RGD肽对内皮细胞在聚酯材料表面粘附、增殖的影响

RGD是许多粘附蛋白结构中的高度保守序列，与细胞在生物材料表面的粘附、增殖密切相关。本研究在聚酯薄膜表面分别预衬纤维粘连蛋白和共价接枝RGD三肽，然后在不同聚酯材料上种植体外培养的人脐静脉内皮细胞，结果显示RGD可明显促进细胞在材料表面的粘附和增殖，与纤维粘连蛋白相比，RGD促进细胞粘附的作用更为明显，而在细胞增殖方面，二者的作用无显著性差异。本研究为改进生物材料的表面设计，促进心血管移植物的内皮化提供了一个切实可行的思路。     

Development of RGD peptides grafted onto silica surfaces: XPS characterization and human endothelial cell interactions.

The attachment of human umbilical vein endothelial cells (HUVECs) on substrates that had been covalently grafted with the cell adhesion peptides Arg-Gly-Asp (RGD) was investigated. This approach was used to provide substrates that are adhesive to cells even in the absence of serum proteins and to cells that have had no prior treatment of the surface with proteins that promote cell adhesion. We wanted to improve control of cellular interactions with cell-adhesive materials by providing fixedly bound adhesion ligands. Silica was examined as a model surface. The peptides were grafted using three different steps: grafting of aminosilane molecules; reaction with a maleimide molecule; and immobilization of cell-binding peptides containing the RGD sequence. The RGD-grafted surface was characterized by X-ray photoelectron spectroscopy (XPS) and contact-angle measurements.     

Corneal epithelial cell growth over tethered-protein/peptide surface-modified hydrogels

In this study, we investigated the corneal epithelial cell growth rate and adhesion to novel hydrogels with (1) extracellular matrix proteins [fibronectin, laminin, substance P, and insulin-like growth factor-1 (IGF-1)] and (2) peptide sequences [RGD and fibronectin adhesion-promoting peptide (FAP)] tethered to their surface on poly(ethylene glycol) (PEG) chains. The growth rate to confluence of primary rabbit cornea epithelial cells was compared for plain polymethacrylic acid-co-hydroxyethyl methacrylate (PHEMA/MAA) hydrogels, PHEMA/MAA hydrogels coated with extracellular matrix proteins or peptides, and PHEMA/MAA hydrogels with tethered extracellular matrix proteins or peptides on the surface. The development of focal adhesions by the epithelial cells grown on the surfaces was determined by F-actin staining. Little to no epithelial cell growth occurred on the plain hydrogel surfaces throughout the 15-day culture period. Of the coated hydrogels, only the fibronectin-coated surfaces showed a significant increase in cell growth compared to plain hydrogels (p < 0.009). However, even these surfaces reached a maximum of only 20% confluence. Laminin, fibronectin adhesion-promoting peptide (FAP), and fibronectin/laminin (1:1) tether-modified hydrogels all achieved 100% confluence by the end of the culture period, although the rates at which confluence was reached differed. F-actin staining showed that focal adhesions were formed for the laminin, FAP, and fibronectin/laminin tether-modified surfaces. The results support the hypothesis that tethering certain extracellular matrix proteins and/or peptides to the hydrogel surface enhances epithelial cell growth and adhesion, compared with that seen for protein-coated or plain hydrogel surfaces.     

Enhancing hepatocyte adhesion by pulsed plasma deposition and polyethylene glycol coupling

Decreased hepatocyte adhesion to polymeric constructs limits the function of tissue engineered hepatic assist devices. We grafted adhesion peptides (RGD and YIGSR) to polycaprolactone (PCL) and poly-L-lactic acid (PLLA) in order to mimic the in vivo extracellular matrix and thus enhance hepatocyte adhesion. Peptide grafting was done by a novel technique in which polyethylene glycol (PEG)-adhesion peptide was linked to allyl-amine coated on the surface of PCL and PLLA by pulsed plasma deposition (PPD). Peptide grafting density, quantified by radio-iodinated tyrosine in YIGSR, was 158 fmol/cm(2) on PLLA and 425 fmol/cm(2) on PCL surfaces. The adhesion of hepatocytes was determined by plating 250,000 hepatocytes/well (test substrates were coated on 12 well plates) and quantifying the percentage of adhered cells after 6 h by MTT assay. Adhesion on PCL surfaces was significantly enhanced (p < 0.05) by both YIGSR (percentage of adhered cells = 53 +/- 7%) and RGD (53 +/- 12%) when compared to control surfaces (31 +/- 8%). Hepatocyte adhesion on PLLA was significantly (p < 0.05) enhanced on PLLA-PEG-RGD surfaces (76 +/- 14%) compared to control surfaces (42 +/- 19%) and more (68 +/- 25%) but not statistically significant (p = 0.15) on PLLA-PEG-YIGSR surfaces compared to control surfaces. These results indicate that hepatocyte adhesion to PCL and PLLA based polymeric surfaces can be enhanced by a novel adhesion peptide grafting technique using pulsed plasma deposition and PEG cross-linking.     

Interactions of corneal epithelial cells and surfaces modified with cell adhesion peptide combinations

In order to facilitate the adhesion of corneal epithelial cells to a poly dimethyl siloxane (PDMS) substrate ultimately for the development of a synthetic keratoprosthesis, PDMS surfaces were modified by covalent attachment of combinations of cell adhesion and synergistic peptides derived from laminin and fibronectin. Peptides studied included YIGSR and its synergistic peptide PDSGR from laminin and the fibronectin derived RGDS and PHSRN. Surfaces were modified with combinations of peptides determined by an experimental design. Peptide surface densities, measured using 125-I labeled tyrosine containing analogs, were on the order of pmol/cm2. Surface density varied as a linear function of peptide concentration in the reaction solution, and was different for the different peptides examined. The lowest surface density at all solution fractions was obtained with GYRGDS, while the highest density was consistently obtained with GYPDSGR. These results provide evidence that the surfaces were modified with multiple peptides. Water contact angles and XPS results provided additional evidence for differences in the chemical composition of the various surfaces. Significant differences in the adhesion of human corneal epithelial cells to the modified surfaces were noted. Statistical analysis of the experimental adhesion results suggested that solution concentration YIGSR, RGDS, and PHSRN as well as the interaction effect of YIGSR and PDSGR had a significant effect on cell interactions. Modification with multiple peptides resulted in greater adhesion than modification with single peptides only. Surface modification with a control peptide PPSRN in place of PHSRN resulted in a decrease in cell adhesion in virtually all cases. These results suggest that surface modification with appropriate combinations of cell adhesion peptides and synergistic peptides may result in improved cell surface interactions.     

Suppression of apoptosis by enhanced protein adsorption on polymer/hydroxyapatite composite scaffolds

Bone tissue engineering is a promising alternative to bone grafting. Scaffolds play a critical role in tissue engineering. Composite scaffolds made of biodegradable polymers and bone mineral-like inorganic compounds have been reported to be advantageous over plain polymer scaffolds by our group and others. In this study, we compared cellular and molecular events during the early periods of osteoblastic cell culture on poly(l-lactic acid)/hydroxyapatite (PLLA/HAP) composite scaffolds with those on plain PLLA scaffolds, and showed that PLLA/HAP scaffolds improved cell survival over plain PLLA scaffolds. Most cells (MC3T3-E1) on PLLA/HAP scaffolds survived the early culture. In contrast, about 50% of the cells initially adhered to the plain PLLA scaffolds were detached within the first 12h and showed characteristics of apoptotic cell death, which was confirmed by TUNEL staining and caspase-3 activation. To investigate the mechanisms, we examined the adsorption of serum protein and adhesion molecules to the scaffolds. The PLLA/HAP scaffold adsorbed more than 1.4 times of total serum protein and much greater amounts of serum fibronectin and vitronectin than pure PLLA scaffolds. Similarly, significantly larger amounts of individual adhesion proteins and peptides (fibronectin, vitronectin, RGD, and KRSR) were adsorbed on the PLLA/HAP scaffolds than on the PLLA scaffolds, which resulted in higher cell density on the PLLA/HAP scaffolds. Furthermore, beta1 and beta3 integrins and phosphorylation of Fak and Akt proteins in the cells on the PLLA/HAP scaffolds were significantly more abundent than those on PLLA scaffolds, which suggest that enhanced adsorption of serum adhesion proteins to PLLA/HAP scaffolds protect the cells from apoptosis possibly through the integrin-FAK-Akt pathway. These results demonstrate that biomimetic composite scaffolds are advantageous for bone tissue engineering.     

Surface modification for controlled studies of cell-ligand interactions

This work describes a method for coupling cell adhesion peptides to hydrophobic materials for the purpose of controlling surface peptide density while simultaneously preventing nonspecific protein adsorption. PEO/PPO/PEO triblock copolymers (Pluronic F108) were equipped with terminal pyridyl disulfide functionalities and used to tether RGD containing peptides to polystyrene (PS). The density of F108 on PS was 1.4 E5 +/- 2.12 E1 molecules/microm2. XPS and ToF SIMS indicated that the F108 coating was homogeneous and that the unmodified and activated F108 distributed evenly on PS. By mixing unmodified F108 with PDS-activated F108 prior to adsorption, it was possible to vary peptide density between 0 and 8.7 E4 +/- 2.66 E3 peptides/microm2, while otherwise, maintaining consistent surface properties. GRGDSY grafted PS supported cell attachment, spreading, and development of cytoskeletal structure, all of which were found to increase with increasing peptide density. Cell proliferation followed this same trend, however, maximal growth occurred at a submaximal peptide density. Cell aspect ratio varied in a biphasic manner with GRGDSY density. F108 coated PS and GRGESY grafted PS were inert to cell adhesion. Cells released from GRGDSY grafted PS upon addition of either a reducing agent or free GRGDSY, which indicates that cell-substrate interactions were mediated solely by the tethered peptides.     

Fibronectin facilitates the migration of human natural killer cells

The interaction of lymphocytes with the extracellular matrix plays an important role in the immune defence against tumor cells and virus-infected cells. We have examined the effect of matrix proteins on the migration of large granular lymphocytes (LGL) through 3-μm pores in Nuclepore filters in a Boyden invasion chamber. Fibronectin bound on the filter surface significantly increased (p < 0.001) the capacity of LGL to migrate, whereas soluble fibronectin did not. In addition, a significantly higher (p < 0.001) percentage of LGL was capable of migration through fibronectin-coated filters than through untreated filters. With fibronectin-coated filters, a strong enrichment of CD16⁺ and CD56⁺CD3^? cells with LGL morphology and reduction of CD3⁺ cells was found among migrating cells when the incubation time was 4 h or less. Later agranular lymphocytes, mainly CD3⁺ T lymphocytes, also started to migrate. Laminin coating of filters also facilitated migration, and when filters were coated with both fibronectin and laminin the increase in migration was equal to the sum of the increases induced by each protein alone. Interactions between cell surface and the Arg-Gly-Asp (RGD) peptide sequence of many matrix proteins had no role in the LGL migration through untreated flters. However, when filters were coated with either fibronectin or laminin, or with both, peptide containing the RGD sequence reduced migration to the level of untreated filter, whereas an Arg-Gly-Glu control peptide had no effect. Our results show that unstimulated LGL/natural killer cells are capable of rapid migration through matrix-coated porous membranes, and that interactions between cell surface receptors and the RGD sequence of fibronectin and probably laminin are utilized in this process.     

Acrylic Acid-RGD as a Biomimetic Surface Modifier for Poly(ethylene terephthalate)

INTRODUCTION　Biomaterials play an importantrole in human disease- treatmentand healing〔1,2〕.Due to the good mechanical property,PET is used to the coating of artificial heartvalve,the film of mending hearts and artificial vessel etc〔3〕.But the imperfection isthe low capability of surface hydrophile leading to the high static and low water ad-sorption〔4〕.In the application,traditional artificial cardiovascular materials( e.g.PET) have blood coagulation,alexin- activation and other…     

Indirect coating of RGD peptides using a poly-L-lysine spacer enhances jaw periosteal cell adhesion, proliferation, and differentiation into osteogenic tissue

The aim of our study was to generate a biofunctionalized, three-dimensional (3D) biomaterial to enhance jaw periosteal cell (JPC) adhesion and differentiation into osteogenic tissue. Therefore, open-cell polylactic acid (OPLA) scaffolds were coated covalently with different RGD peptides (a conserved recognition sequence of the most ECM proteins--arginine-glycine-asparagine) and different coating variants. The linear and cyclic RGD peptides were either applied directly or indirectly via a poly-L-lysine (PLL) spacer. JPCs were analyzed on coated constructs in 2D and 3D cultures and showed enhanced rates for indirectly coated scaffolds using the PLL spacer. By gene expression, we detected significantly increased levels of osteogenic marker genes, such as alkaline phosphatase, RUNX2, and AMELY in JPCs seeded onto PLL/linear RGD constructs compared to the otherwise-coated constructs. An analysis of the JPC mineralization capacity revealed the highest amounts of calcium-phosphate precipitates in cells growing within the PLL/linear scaffolds. Additionally, the JPC adhesion behavior on OPLA scaffolds seems to be mediated by ITGB3, ITGB1, and ITGAV, as shown by blocking assays. We concluded that coating of OPLA constructs with linear RGD peptides via PLL represents a suitable approach for functionalizing the polymer surface and enhancing adhesion, proliferation, and mineralization of JPCs.     

Paracoccidioides brasiliensis conidia recognize fibronectin and fibrinogen which subsequently participate in adherence to human type II alveolar cells: involvement of a specific adhesin

We examined the ability of Paracoccidioides brasiliensis conidia to interact with fibronectin, fibrinogen and with A549 cells, in order to establish the nature of the molecules involved. Conidia bound to immobilized proteins in a concentration-dependent manner. Antibodies against fibronectin and fibrinogen inhibited the fungal adherence to the corresponding proteins; as did laminin and fibronectin, but not fibrinogen when added in soluble form; however, the fibrinogen fragment D interfered with adhesion in a significant manner. Various monosaccharides and RGD/RGDS peptides had no effect on adherence to fibronectin or fibrinogen, while N-acetylneuraminic acid (NANA) abolished adherence to both proteins. Additionally, these proteins were detected on the surface of A549 cells. Inhibition assays showed a significant decrease in fungal adherence when A549 cells were treated with anti-fibrinogen, anti-fibronectin antibodies and a purified adhesin of P. brasiliensis (32-kDa protein); or when conidia were treated with these soluble proteins, mAb anti-32-kDa protein, RGD peptides and NANA. These results suggest that fibrinogen and fibronectin facilitate the adherence of conidia to A549 cells probably through the interaction with adhesin-type molecules or a sialic acid based recognition system. These interactions appear to play a role in the initial fungal attachment to the lung, and consequently, also in the pathogenesis of paracoccidioidomycosis.     

Mediating specific cell adhesion to low-adhesive diblock copolymers by instant modification with cyclic RGD peptides

One promising strategy to control the interactions between biomaterial surfaces and attaching cells involves the covalent grafting of adhesion peptides to polymers on which protein adsorption, which mediates unspecific cell adhesion, is essentially suppressed. This study demonstrates a surface modification concept for the covalent anchoring of RGD peptides to reactive diblock copolymers based on monoamine poly(ethylene glycol)-block-poly(D,L-lactic acid) (H(2)N-PEG-PLA). Films of both the amine-reactive (ST-NH-PEG(2)PLA(20)) and the thiol-reactive derivative (MP-NH-PEG(2)PLA(40)) were modified with cyclic alphavbeta3/alphavbeta5 integrin subtype specific RGD peptides simply by incubation of the films with buffered solutions of the peptides. Human osteoblasts known to express these integrins were used to determine cell-polymer interactions. The adhesion experiments revealed significantly increased cell numbers and cell spreading on the RGD-modified surfaces mediated by RGD-integrin-interactions.     

Biological evaluation of RGD peptidomimetics, designed for the covalent derivatization of cell culture substrata, as potential promotors of cellular adhesion.

Our aim was to replace the proteins and peptides, generally used for the biocompatibilization of polymer substrata, with synthetic molecules mimicking the RGD (Arg-Gly-Asp) active sequence. Based on the (L)-tyrosine template, RGD peptidomimetics were constructed; one molecule 3 was equipped with an anchorage arm that allowed its covalent grafting on a culture substratum made from poly(ethylene terephthalate) (PET) microporous membrane. The amount of fixed molecules was readily determined by XPS, using a fluorine tag incorporated in the peptidomimetic structure. The binding of peptidomimetics 1-3 to the vitronectin (VN) and fibronectin (FN) receptors could not be revealed in a test of inhibition of MSC 80 cells adhesion, by the synthetic compounds in solution placed in competition with the adhesive proteins (VN and FN) coating polystyrene plates. However, the cell-attachment activity of peptidomimetic 3 was shown by culturing CaCo2 cells, in the absence of serum, on the PET substratum grafted with 3. The performance of this support was similar to that of PET grafted with the reference peptide RGDS (Arg-Gly-Asp-Ser), and only reduced by half comparatively to the PET grafted with FN.     

The effect of the addition of a polyglutamate motif to RGD on peptide tethering to hydroxyapatite and the promotion of mesenchymal stem cell adhesion

Mimicking endogenous bone-binding proteins, RGD peptides have been synthesized with polyacidic amino acid domains in order to ionically tether the peptides to bone-like synthetic biomaterials, including hydroxyapatite (HA). However, a direct comparison of unmodified RGD with polyacidic-conjugated RGD has not been performed, and thus a benefit for the acidic domain has not been established. We evaluated the peptide/HA bond of RGD peptides with and without an attached polyglutamate sequence (E(7)), as well as examined mesenchymal stem cell (MSC) adhesion and morphology as they were affected by the conjugated peptide. We found that significantly more E(7)RGD was bound to HA than RGD at all coating concentrations tested, and moreover, more E(7)RGD was retained on the HA surface even after extended washing in serum-free media. Consistent with in vitro results, higher levels of E(7)RGD than RGD remained on HA that had been implanted in vivo for 24 h, indicating that the polyacidic domain improved peptide-binding efficiency. At several peptide concentrations, E(7)RGD increased cell adhesion compared to RGD surfaces, establishing a biological benefit for the E(7) modification. In addition, HA pre-coated sequentially with low-density E(7)RGD (1-10 microg/ml) and serum (FBS) stimulated cell adhesion and spreading, compared to either coating alone, suggesting that an ionic linkage allows for the potential adsorption of serum proteins to unoccupied sites, which may be important for bone formation in vivo. Collectively, these results suggest that tethering peptides to HA via a polyglutamate domain is an effective method for improving the peptide/HA bond, as well as for enhancing MSC adhesion.     

Human endothelial cell interactions with surface-coupled adhesion peptides on a nonadhesive glass substrate and two polymeric biomaterials.

The attachment, spreading, spreading rate, focal contact formation, and cytoskeletal organization of human umbilical vein endothelial cells (HUVECs) were investigated on substrates that had been covalently grafted with the cell adhesion peptides Arg-Gly-Asp (RGD) and Tyr-Ile-Gly-Ser-Arg (YIGSR). This approach was used to provide substrates that were adhesive to cells even in the absence of serum proteins and with no prior pretreatment of the surface with proteins of the cell adhesion molecule (CAM) family. This approach was used to dramatically enhance the cell-adhesiveness of substrates that were otherwise cell-nonadhesive and to improve control of cellular interactions with cell-adhesive materials by providing stably bound adhesion ligands. Glycophase glass was examined as a model cell-nonadhesive substrate prior to modification, and polyethylene terephthalate (PET) and polytetrafluoroethylene (PTFE) were examined as representative materials for biomedical applications. The peptides were surface-coupled by their N-terminal amine to surface hydroxyl moieties using tresyl chloride chemistry. Prior to peptide grafting, the PET and PTFE were surface hydroxylated to yield PET-OH and PTFE-OH. The PET-OH was less cell-adhesive and the PTFE-OH was much more cell-adhesive than the native polymers. Radioiodination of a C-terminal tyrosine residue was used to quantify the amount of peptide coupled to the surface, and these amounts were 12.1 pmol/cm2 on glycophase glass, 139 fmol/cm2 on PET-OH, and 31 fmol/cm2 on PTFE-OH. Although the glycophase glass did not support adhesion or spreading even in the presence of serum, the RGD- and YIGSR-grafted glycophase glass did support adhesion and spreading, even when the only serum protein that was included was albumin. Although PET and PTFE-OH supported adhesion when incubated in serum-supplemented medium, neither of these materials supported adhesion with only albumin present, indicating that cell adhesion is mediated by adsorbed CAM proteins. When these materials were peptide-grafted, however, extensive adhesion and spreading did occur even when only albumin was present. Since the peptide grafting is quite easily controlled and is temporally stable, while protein adsorption is quite difficult to precisely control and is temporally dynamic, peptide grafting may be advantageous over other approaches employed to improve long-term cell adhesion to biomaterials.     

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

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

Immunofluorescence and flow cytometry analysis of fibronectin and laminin binding to Sporothrix schenckii yeast cells and conidia

The adherence of Sporothrix schenckii yeast cells to several extracellular matrix (ECM) components has already been demonstrated, but the mechanisms of these interactions remained to be defined. In indirect immunofluorescence assays with polyclonal antibodies directed towards the ECM proteins, both hyphae and yeast cells of S. schenckii exhibited the ability to bind laminin and fibronectin. Flow cytometry confirmed the binding of these proteins, and revealed a significant greater binding capability for the yeast cells than for the conidia. Fibronectin and laminin binding was dose-dependent and specific. In addition, competition experiments with synthetic peptides mimicking the adhesive sequences of these proteins, or with cell wall fractions and carbohydrates constitutive of their sugar chains, were performed in order to specify the peptide or carbohydrate motifs involved in the recognition process. A 50% reduction was noticed in fibronectin binding in the presence of the synthetic peptide RGD, and a 38% reduction in laminin binding with the peptide YIGSR. Some carbohydrate-containing fractions of the yeast cell wall also inhibited the binding of fibronectin, but had no significant effect on laminin binding. Together, these results suggest the presence at the yeast surface of distinct receptors for laminin and fibronectin.     

Outcome and perspectives in bioactive coatings: What''s new, what''s coming

The main interest of calcium phosphate (CaP) coating is osteoconduction and faster and better osteogenic cells spreading and bone ingrowth onto the dental or orthopaedic surface implant. CaP coating are bioactive materials that have been shown to promote early bone apposition at the surface of metallic implants. However, every CaP is more or less resorbable; in this way, new technologies are developed to promote and/or induce osteogenic activity. These evolutions tend to control the chemical nature of CaP ceramic, the porosity and the thickness of the coating; and/or to integrate in the coating osteoinductive or therapeutic agents (biphosphonates, antibiotics); and/or to immobilize constitutional elements of bone on implants surface (growth factors (BMP, TGF-β), adhesion proteins (collagen, fibronectin, laminin, vitronectin) or peptides (RGD sequence). All these technological and biologic progress should be evaluated compared to possible side effects and the long-term benefit for the patients.     

Protein-coated poly(L-lactic acid) fibers provide a substrate for differentiation of human skeletal muscle cells

Tissue engineering represents a potential method for repairing damaged skeletal muscle tissue. Extracellular matrix (ECM) proteins were evaluated for their ability to aid in cell attachment, whereas a poly(L-lactic acid) (PLLA) fiber scaffold was tested as a substrate for the differentiation of human skeletal muscle cells. In comparison to uncoated or gelatin-coated PLLA films, cell attachment increased significantly (p < 0.001) on PLLA films coated with ECM gel, fibronectin, or laminin. Myoblasts differentiated into multinucleated myofibers on ECM gel-coated PLLA fibers, and expressed muscle markers such as myosin and alpha-actinin. Oligonucleotide microarray analysis showed similar gene expression profiles for human skeletal muscle cells on ECM gel-coated PLLA fibers as to that observed for myofibers on tissue culture plates. Therefore, PLLA fibers coated with ECM proteins provide a scaffold for the development of skeletal muscle tissue for tissue engineering and cell transplantation applications.     

Behavior of osteoblasts on a type I atelocollagen grafted ozone oxidized poly L-lactic acid membrane

With oxidizing poly-L-lactic acid (PLLA) surface by ozone, peroxide groups are easily generated on the surface. Those peroxides are broken down by redox-coupling reaction, and provide active species that initiate grafting by reaction with the collagen molecules. The surface density of generated peroxide on a PLLA surface was determined by an iodide method. The maximum concentration of peroxide was about 2.87 x 10(-8) mol/cm2 when ozone oxidation was performed at 60 V for 60 min. After the surface oxidation, type I atelocollagen was grafted onto PLLA surface. All physical measurements on the collagen-grafted surface indicated that the PLLA surface was effectively grafted with type I atelocollagen. Behavior of rat calvaria osteoblasts on type I atelocollagen grafted PLLA (PLLA + COL) surface was observed. Initial attachment of osteoblasts on the surface was significantly enhanced, and it is assumed that the atelocollagen matrix supported the initial attachment and growth of cells. Collagenous protein synthesis of osteoblasts was maintained at relatively low level in the early stage of proliferation due to the primarily existing grafted type I atelocollagen, and then increased in 7 days as the osteoblast differentiated. After 7 days, collagenous protein synthesis in osteoblasts was activated. Alkaline phosphatase (ALPase) activity and mineralization by osteoblasts were promoted on PLLA + COL surface. In comparison with PLLA + COL, non-treated PLLA and tissue culture plate (TCPS) did not show any feature expressed in osteoblasts' maturation up to 9 days in this experiment. The grafted type I atelocollagen provided a favorable matrix for cell migration in relation with collagenase expression. Ozone oxidation might be a favorable method for surface modification of PLLA membranes by collagen grafting, and cell behavior could be modulated by the grafted collagen.