Incorporation of basic fibroblast growth factor by a layer-by-layer assembly technique to produce bioactive substrates

Basic fibroblast growth factor (bFGF) was immobilized onto quartz slides and collagen films by assembly with chondroitin sulfate (CS) in a layer-by-layer (LBL) manner. First, the LBL-deposition process on the amino-silanized quartz slides was monitored by UV-vis spectroscopy and water contact angle measurement. By substituting the normal bFGF with rhodamine-labeled one (Rd-bFGF), a linear increase of the absorbance versus bilayer number was recorded. The water contact angle oscillated between the odd CS and the even bFGF layers, demonstrating the alternating change of the surface chemistry. Scanning force microscopy (SFM) revealed that the surface topography was altered slightly after multilayer assembly. In vitro incubation of the CS/bFGF multilayers in PBS showed that approximately 30% of the incorporated bFGF was released within 8 days. In vitro cell culture found that the fibroblasts showed star-like morphology with plenty of pseudopods on the bFGF-incorporated collagen film after cultured for 1 day, and the collagen films assembled with bFGF possess improved bioactivity than that of the virgin one and the bFGF control. Since the immobilized growth factors can maximally retain their bioactivity, the LBL assembly would be a potential approach to construct a bioactive substrate for biomedical applications.     

Layer-By-Layer Assembly of Collagen Thin Films: Controlled Thickness and Biocompatibility

The extracellular matrix molecular collagen is the one of the most widely utilized scaffolding materials in tissue engineering. However, obtaining uniform bioactive collagen films in the nanoscale range and precisely controlling the physical and chemical properties of these biological films is still a challenge for biomedical engineering. Layer-by-layer assembly, i.e., sequential adsorption of oppositely charged macromolecular species, is a powerful new film preparation technique that can be applied to the design of versatile biomaterials, with well-controlled interfacial, mechanical and biological functions. To demonstrate the feasibility of biomaterial design by means of layer-by-layer assembly, type-I collagen thin films were prepared by using this technique with poly(styrene) sulfonate as a partner polyelectrolyte. The gradual build-up of the collagen films was confirmed by UV-vis spectroscopy and ellipsometry, while their surface morphology was assessed by atomic force microscopy. The thickness of the collagen layers can be changed by increasing the number of bilayers adsorbed with an increment of 13 nm. It was found that the layer-by-layer assembled collagen scaffolds can support the attachment and growth of C2C12 myoblast cells and PC12 pheochromocytoma cells. Accurate thickness control and compatibility with nerve cell precursors indicate the utility of layer-by-layer assembled films in neuroprosthesis.     

Surface engineering of titanium thin films with silk fibroin via layer-by-layer technique and its effects on osteoblast growth behavior

The objective of the present study was to surface modify the titanium thin films to improve its biocompatibility. A layer-by-layer (LBL) self-assembly technique, based on the electrostatic interactions mediated adsorption of chitosan (Chi) and silk fibroin (SF), was used leading to the formation of multilayers on the titanium thin film surfaces. The surface chemistry and wettability of LBL films were investigated by X-ray photoelectron spectroscopy (XPS), water contact angle measurement, and atomic force microscopy, respectively. XPS and contact angle measurement results indicated that a full SF/Chi pair film was formed after the deposition layers of PEI/(SF/Chi)(2) on the titanium film surfaces. The topographies of multilayered films were directly related to the corresponding outmost layer components. The build-up of such SF/Chi pair films on titanium films may in turn affect the biocompatibility of the modified titanium films. Therefore, an in vitro investigation was performed to confirm this hypothesis. Cell proliferation, cell viability, DNA synthesis as well as differentiation function (alkaline phosphatase) of osteoblasts on LBL-modified titanium films and control samples were investigated, respectively. Osteoblasts cultured on modified titanium films was found to be higher proliferation tendency than that on control (p < 0.05). Cell viability, alkaline phosphatase as well as DNA synthesis measurement indicated that osteoblasts on LBL-modified films were greater (p < 0.05 or p < 0.01) than the control, respectively. These results suggest that surface engineering of titanium was successfully achieved via LBL deposition of Chi/SF pairs, and enhanced its cell biocompatibility. The approach presented in the study may be exploited as an efficient alternative for surface engineering of titanium-based implants.     

Surface modifications and cell-materials interactions with anodized Ti

The objective of this study was to investigate in vitro cell-materials interactions using human osteoblast cells on anodized titanium. Titanium is a bioinert material and therefore becomes encapsulated after implantation into the living body by a fibrous tissue that isolates it from the surrounding tissues. In this work, a bioactive TiO(2) layer was grown on commercially pure titanium substrate by an anodization process using different electrolyte solutions, namely H(3)PO(4), HF and H(2)SO(4). These electrolytes produced bioactive TiO(2) films with a nonporous structure showing three distinctive surface morphologies. Human osteoblast cell growth behavior was studied with as-received and anodized surfaces using an osteoprecursor cell line (OPC 1) for 3, 5 and 11days. When anodized surfaces were compared for cell-materials interaction, it was noticed that each of the surfaces has different surface properties, which led to variations in cell-materials interactions. Colonization of the cells was noticed with a distinctive cell-to-cell attachment in the HF anodized surface. Good cellular adherence with extracellular matrix extensions in between the cells was noticed for samples anodized with H(3)PO(4) electrolyte. The TiO(2) layer grown in H(2)SO(4) electrolyte did not show significant cell growth on the surface, and some cell death was also noticed. Cell adhesions and differentiation were more pronounced with vinculin protein and alkaline phosphatase, respectively, on anodized surfaces. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assays also showed an increase in living cell density and proliferation with anodized surfaces. It was clear that rough surface morphology, high surface energy and low values of contact angles were important factors for better cell materials interaction. A mineralization study was done in simulated body fluid with ion concentrations nearly identical to those of human blood plasma to further understand biomimetic apatite deposition behavior. Similar to cell-materials interaction, variations in mineral deposition behavior were also noticed for films grown with different electrolytes.     

Silk film biomaterials for cornea tissue engineering

Biomaterials for corneal tissue engineering must demonstrate several critical features for potential utility in vivo, including transparency, mechanical integrity, biocompatibility and slow biodegradation. Silk film biomaterials were designed and characterized to meet these functional requirements. Silk protein films were used in a biomimetic approach to replicate corneal stromal tissue architecture. The films were 2 μm thick to emulate corneal collagen lamellae dimensions, and were surface patterned to guide cell alignment. To enhance trans-lamellar diffusion of nutrients and to promote cell–cell interaction, pores with 0.5–5.0 μm diameters were introduced into the silk films. Human and rabbit corneal fibroblast proliferation, alignment and corneal extracellular matrix expression on these films in both 2D and 3D cultures were demonstrated. The mechanical properties, optical clarity and surface patterned features of these films, combined with their ability to support corneal cell functions suggest that this new biomaterial system offers important potential benefits for corneal tissue regeneration.     

Biomimetic materials for tissue engineering

The development of biomaterials for tissue engineering applications has recently focused on the design of biomimetic materials that are capable of eliciting specific cellular responses and directing new tissue formation mediated by biomolecular recognition, which can be manipulated by altering design parameters of the material. Biomolecular recognition of materials by cells has been achieved by surface and bulk modification of biomaterials via chemical or physical methods with bioactive molecules such as a native long chain of extracellular matrix (ECM) proteins as well as short peptide sequences derived from intact ECM proteins that can incur specific interactions with cell receptors. The biomimetic materials potentially mimic many roles of ECM in tissues. For example, biomimetic scaffolds can provide biological cues for cell-matrix interactions to promote tissue growth, and the incorporation of peptide sequences into materials can also make the material degradable by specific protease enzymes. This review discusses the surface and bulk modification of biomaterials with cell recognition molecules to design biomimetic materials for tissue engineering. The criteria to design biomimetic materials such as the concentration and spatial distribution of modified bioactive molecules are addressed. Recent advances for the development of biomimetic materials in bone, nerve, and cardiovascular tissue engineering are also summarized.     

Polysaccharide-protein surface modification of titanium via a layer-by-layer technique: characterization and cell behaviour aspects

To improve the surface biocompatibility of titanium films, a layer-by-layer (LBL) self-assembly technique, based on the polyelectrolyte-mediated electrostatic adsorption of chitosan (Chi) and gelatin (Gel), was used leading to the formation of multilayers on the titanium thin film surfaces. The film growth was initialized by deposition of one layer of positively charged poly(ethylene imine) (PEI). Then the thin film was formed by the alternate deposition of negatively charged Gel and positively charged Chi utilizing electrostatic interactions. The LBL film growth was monitored by several techniques. The chemical composition, surface topography as well as wettability were investigated by using X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), confocal laser scanning microscopy (CLSM) and water contact angle measurement, respectively. Quantitative XPS analysis showed the alternative change of C/N ratio after four sequential cycles coating of Ti/PEI/Gel/Chi/Gel, which indicated the discrete layer structure of coatings. Uncoated titanium (control sample) displayed a smooth surface morphology (root mean square (RMS) roughness was around 2.5 nm). A full coverage of coating with Gel/Chi layers was achieved on the titanium surface only after the deposition layers of PEI/(Gel/Chi)2. The PEI/Gel/(Chi/Gel)3 layer displayed a rough surface morphology with a tree-like structure (RMS roughness is around 82 nm). These results showed that titanium films could be modified with Chi/Gel which may affect the biocompatibility of the modified titanium films. To confirm this hypothesis, cell proliferation and cell viability of osteoblasts on LBL-modified titanium films as well as control samples were investigated in vitro. The proliferation of osteoblasts on modified titanium films was found to be greater than that on control (p<0.05) after 1 and 7 days culture, respectively. Cell viability measurement showed that the Chi/Gel-modified films have higher cell viability (p<0.05) than the control. These data suggest that Chi/Gel were successfully employed to surface engineer titanium via LBL technique, and enhanced its cell biocompatibility. The approach presented here may be exploited for fabrication of titanium-based implant surfaces.     

The use of layer by layer self-assembled coatings of hyaluronic acid and cationized gelatin to improve the biocompatibility of poly(ethylene terephthalate) artificial ligaments for reconstruction of the anterior cruciate ligament

In this study layer by layer (LBL) self-assembled coatings of hyaluronic acid (HA) and cationized gelatin (CG) were used to modify polyethylene terephthalate (PET) artificial ligament grafts. Changes in the surface properties were characterized by scanning electron microscopy, attenuated total reflection Fourier transform infrared spectroscopy, energy-dispersive X-ray spectroscopy, and contact angle and biomechanical measurements. The cell compatibility of this HA–CG coating was investigated in vitro on PET films seeded with human foreskin dermal fibroblasts over 7 days. The results of our in vitro studies demonstrated that the HA–CG coating significantly enhanced cell adhesion, facilitated cell growth, and suppressed the expression of inflammation-related genes relative to a pure PET graft. Furthermore, rabbit and porcine anterior cruciate ligament reconstruction models were used to evaluate the effect of this LBL coating in vivo. The animal experiment results proved that this LBL coating significantly inhibited inflammatory cell infiltration and promoted new ligament tissue regeneration among the graft fibers. In addition, the formation of type I collagen in the HA–CG coating group was much higher than in the control group. Based on these results we conclude that PET grafts coated with HA–CG have considerable potential as substitutes for ligament reconstruction.     

Antimicrobial polypeptide multilayer nanocoatings

A multilayer coating (or film) of nanometer-thick layers can be made by sequential adsorption of oppositely charged polyelectrolytes on a solid support. The method is known as layer-by-layer assembly (LBL). No special apparatus is required for LBL and nanofilms can be prepared under mild, physiological conditions. A multilayer nanofilm in which at least one of the constituent species is a polypeptide is a polypeptide multilayer nanofilm. The present work was aimed at assessing whether polypeptide multilayer nanofilms with specific antimicrobial properties could be prepared by incorporation of a known antimicrobial agent in the film structure, in this case the edible protein hen egg white lysozyme (HEWL). The chicken enzyme is widely employed as a human food preservative. An advantage of LBL in this context is that the nanofilm is fabricated directly on the surface of interest, eliminating the need to incorporate the antimicrobial in other packaging materials. Here, nanofilms were made of poly(L-glutamic acid) (PLGA), which is highly negatively charged in the mildly acidic pH range, and HEWL, which has a high net positive charge at acidic pH. We show that PLGA/HEWL nanofilms inhibit growth of the model microbe Microccocus luteus in the surrounding liquid medium. The amount of HEWL released from PLGA/HEWL films depends on the number of HEWL layers and therefore on the total quantity of HEWL in the films. This initial study provides a sketch of the scope for further development of LBL in the area of antimicrobial polypeptide multilayer films. Potential applications of such films include strategies for food preservation and coatings for implant devices.     

Antimicrobial polypeptide multilayer nanocoatings

A multilayer coating (or film) of nanometer-thick layers can be made by sequential adsorption of oppositely charged polyelectrolytes on a solid support. The method is known as layer-by-layer assembly (LBL). No special apparatus is required for LBL and nanofilms can be prepared under mild, physiological conditions. A multilayer nanofilm in which at least one of the constituent species is a polypeptide is a polypeptide multilayer nanofilm. The present work was aimed at assessing whether polypeptide multilayer nanofilms with specific antimicrobial properties could be prepared by incorporation of a known antimicrobial agent in the film structure, in this case the edible protein hen egg white lysozyme (HEWL). The chicken enzyme is widely employed as a human food preservative. An advantage of LBL in this context is that the nanofilm is fabricated directly on the surface of interest, eliminating the need to incorporate the antimicrobial in other packaging materials. Here, nanofilms were made of poly(L-glutamic acid) (PLGA), which is highly negatively charged in the mildly acidic pH range, and HEWL, which has a high net positive charge at acidic pH. We show that PLGA/HEWL nanofilms inhibit growth of the model microbe Microccocus luteus in the surrounding liquid medium. The amount of HEWL released from PLGA/HEWL films depends on the number of HEWL layers and therefore on the total quantity of HEWL in the films. This initial study provides a sketch of the scope for further development of LBL in the area of antimicrobial polypeptide multilayer films. Potential applications of such films include strategies for food preservation and coatings for implant devices.     

Characterization and cell behavior of titanium surfaces with PLL/DNA modification via a layer-by-layer technique

This study describes the fabrication of two types of multilayered films onto titanium by layer-by-layer (LBL) self-assembly, using poly-L-lysine (PLL) as the cationic polyelectrolyte and deoxyribonucleic acid (DNA) as the anionic polyelectrolyte. The assembling process of each component was studied using atomic force microscopy (AFM) and quartz crystal balance (QCM). Zeta potential of the LBL-coated microparticles was measured by dynamic light scattering. Titanium substrates with or without multilayered films were used in osteoblast cell culture experiments to study cell proliferation, viability, differentiation, and morphology. Results of AFM and QCM indicated the progressive build-up of the multilayered coatings. The surface morphology of three types of multilayered films showed elevations in the nanoscale range. The data of zeta potential showed that the surface terminated with PLL displayed positive charge while the surface terminated with DNA displayed negative charge. The proliferation of osteoblasts on modified titanium films was found to be greater than that on control (p < 0.05) after 3 and 7 days culture, respectively. Alamar blue measurement showed that the PLL/DNA-modified films have higher cell viability (p < 0.05) than the control. Still, the alkaline phosphatase activity assay revealed a better differentiated phenotype on three types of multilayered surfaces compared to noncoated controls. Collectively our results suggest that PLL/DNA were successfully used to surface engineer titanium via LBL technique, and enhanced its cell biocompatibility.     

Layer-by-layer microfluidics for biomimetic three-dimensional structures

Due to the complex structures of living systems, with size scales spanning from the micron to millimeter range, the use of microtechnology to recreate in vivo-like architecture has exciting potential applications. However, most microscale systems are two-dimensional, and few three-dimensional (3-D) systems are being explored. We have developed a versatile technique, combining surface engineering with layer-by-layer microfluidics technology, to create a 3-D microscale hierarchical tissue-like structure. The process involves immobilization of a cell-matrix assembly, cell-matrix contraction, and pressure-driven microfluidic delivery. An aminopropyltriethoxysilane-glutaraldehyde activated chip is used to effectively immobilize the cell-matrix assemblies while maintaining cell viability. Pressure-driven microfluidics is applied to transport cells-matrices with controlled flow rates, determined from dynamic flow imaging. By taking advantage of the contraction of the biopolymer matrices by cells, layer-by-layer microfluidics can be used to build multilayers of cell-matrix inside a microchannel and the thickness of each layer can be controlled down to microscale dimensions. Confocal and electron microscopy images of the final structure show a hierarchical layered cellular configuration composed of heterogeneous biomimetic materials. For a model system, a biomimetic arterial structure is formed using three types of vascular cells to mimic the 3-tunic structure found in vivo. This approach provides solutions to fabricate hierarchical "neotissues" with controlled microarchitectures and 3-D configurations of multiple cell types.     

Preparation and characterization of bioactive mesoporous calcium silicate-silk fibroin composite films

Composite films of bioactive mesoporous calcium silicate (MCS)/silk fibroin (SF) and conventional calcium silicate (CS)/SF were fabricated by the solvent casting method, and the structures and properties of the composite films were characterized. Results of field emission scanning electron microscope (FESEM) indicated that MCS or CS was uniformly dispersed in the SF films. The measurements of the water contact angles suggested that the incorporation of either MCS or CS into SF could improve the hydrophilicity of the composite films, and the former was more effective than the later. The bioactivity of the composite films was evaluated by soaking in a simulated body fluid (SBF), and the formation of a hydroxycarbonate apatite (HCA) layer was determined by XRD and FT-IR. The results showed that the MCS/SF composite films have significantly enhanced apatite-forming bioactivity compared with the CS/SF composite films owing to the highly specific surface area and pore volume of MCS. In vitro cell attachment and proliferation tests showed that the MCS/SF composite film was a good matrix for the growth of MG63 cells. Consequently, the MCS/SF composite film possessed excellent physicochemical and biological properties, indicating its potential application for bone tissue engineering by designing 3D scaffolds according to its corresponding composition.     

The effect of acetylcholine-like biomimetic polymers on neuronal growth

Driven by clinical needs, nerve regeneration studies have recently become the focus of research and area of growth in tissue engineering. Biomimetic polymer synthesis and functional interface construction is a promising solution to induce neuritic sprouting and guide the regenerating nerve. However, few studies have been made on primary hippocampal neurons. In this study, a new type of acetylcholine-like biomimetic polymers for their potential in biomaterial-modulated nerve regeneration application is synthesized using click chemistry and free radical polymerization. The structure of the synthesized polymers includes a "bioactive" unit (acetylcholine-like unit) and a "bioinert" unit [poly(ethylene glycol) unit]. To explore the effects of the bioactive unit and the bioinert unit on neuronal growth, different ratios of the two initial monomers poly(ethylene glycol) monomethyl ether-glycidyl methacrylate (MePEG-GMA) and dimethylaminoethyl methacrylate (DMAEMA) were employed and five different polymers were synthesized. Their chemical structures were characterized using (1)H nuclear magnetic resonance and Fourier-transform infrared spectroscopy, and their physical properties (including molecular weight, polydispersity, glass transition temperature, and melting point) were determined using gel permeation chromatography and differential scanning calorimetry. Culturing of the primary rat hippocampal neurons on the polymeric surfaces show that the ratio of the two initial monomers utilized for polymer synthesis significantly affects neuronal growth. Rat hippocampal neurons show different growth morphologies on different polymeric surfaces. The polymeric surface prepared with 1:60 (mol/mol) of MePEG-GMA to DMAEMA induces neuronal regenerative responses similar to that on poly-l-lysine, a very common benchmark material for nerve cell cultures. These results suggest that acetylcholine-like biomimetic polymers are potential biomaterials for neural engineering applications, particularly in modulating the growth of hippocampal neurons.     

Cellular response to nanoscale elastin-like polypeptide polyelectrolyte multilayers

Ionic elastin-like polypeptide (ELP) conjugates are a new class of biocompatible, self-assembling biomaterials. ELPs composed of the repeat unit (GVGVP)(n) are derived from the primary sequence of mammalian elastin and produced in Escherichia coli. These biopolymers exhibit an inverse transition temperature that renders them extremely useful for applications in cell-sheet engineering. Cationic and anionic conjugates were synthesized by the chemical coupling of ELP to polyethyleneimine (PEI) and polyacrylic acid (PAA). The self-assembly of ELP-PEI and ELP-PAA using the layer-by-layer deposition of alternately charged polyelectrolytes is a simple, versatile technique to generate bioactive and biomimetic surfaces with the ability to modulate cell-substratum interactions. Our studies are focused on cellular response to self-assembled multilayers of ionic (GVGVP)(40) incorporated within the polymeric sequence H(2)N-MVSACRGPG-(GVGVP)(40)-WP-COOH. Angle-dependent XPS studies indicated a difference in the chemical composition at the surface ( approximately 10A below the surface) and subsurface regions. These studies provided additional insight into the growth of the nanoscale multilayer assembly as well as the chemical environment that the cells can sense. Overall, cellular response was enhanced on glass substrata coated with ELP conjugates compared with uncoated surfaces. We report significant differences in cell proliferation, focal adhesions and cytoskeletal organization as a function of the number of bilayers in each assembly. These multilayer assemblies have the potential to be successfully utilized in the rational design of coatings on biomaterials to elicit a desired cellular response.     

A Biomimetic Material with a High Bio-responsibility for Bone Reconstruction and Tissue Engineering

A biomimetic composite was prepared using type-I collagen as the matrix, and particles of sol–gel-derived bioactive glass (58S), hyaluronic acid and phosphatidylserine as additives. The material has an interconnected 3-D porous structure with a porosity > 85%. When incubated in simulated body fluid (SBF), the composite induced the formation of microcrystals of bone-like hydroxyapatite (HA), suggesting good bioactive properties. During the in vitro cell-culture experiment, MC3T3-E1 cells adhered to, migrated and spread on the surface of the porous composite. The material was employed to repair a 10-mm defect in a rabbit's radius. The composite was gradually degraded within 8 weeks and replaced by new bone. After 12 weeks, the bone marrow cavity was restored and the Haversian canal was noted from the histological observation. The biomimetic composite is a potential scaffold material for bone reconstruction and bone tissue engineering.     

Development of biodegradable polyurethane and bioactive glass nanoparticles scaffolds for bone tissue engineering applications

The development of polymer/bioactive glass has been recognized as a strategy to improve the mechanical behavior of bioactive glass-based materials. Several studies have reported systems based on bioactive glass/biopolymer composites. In this study, we developed a composite system based on bioactive glass nanoparticles (BGNP), obtained by a modified St?ber method. We also developed a new chemical route to obtain aqueous dispersive biodegradable polyurethane. The production of polyurethane/BGNP scaffolds intending to combine biocompatibility, mechanical, and physical properties in a material designed for tissue engineering applications. The composites obtained were characterized by structural, biological, and mechanical tests. The films presented 350% of deformation and the foams presented pore structure and mechanical properties adequate to support cell growth and proliferation. The materials presented good cell viability and hydroxyapatite layer formation upon immersion in simulated body fluid.     

Fabrication, characterization, and biological assessment of multilayer DNA coatings on sandblasted-dual acid etched titanium surface

As local gene therapy has received attention, immobilizing functional gene onto irregular oral implant surface has become an advanced challenge. Electrostatic layer-by-layer (LBL) assembly technique could achieve this goal and allow local and efficient administration of genes to the target cells. In this study, multilayers of cationic lipid/plasmid DNA (pEGFP-C1) complex (LDc) and anionic hyaluronic acid were assembled onto sandblasted-dual acid etched titanium disks by the LBL technique. Surface characteristics of the coatings were performed by x-ray photospectroscopy (XPS), contact angle measurements, and scanning electron microscopy (SEM). The cell biological characteristics of the coatings were evaluated by in vitro experiments. SEM results demonstrated that the porous titanium surface was gradually flattened with the increase of the multilayer. The XPS survey indicated that the N element was found from the coating. The coating degradation and pEGFP-C1 releasing kinetics showed that the more assembled layer numbers were, the larger the amount of DNA released in the first 30 h. MC3T3-E1 cells were cultured directly on the DNA-loaded surface. Higher enhanced green fluorescent protein (EGFP) expression efficiency was achieved by increasing the number of layers when cells were cultured after 24 or 72 h. The MC3T3-E1 cell viability on the surface of multilayer DNA coatings was significantly higher than that on control porous titanium surface. It was concluded that the approach established by the LBL technique had great potential in immobilizing gene coatings onto the porous titanium surface and subsequently influenced the function of the cultured cell.     

Cell-adhesive and blood-coagulant properties of ultrathin poly(methyl methacrylate) stereocomplex films

Isotactic (it) and syndiotactic (st) poly(methyl methacrylate)s (PMMAs) were assembled on a poly(ethylene terephthalate) cell disk (as a solid substrate), which is normally used for cell culture, to produce ultrathin stereocomplex films. Static contact angles of the films using air bubbles in aqueous phase revealed the stepwise assembly of both polymers. More L929 fibroblast cells adhered and were flattened on the ultrathin stereocomplex films, compared to homogeneous PMMA films and to spin-coated films comprised of a mixed solution of it- and st-PMMAs with a molar ratio of 1 : 2, which is the stoichiometry of a stereocomplex. The difference in the number of adherent cells was greatest after the first 3 h of incubation. Pre-adsorption of proteins, which are related to cell adhesion, onto the stereocomplex film potentially modulated the cell-adhesive properties. Fresh whole human blood did not coagulate on the complex film for 20 min, although blood coagulated after 15 min on the homogeneous films and on the 1 : 2 mixed film. Platelets did not adhere onto the complex films after 15 min of incubation, which was consistent with the results of blood coagulation. These observations indicate that the stereocomplex formation of PMMAs on the surface of films strongly affects the bioactivity of these films.