathway.     

The influence of surface roughness of titanium on beta1- and beta3-integrin adhesion and the organization of fibronectin in human osteoblastic cells

Mechanisms of cell adhesion and extracellular matrix formation are primary processes in the interaction with the material surface of an implant which are controlled by integrin receptors. The aim of our study was to find out whether beta1- and beta3-integrins of osteoblastic cells sense the surface topography of titanium, and if structural alterations of integrin adhesions were involved in the organization of fibronectin. Pure titanium surfaces were modified by polishing (P), machining (NT), blasting with glass spheres (GB), and blasting with corundum particles (CB) resulting in increasing roughness. Confocal microscopic investigations revealed fibrillar adhesions of beta1- and alpha5-integrins on P, NT, and GB, but on CB with its sharp edges these integrin subunits did not form fibrillar adhesions. beta3 generally appeared in focal adhesions. We observed aligned fibrillar structures of fibronectin on NT not only on the basal site but interestingly, also on the apical cell surface. In contrast, on CB, fibronectin appeared apically clustered. We suggest that this alignment of fibronectin fibrils depends on the directed actin cytoskeleton and in particular, on the capability of the beta1-integrins to form fibrillar adhesions, which is affected by the surface roughness of titanium.     

The effect of the RACK1 signalling protein on the regulation of cell adhesion and cell contact guidance on nanometric grooves

A wide variety of different cell types have been shown to respond to nanofabricated growth surfaces via the process of contact guidance, however little is known about the intracellular mechanisms that control these events. In the present study we have identified the multi-functional signalling adaptor protein, RACK1, as a novel negative regulator of contact guidance on custom-engineered nanometric grooves. We found that over-expression of RACK1 in human breast cancer cells leads to a pro-adherent morphology characterised by the formation of stress fibres and focal adhesions. Enforced expression of RACK1 also limits the response of cells to contact guidance on nanometric grooves. In contrast, ablation of RACK1 protein with specific anti-sense oligonucleotides led to a dramatic enhancement of bi-directional extension of cells on nanometrically deep grooved surfaces, with a corresponding loss of focal adhesions and stress fibres. RACK1 therefore exerts a tonic inhibitory effect on cell contact guidance, while positively promoting an adhesive phenotype. This is the first example of an intracellular signalling molecule involved in the regulation of cell contact guidance on nanometric growth surfaces.     

Human endothelial cell interaction with biomimetic surfactant polymers containing Peptide ligands from the heparin binding domain of fibronectin

Biomimetic materials that mimic the extracellular matrix (ECM) provide a means to control cellular functions such as adhesion and growth, which are vital to successful engineering of tissue-incorporated biomaterials. Novel "ECM-like" biomimetic surfactant polymers consisting of a poly(vinyl amine) backbone with pendant cell-adhesive peptides derived from one of the heparin-binding domains of fibronectin were developed to improve endothelial cell adhesion and growth on vascular biomaterials. Heparin-binding peptide (HBP) sequences, alone and in combination with RGD peptides, were examined for their ability to promote human pulmonary artery endothelial cell (HPAEC) adhesion and growth (HBP1, WQPPRARI; HBP2, SPPRRARVT; HBP1:RGD; and HBP2:RGD) and compared with cell adhesion and growth on fibronectin and on negative control polymer surfaces in which alanines were substituted for the positively charged arginine residues in the two peptides. The results showed that HPAECs adhered and spread equally well on all HBP-containing polymers and the positive fibronectin control, showing similar stress fiber and focal adhesion formation. However, the HBP alone was unable to support long-term HPAEC growth and survival, showing a loss of focal adhesions and cytoskeletal disorganization by 24 h after seeding. With the addition of RGD, the surfaces behaved similarly or better than fibronectin. The negative control polymers showed little to no initial cell attachment, and the addition of soluble heparin to the medium reduced initial cell adhesion on both the HBP2 and HBP2:RGD surfaces. These results indicate that the HBP surfaces promote initial HPAEC adhesion and spreading, but not long-term survival.     

Substrate chemistry influences the morphology and biological function of adsorbed extracellular matrix assemblies

In addition to mediating cell signalling events, native extracellular matrix (ECM) assemblies interact with other ECM components, act as reservoirs for soluble signalling molecules and perform structural roles. The potential of native ECM assemblies in the manufacture of biomimetic materials has not been fully exploited due, in part, to the effects of substrate interactions on their morphology. We have previously demonstrated that the ECM components, fibrillin and type VI collagen microfibrils, exhibit substrate dependent morphologies on chemically and topographically variable heterogeneous surfaces. Using both cleaning and coating approaches on silicon wafers and glass coverslips we have produced chemically homogeneous, topographically similar substrates which cover a large amphiphilic range. Extremes of substrate amphiphilicity induced morphological changes in periodicity, curvature and lateral spreading which may mask binding sites or disrupt domain structure. Biological functionality, as assayed by the ability to support cell spreading, was significantly reduced for fibrillin microfibrils adsorbed on highly hydrophilic substrates (contact angle 20.7 degrees) compared with less hydrophilic (contact angle 38.3 degrees) and hydrophobic (contact angle 92.8 degrees) substrates. With an appropriate choice of surface chemistry, multifunctional ECM assemblies retain their native morphology and biological functionality.     

Substrate affects the initial attachment and subsequent behavior of human osteoblastic cells (Saos-2).

Interaction between implant materials and bone cells contributes to the clinical success of dental implants. The object of this study was to investigate the initial attachment and subsequent behavior of human osteoblastic cells (Saos-2) to pure titanium (Ti), hydroxyapatite (HA), and glass. We, therefore, performed a time-course study for examining the area, attachment rate, distribution of focal adhesion kinase (FAK) vinculin, and actin, and the motility of Saos-2 cells on the materials. On Ti, cell area increased gradually, whereas on HA, cells spread quickly, but quitted spreading at 12 h after cell seeding. The number of cells on HA was greater than on the other materials. On Ti, the numbers of FAK- and vinculin-positive focal adhesions increased continuously. On HA, although the number of FAK-positive focal adhesions also increased continuously, the number of vinculin-positive focal adhesions decreased. Furthermore, actin staining showed that the cells on HA poorly formed stress fibers with weak polarity, whereas the cell on Ti possessed well-defined polarized stress fibers. On HA, cells started extension earlier than on Ti, motility was inactive, and the cells settled on the materials. These results suggest that the earlier settling of osteoblasts on HA might result in earlier osteogenesis on HA than other materials.     

Laser processing of bioactive tricalcium phosphate coating on titanium for load-bearing implants

Laser-engineered net shaping (LENS), a commercial rapid prototyping (RP) process, was used to coat titanium with tricalcium phosphate (TCP) ceramics to improve bone cell-materials interactions. During LENS coating process, the Nd:YAG laser melts the top surface of Ti substrate in which calcium phosphate powder is fed to create a TCP-Ti composite layer. It was found that an increase in laser power and/or powder feed rate increases the thickness of the coating. However, coating thickness decreased with increasing laser scan speed. TCP coating showed columnar titanium grains at the substrate side of the coating and transitioned to equiaxed titanium grains at the outside. When the scan speed was reduced from 15 to 10mms(-1), coating hardness increased from 882+/-67 to 1049+/-112Hv due to an increase in the volume fraction of TCP in the coating. Coated surfaces showed uniformly distributed TCP particles and X-ray diffraction data confirmed the absence of any undesirable phases, while maintaining a high level of crystallinity. The effect of TCP coating on cell-material interaction was examined by culturing osteoprecursor cells (OPC1) on coated surfaces. The results indicated that TCP coating had good biocompatibility where OPC1 cells attached and proliferated on the coating surface. The coating also initiated cell differentiation, ECM formation and biomineralization.     

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.     

The use of poly(ethylene glycol) hydrogels to investigate the impact of ECM chemistry and mechanics on smooth muscle cells

Hydrogels based on poly(ethylene glycol) (PEG) are of increasing interest for regenerative medicine applications and are ideal materials to direct cell function due to the ability to confer key functionalities of native extracellular matrix (ECM) on PEG's otherwise inert backbone. Given extensive recent evidence that ECM compliance influences a variety of cell functions, PEG-based hydrogels are also attractive due to the ease with which their mechanical properties can be controlled. In these studies, we exploited the chemical and mechanical tunability of PEG-based gels to study the impact of ECM chemistry and mechanics on smooth muscle cells (SMCs) in both 2-D and 3-D model systems. First, by controlling the extent of crosslinking and therefore the mechanical properties of PEG-based hydrogels (tensile moduli from 13.7 to 423.9kPa), we report here that the assembly of F-actin stress fibers and focal adhesions, indicative of the state of actin contractility, were influenced by the compliance of 2-D PEG gels functionalized with either short adhesive peptides or full-length ECM proteins. Varying ECM ligand density and identity independent of gel compliance affected the physical properties of the focal adhesions, and also influenced SMC spreading in 2-D. Furthermore, SMCs proliferated to a greater extent as gel stiffness was increased. In contrast, the degree of SMC differentiation, which was qualitatively assessed by the extent of smooth muscle alpha-actin bundling and the association of calponin and caldesmon with the alpha-actin fibrils, was found to decrease with substrate stiffness in 2-D cultures. In 3-D, despite the fact that their viability and degree of spreading were greatly reduced, SMCs did express some contractile markers indicative of their differentiated phenotype when cultured within PEG-RGDS constructs. Combined, these data suggest that the mechanical and chemical properties of PEG hydrogels can be tuned to influence SMC phenotype in both 2-D and 3-D.     

Tuning the material-cytoskeleton crosstalk via nanoconfinement of focal adhesions

Material features proved to exert a potent influence on cell behaviour in terms of adhesion, migration and differentiation. In particular, biophysical and biochemical signals on material surfaces are able to affect focal adhesion distribution and cytoskeletal assemblies, which are known to regulate signalling pathways that ultimately influence cell fate and functions. However, a general, unifying model that correlates cytoskeletal-generated forces with genetic events has yet to be developed. Therefore, it is crucial to gain a better insight into the material-cytoskeleton crosstalk in order to design and fabricate biomaterials able to govern cell fate more accurately. In this work, we demonstrate that confining focal adhesion distribution and growth dramatically alters the cytoskeleton's structures and dynamics, which in turn dictate cellular and nuclear shape and polarization. MC3T3 preosteoblasts were cultivated on nanograted polydimethylsiloxane substrates and a thorough quantification – in static and dynamic modes – of the morphological and structural features of focal adhesions and cytoskeleton was performed. Nanoengineered surfaces provided well-defined zones for focal adhesions to form and grow. Unique cytoskeletal structures spontaneously assembled when focal adhesions were confined and, in fact, they proved to be very effective in deforming the nuclei. The results here presented provide elements to engineer surfaces apt to guide and control cell behaviour through the material-cytoskeleton-nucleus axis.     

Functionalization of dynamic culture surfaces with a cartilage extracellular matrix extract enhances chondrocyte phenotype against dedifferentiation

Culture on silicone rubber surfaces has been shown to partially overcome the chondrocyte dedifferentiation characteristic of standard culture on rigid polystyrene. These methods typically involve functionalization of culture surfaces with proteins. Collagen type I is often used, but more cartilage-specific proteins may be more appropriate for chondrocytes. To explore this hypothesis, a twofold experimental design was applied. First, chondrocytes were cultured in rigid Petri dishes coated with silicone rubber ("static silicone" or SS culture) functionalized with either cartilage extracellular matrix (ECM) extract or collagen type I. Second, chondrocytes were cultured on monotonically expanded high extension silicone rubber dishes ("continuous expansion" or CE culture) functionalized with ECM extract and compared to cells grown in SS culture. There were no differential effects of surface functionalization with the ECM extract vs. collagen type I on chondrocyte morphology, viability, proliferation or apoptosis in SS culture. However, chondrocyte growth on the ECM extract was associated with significantly reduced collagen types I and X gene expression and significantly increased glycosaminoglycan (GAG) secretion. After 3 passages (P3) on ECM-coated SS culture, chondrocyte phenotype and GAG secretion was enhanced compared to cells passaged on collagen type I. Pellet cultures from P3 SS culture displayed enhanced collagen type II content when ECM extract was used for functionalization rather than collagen type I. In CE culture with ECM functionalization, chondrocyte dedifferentiation was significantly inhibited vs. SS cultures, as evidenced by both gene expression and pellet cultures. Functionalization of extendable culture surfaces with cartilage ECM extract therefore supports enhanced preservation of chondrocyte phenotype.     

The initial attachment and subsequent behavior of osteoblastic cells and oral epithelial cells on titanium

The interaction between implant materials and bone cells or oral epithelial (OE) cells contributes to the clinical success of dental implants. The functional activity of cells in contact with an implant is determined by its surface properties. Before cells attach, extracellular matrix (ECM) in the serum deposits on the substrate; rounded cells then attach and spread upon it. Cells form focal adhesions and polarize, then start to migrate or proliferate to form colonies. Comparison of the attachment and behavior of osteoblastic cells on titanium (Ti) and hydroxyapatite (HA) revealed that more cells attached on HA and that these spread more rapidly than on Ti. In contrast, cells did not form good stress fibers or vinculin-positive focal adhesions on HA, whereas the cells on Ti possessed well-defined and polarized stress fibers. The initial attachment of OE cells to Ti was inferior to that on polystyrene culture dish or glass, and the OE cell migration area, indicated by the deposition of LN5, was smaller on Ti than on the other materials. This review summarizes data on the attachment and behavior of osteoblastic cells and OE cells on biomaterials, which may suggest future improvements in surface properties.     

Facile surface functionalization with glycosaminoglycans by direct coating with mussel adhesive protein

The use of mussel adhesive proteins (MAPs) as a surface coating for cell adhesion has been suggested due to their unique properties of biocompatibility and effective adhesion on diverse inorganic and organic surfaces. The surface functionalization of scaffolds or implants using extracellular matrix (ECM) molecules is important for the enhancement of target cell behaviors such as proliferation and differentiation. In the present work, we suggest a new, simple surface functionalization platform based on the charge interactions between the positively charged MAP linker and negatively charged ECM molecules, such as glycosaminoglycans (GAGs). MAP was efficiently coated onto a titanium model surface using its adhesion ability. Then, several GAG molecules, including hyaluronic acid (HA), heparin sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS), were effectively immobilized on the MAP-coated surfaces by charge interactions. Using HA as a model GAG molecule, we found that the proliferation, spreading, and differentiation behaviors of mouse preosteoblast cells were all significantly improved on MAP/HA-layered titanium. In addition, we successfully constructed a multilayer film on a titanium surface with oppositely charged layer-by-layer coatings of MAP and HA. Collectively, our simple MAP-based surface functionalization strategy can be successfully used for the efficient surface immobilization of negatively charged ECM molecules in various tissue engineering and medical implantation applications.     

The role of surface wettability on hepatocyte adhesive interactions and function

In this paper the effect of surface wettability on hepatocyte morphology and function was studied, using clean and octadecylsylane (ODS)-coated glass as a model for hydrophilic and hydrophobic surfaces, respectively. C3A cells - a hepatoblastoma cell line, and freshly obtained porcine hepatocytes were cultured for a short-time period of up to 4 days on the above substrata. Hepatocyte adhesive interactions were characterized monitoring the initial cell attachment, the overall cell morphology, the formation of focal adhesions, and actin filaments. Since hepatocytes showed a clear tendency for homotypic adhesion on ODS, specific E-cadherin staining was used to visualize the intercellular contacts by immunofluorescence microscopy. Additionally, functional assays were carried out to monitor proliferation, metabolic activity, and albumin synthesis of C3A cells. It could be shown that both C3A cells and normal porcine hepatocytes spread better on hydrophilic glass; spreading being accompanied by the development of pronounced actin stress fibers and focal adhesion contacts. In contrast, on hydrophobic substrata predominant cell-cell interactions took place which led to intense E-cadherin staining in the intercellular contacts of porcine hepatocytes but not in C3A cells. On the other hand, metabolic activity and growth of C3A cells were reduced on hydrophobic ODS, but albumin synthesis was similar on both surfaces. It was concluded that the wettability of materials has a strong influence on the attachment and morphology of hepatocytes while the influence of surface properties on the functional activity of hepatocytes still remains to be elucidated.     

The role of surface wettability on hepatocyte adhesive interactions and function

In this paper the effect of surface wettability on hepatocyte morphology and function was studied, using clean and octadecylsylane (ODS)-coated glass as a model for hydrophilic and hydrophobic surfaces, respectively. C3A cells--a hepatoblastoma cell line, and freshly obtained porcine hepatocytes were cultured for a short-time period of up to 4 days on the above substrata. Hepatocyte adhesive interactions were characterized monitoring the initial cell attachment, the overall cell morphology, the formation of focal adhesions, and actin filaments. Since hepatocytes showed a clear tendency for homotypic adhesion on ODS, specific E-cadherin staining was used to visualize the intercellular contacts by immunofluorescence microscopy. Additionally, functional assays were carried out to monitor proliferation, metabolic activity, and albumin synthesis of C3A cells. It could be shown that both C3A cells and normal porcine hepatocytes spread better on hydrophilic glass; spreading being accompanied by the development of pronounced actin stress fibers and focal adhesion contacts. In contrast, on hydrophobic substrata predominant cell-cell interactions took place which led to intense E-cadherin staining in the intercellular contacts of porcine hepatocytes but not in C3A cells. On the other hand, metabolic activity and growth of C3A cells were reduced on hydrophobic ODS, but albumin synthesis was similar on both surfaces. It was concluded that the wettability of materials has a strong influence on the attachment and morphology of hepatocytes while the influence of surface properties on the functional activity of hepatocytes still remains to be elucidated.