Adsorbed Collagen Layers Research Articles

Osteoblastic Behavior of Human Bone Marrow Cells Cultured Over Adsorbed Collagen Layer, Over Surface of Collagen Gels, and Inside Collagen Gels

Osteoblastic Behavior of Human Bone Marrow Cells Cultured Over Adsorbed Collagen Layer, Over Surface of Collagen Gels, and Inside Collagen Gels

MORPHOLOGY OF ADSORBED COLLAGEN LAYERS OBSERVED BY ATOMIC FORCE MICROSCOPY

The interaction between cells and biomaterials strongly depends on the assembled structure of collagen adsorption upon the solid surface. Due to its self-assembling property, Type I collagen may aggregate and form fibrils in vivo and in vitro. This study utilizes an atomic force microscope to investigate nanometer-scale organization of adsorbed Type I collagen layers on mica and on poly(methyl methacrylate) (PMMA). We have observed various film morphologies, depending on substrate hydrophobicity and the state of collagen solution used. On mica, the atomic force microscopy (AFM) study obtains dense felt-like structures of randomly distributed assemblies. Images of network-like assemblies composed of interwoven fibrils appear on PMMA. According to the above results, we believe that these assemblies are associated at the interface rather than aggregated in the solution. This work also investigates the adsorbed collagen structure on PMMA after collagen aggregation in solution, to realize the relation between adsorption and aggregation. Consequently, the result exhibits a dendritic fibrillar structure adsorbed on PMMA, following collagen molecule aggregation, to form a fibrillar structure in the solution. This result suggests that the adsorption of aggregates preformed in the solution is preferable to collagen molecules adsorption. This research created all assembled structures of adsorbed collagen layers in nanometer-scale thickness.

Osteoblastic Behavior of Human Bone Marrow Cells Cultured Over Adsorbed Collagen Layer, Over Surface of Collagen Gels, and Inside Collagen Gels

While collagen type I is often used as a substrate for cell culturing and as a coating in biomedical implants, as far as we know a simple systematic study comparing the effects of the different presentations of collagen type I on the osteoblastic behavior of cells is missing. In this work, human bone marrow cells (hBMCs) were cultured under osteoblastic-inducing conditions, for 21 days, over a layer of adsorbed collagen (monomeric) and on the surface and inside collagen gels (fibrillar). Comparison was made based on three classical parameters; cell proliferation/viability, alkaline phosphatase (ALP) activity, and production of mineral deposits. The three types of collagen type I substrates allowed the adhesion, proliferation, and the osteoblastic differentiation of cells. However, hBMCs behavior was influenced by the monomeric/fibrillar and 2-/3-dimensional nature of the collagen substrates, namely: monomeric collagen favored cell attachment; cells on 2D substrates presented higher proliferation rates during the exponential phase of growth with formation of spiral-like multilayered structures; cells seeded inside 3D collagen gels formed a regular dense cellular mesh and had a low proliferating rate; cells cultured over or inside fibrillar collagen differentiated faster, with the 3D cultures presenting higher levels of ALP activity; and the extension of mineralization was greater for the cultures done over or inside fibrillar collagen. Thus, cells cultured over collagen gels showed both the ability for cell proliferation and for earlier differentiation, a fact that can be exploited in the biomaterials field.

Network structure of collagen layers absorbed on LB film

Elucidating the assembly mechanism of the collagen at interfaces is important. In this work, the structures of type I collagen molecules adsorbed on bare mica and on LB films of propanediyl-bis(dimethyloctadecylammonium bromide) transferred onto mica at zero surface pressure was characterized by AFM. On mica, the granular morphologies randomly distributed as elongated structures were observed, which were resulted from the interlacement of the adsorbed collagen molecules. On the LB films, the topographical evolution of the adsorbed collagen layers upon the increasing adsorption time was investigated. After 30 s, the collagen assembled into network-like structure composed of the interwoven fibrils, called as the first adlayer, which was attributed to its adsorption on the LB film by means of a limited number of contact points followed by the lateral association. One minute later, the second adlayer was observed on the top of the first adlayer. Up to 5 min, collagen layers, formed by inter-twisted fibrils, were observed. Under the same conditions after 1 min adsorption on LB film, the AFM image of the layer obtained in the diluted hydrochloric acid solution is analogous to the result of the sample dried in air, indicating that it is the LB film that leads to the formation of the network structure of collagen and the formation of the network structures of collagen layers is tentatively ascribed to the self-assembly of type I collagen molecules on LB film, not to the dewetting of the collagen solution during drying.

A dewetting process to nano-pattern collagen on hydroxyapatite

Nano-patterned collagen layers on the sintered hydroxyapatite (HAp) disk with a mirror surface were fabricated by the dewetting methods. The surface wettability, energy and tension were of great importance for the formation of nanostructured collagen layers. The maximum adsorption amount and binding affinity of collagen onto the sintered HAp particle were analyzed by the adsorption isotherm curve to elucidate the formation mechanism of nano-patterned collagen layers. The slow evaporation of solvent from the adsorbed collagen layers can produce netlike structures of collagen with an average pore diameter of 80–90 nm, and its height of rims was 2–3 nm from atomic force microscopy.

Resolution of the Vertical and Horizontal Heterogeneity of Adsorbed Collagen Layers by Combination of QCM-D and AFM

Collagen (type I from calf skin) adsorption on polystyrene (PS) and plasma-oxidized polystyrene (PSox) was studied, using a quartz crystal microbalance with energy dissipation measurements (QCM-D) and atomic force microscopy (AFM) in tapping mode. Radio-labeled collagen was used to measure the adsorbed amount and the ability of adsorbed collagen to exchange with molecules in the solution. The results show that the collagen adlayer consists of two parts: a dense and thin sheet in which fibrils are formed (directly observed by AFM) and an overlying thick layer (up to 200 nm) containing protruding molecules or bundles which are in very low concentration but modify noticeably the local viscosity. The thickness and viscosity of the semi-liquid adlayer both increase with adsorption time and collagen concentration. Fibril formation near the surface also increases with time and collagen concentration and occurs more readily on PS compared to PSox. Radiochemical measurements show that this may be related to the larger mobility of molecules adsorbed on PS, presumably owing to a smaller number of binding points.

Surface engineering of stainless steel materials by covalent collagen immobilization to improve implant biocompatibility

It was shown recently that the deposition of thin films of tantalum and tantalum oxide enhanced the long-term biocompatibility of stainless steel biomaterials due to an increase in their corrosion resistance. In this study, we used this tantalum oxide coating as a basis for covalent immobilization of a collagen layer, which should result in a further improvement of implant tissue integration. Because of the high degradation rate of natural collagen in vivo, covalent immobilization as well as carbodiimide induced cross-linking of the protein was performed. It was found that the combination of the silane-coupling agent aminopropyl triethoxysilane and the linker molecule N, N′-disulphosuccinimidyl suberate was a very effective system for collagen immobilizing. Mechanical and enzymatic stability testing revealed a higher stability of covalent bound collagen layers compared to physically adsorbed collagen layers. The biological response induced by the surface modifications was evaluated by in vitro cell culture with human mesenchymal stem cells as well as by in vivo subcutaneous implantation into nude mice. The presence of collagen clearly improved the cytocompatibility of the stainless steel implants which, nevertheless, significantly depended on the cross-linking degree of the collagen layer.

Influence of the aggregation state in solution on the supramolecular organization of adsorbed type I collagen layers

In the last years, adsorbed collagen was shown to form layers with a supramolecular organization depending on the substrate surface properties and on the preparation procedure. If the concentration of collagen and the duration of adsorption are sufficient, fibrillar collagen structures are formed, corresponding to assemblies of a few molecules. This occurs more readily on hydrophobic compared to hydrophilic surfaces. This study aims at understanding the origin of such fibrillar structures and in particular at determining whether they result from the deposition of fibrils formed in solution or from the building of assemblies at the interface. Therefore, type I collagen solutions with an increasing degree of aggregation were prepared, using the “neutral-start” approach, by ageing pH 5.8 solutions at 37 °C for 15 min, 2 or 7 days. The obtained solutions were used to investigate the influence of collagen aggregation in solution on the supramolecular organization of adsorbed collagen layers, which was characterized by X-ray photoelectron spectroscopy and atomic force microscopy. Polystyrene and plasma-oxidized polystyrene were chosen as substrates for the adsorption. The size and the density of collagen fibrils at the interface decreased upon increasing the degree of aggregation of collagen in solution. This is explained by a competitive adsorption process between monomers and aggregates of the solution, turning at the advantage of the monomers. More aggregated solutions, which are thus depleted in free monomers, behave like less concentrated solutions, i.e. lead to a lower adsorbed amount and less fibril formation at the interface. This study shows that the supramolecular fibrils observed in adsorbed collagen layers, especially on hydrophobic substrates, are not formed in the solution, prior to adsorption, but are built at the interface, through the assembly of free segments of adsorbed molecules.

AFM Study of the Interaction of Collagen with Polystyrene and Plasma-Oxidized Polystyrene

Adsorption of collagen on polystyrene (PS) and plasma-oxidized PS (PSox) was investigated using atomic force microscopy (AFM) as the main tool. The extent of alteration produced by the AFM tip upon scanning at controlled loading forces in water was used to obtain semiquantitative information on the properties of the interfacial phase. This information was completed by observing the extent of reorganization of the adsorbed collagen layer after drying. The PSox surface was altered by scanning at high loading force in water. This behavior, which was not observed on PS, was attributed to the presence of a solvated polyelectrolyte layer at the surface of PSox, as demonstrated by previous works. Collagen adsorbed on PS gathered together under the action of the AFM tip while no perturbation was observed for collagen adsorbed on PSox. After drying at low rate of a thin adsorbed collagen layer, a netlike structure was formed on PS, in contrast with PSox. These differences were attributed to the formation of a dense, mechanically strong mat of collagen molecules adsorbed on PSox and/or to anchoring of collagen molecular ends in the swollen surface layer of PSox, in contrast with PS.

Modulable Nanometer-Scale Surface Architecture Using Spin-Coating on an Adsorbed Collagen Layer

A strategy was developed to create a modulable polymer surface architecture (topography, chemical composition) at the nanometer scale. Therefore, collagen was first adsorbed on a poly(methyl methacrylate) (PMMA) substrate and dried at high or low rate to produce continuous or discontinuous layers, respectively. Solutions of PMMA in chlorobenzene were then spin-coated on top of these collagen layers. The obtained surfaces were investigated using atomic force microscopy and X-ray photoelectron spectroscopy. Spin-coating with pure chlorobenzene on the continuous collagen layer produced pits in the PMMA substrate through the collagen layer; spin-coating with PMMA solutions of increasing concentrations progressively led to the formation of a surface covered by particles made of PMMA and collagen, resulting from a combination of dissolution of PMMA from the substrate and deposition of PMMA from the solution. Spin-coating with pure chlorobenzene on the discontinuous collagen layer led to dissolution of PMMA and to its redeposition on the collagen pattern, which served as a template. This provided a surface entirely composed of PMMA, with cavities in the range of 0.1-1 mum diameter and 50-250 nm depth. In this case, the surface relief was independent of the PMMA concentration of the spin-coated solution, the substrate PMMA dissolution and redeposition being the dominant processes.

Probing the organization of adsorbed protein layers: complementarity of atomic force microscopy, X-ray photoelectron spectroscopy and radiolabeling

Atomic force microscopy (AFM) has been used to investigate the organization of the collagen layer present on polymer substrata after adsorption and drying, while the adsorbed amount was monitored using X-ray photoelectron spectroscopy (XPS) and radiochemical measurements. Differences in the organization of the adsorbed collagen layer (surface coverage, layer thickness) observed by AFM fitted well with those found by models obtained from XPS and radiolabeling data.

DECREASED INTRACELLULAR PROTEOLYSIS CORRELATES WITH THE MAINTENANCE OF A SPECIFIC ISOENZYME OF CYTOCHROME P-450

The rates of intracellular protein degradation, of identically labelled populations of proteins, were compared in hepatocytes cultured at 37° (on an adsorbed collagen layer) and in cells preserved on gelatin gels at 10°C. The half-lives of the long-lived proteins were 35.4±8.6h (N=4) and 692.9±216.9h (N=4) respectively. Proteolysis was substantially decreased at 10°C but the rate of decrease remained constant. Hepatocytes rapidly removed resorufin from the culture medium. The resorufin was not being conjugated or accumulated within the cells. Dicumarol, a potent inhibitor of quinone oxidoreductase, at high concentration (500μm ) caused only a 72% decrease in the utilization of resorufin. The microsomal detoxifying enzyme, cytochrome P-450 1A1 remained at a constant level in the preserved hepatocyte monolayers. The results of this study strongly favour storing hepatocytes at 10°C rather than at 4° or 37°C.

Collagen at interfaces. I. In situ collagen adsorption at solution/air and solution/polymer interfaces.

Collagen was isolated from rat tail tendons and acetylated with 1-14C acetic anhydride. In situ adsorption of this collagen from a buffer solution (pH = 2.7) was measured at the interfaces to air, polyethylene and polyethylene grafted with poly(maleic acid), respectively. The kinetics of adsorption were recorded for all surfaces studied and the corresponding diffusion coefficients for collagen in solution with various protein concentrations were calculated. The desorption of collagen from polymer surfaces was also studied. These experiments reveal the existence of both a reversibly and an irreversibly adsorbed collagen layer on the polymers tested. The desorption/adsorption ratio for the polyethylene is higher than that for the grafted polyethylene indicating stronger interactions of collagen with the grafted surface than with the non-modified polyethylene.