Effects of Poly(L-lysine), Poly(acrylic acid) and Poly(ethylene glycol) on the Adhesion,Proliferation and Chondrogenic Differentiation of Human Mesenchymal Stem Cells

Microenvironments, composed of many kinds of cytokines and growth factors plus extracellular matrices with diverse electrostatic properties, play key roles in controlling cell functions in vivo. In this study, three kinds of water-soluble polymers, positively charged poly(L-lysine) (PLL), negatively charged poly(acrylic acid) (PAAc) and neutral poly(ethylene glycol) (PEG), were compared based on their effects on the adhesion, spread, proliferation and chondrogenic differentiation of human mesenchymal stem cells (MSCs). The MSCs were seeded and cultured in the presence of polymers of different concentrations applied by methods using coating, mixing or covering. The effects of the water-soluble polymers depended on their electrostatic properties and method of application. The methods were in the order of coating, mixing and covering in terms of high to low influence. A low concentration of PLL promoted MSC adhesion, spread, proliferation and chondrogenic differentiation, while a high concentration of PLL was toxic. The PEG-coated surface facilitated cell aggregation and spheroid formation by inhibiting cell adhesion. A high concentration of mixed PEG (10 μg/ml) promoted cell proliferation in serum-free medium. PAAc showed no obvious effects on MSC adhesion, spread, proliferation, or chondrogenic differentiation.     

Porous polymer scaffolds surface-modified with arginine-glycine-aspartic acid enhance bone cell attachment and differentiation in vitro

This study was designed to determine if the surface modification of porous poly(lactic acid) (PLA) scaffolds would enhance osteogenic precursor cell (OPC) attachment, growth, and differentiation. A covalently grafted amino group (-NH(2)), poly(L-lysine) (PLL), and the peptide arginine-glycine-aspartic acid (RGD) were selected for the evaluation. The hypothesis was that surface modification would have a positive impact on cell-substratum interactions. The experiment was performed by OPC cells being placed on PLA films and scaffolds modified with NH(2), PLL, or RGD in tissue culture media. OPC attachment to PLA films was assessed after 24 h of incubation. The growth and differentiation of the adherent OPCs on porous PLA scaffolds were assessed after 14 and 28 days for alkaline phosphatase (APase) activity and calcium levels, both of which increase as OPCs differentiate into mature bone cells. All assays were accomplished in triplicate, and data were tested with post hoc orthogonal contrasts (i.e., Fisher's least significant difference) at p < or = 0.05. The PLA film surface-modified with RGD showed better OPC cell attachment than the other films. The cells on the PLA scaffolds surface-modified with RGD also exhibited an increase in APase activity and calcium levels in comparison with those on other scaffolds. This difference was apparent at both time intervals and was especially evident in the tissue culture media containing an osteogenic supplement. The results of this study indicate that modifying the surface of PLA polymer scaffolds with RGD enhances bone cell attachment and differentiation and may improve their ability to regenerate bone tissue more efficiently in wound models.     

Modulation of marrow stromal cell function using poly(D,L-lactic acid)-block-poly(ethylene glycol)-monomethyl ether surfaces.

The adhesion of marrow stromal osteoblasts and the adsorption of fetal bovine serum (FBS) proteins to end-capped poly(D,L-lactic acid) 50:50 (PLA50) of molecular weight 17,000 (PLA5017), non-end-capped PLA50 of molecular weight 11,000 (PLA5011h), and a diblock copolymer made of poly(ethylene glycol)-monomethyl ether of molecular weight 5,000 and PLA50 of molecular weight 20,000 (Me. PEG5-PLA20) were investigated. Cell attachment and proliferation on both PLA50 polymers were equally good. The block copolymer did not allow the proliferation of cells. However, the attached cells were highly differentiated and metabolically active in contrast to the cells on PLA50. Moreover, surface analysis studies using electron spectroscopy revealed that FBS proteins adsorbed well from aqueous solutions to the PLA50 surfaces while they adsorbed substantially less to the block copolymer. These results suggest that Me.PEG-PLA block copolymers may be used to regulate protein adsorption and, therefore, cell adhesion by varying the block composition of the copolymer.     

Poly(ethylene oxide)-graft-poly(L-lysine) copolymers to enhance the biocompatibility of poly(L-lysine)-alginate microcapsule membranes.

A graft copolymer having poly(L-lysine) (PLL) as the backbone and monomethoxy poly(ethylene glycol) (MPEG) as pendent chains was synthesized. This polycationic copolymer was used to form microcapsules with sodium alginate, a polyanion. Microcapsules and model surfaces formed with PLL-graft-MPEG demonstrated reduced protein adsorption, complement binding and cell adhesion in vitro compared to materials with unmodified PLL. Microcapsules with PLL-g-MPEG on the surface were seen to be much more biocompatible than the widely used alginate/PLL/alginate microcapsule in a mouse intraperitoneal implant model. The graft copolymers demonstrated a lower affinity for alginate and increased microcapsule permeability more than PLL. To correct this, pentalayered alginate/PLL/alginate/PLL-g-MPEG/alginate microcapsules were fabricated, and these demonstrated both appropriate permselectivity and enhanced biocompatibility.     

Control of cell adhesion on poly(methyl methacrylate)

Keratoprostheses have been constructed from a wide variety of transparent materials, including poly(methyl methacrylate) (PMMA). However, the success of keratoprosthesis has been plagued by numerous shortcomings that include the weakening of the implant-host interface due to weak cell adhesion and opaque fibrous membrane formation over the inner surface of the implant due to fibroblast attachment. An effective solution requires a surface modification that would selectively allow enhanced cell attachment at the implant-host interface and reduced cell attachment over the interior surface of the implant. Here, we have developed a novel and simple peptide conjugation scheme to modify PMMA surfaces, which allowed for region-specific control of cell adhesion. This method uses di-amino-PEG, which can be grafted onto PMMA using hydrolysis or aminolysis method. PEG can resist cell adhesion and protein adsorption. The functionalization of grafted di-amino-PEG molecules with RGD peptide not only restored cell adhesion to the surfaces, but also enhanced cell attachment and spreading as compared to untreated PMMA surfaces. Long-term cell migration and micropatterning studies clearly indicated that PEG-PMMA surfaces with and without RGD conjugation can be used to differentiate cell adhesion and control cell attachment spatially on PMMA, which will have potential applications in the modification of keratoprostheses.     

Polyelectrolyte multilayer films with pegylated polypeptides as a new type of anti-microbial protection for biomaterials

Adhesion of bacteria at the surface of implanted materials is the first step in microbial infection, leading to post-surgical complications. In order to reduce this adhesion, we show that poly(L-lysine)/poly(L-glutamic acid) (PLL/PGA) multilayers ending by several PLL/PGA-g-PEG bilayers can be used, PGA-g-PEG corresponding to PGA grafted by poly(ethylene glycol). Streaming potential and quartz crystal microbalance-dissipation measurements were used to characterize the buildup of these films. The multilayer films terminated by PGA and PGA-g-PEG were found to adsorb an extremely small amount of serum proteins as compared to a bare silica surface but the PGA ending films do not reduce bacterial adhesion. On the other hand, the adhesion of Escherichia coli bacteria is reduced by 72% on films ending by one (PLL/PGA-g-PEG) bilayer and by 92% for films ending by three (PLL/PGA-g-PEG) bilayers compared to bare substrate. Thus, our results show the ability of PGA-g-PEG to be inserted into multilayer films and to drastically reduce both protein adsorption and bacterial adhesion. This kind of anti-adhesive films represents a new and very simple method to coat any type of biomaterials for protection against bacterial adhesion and therefore limiting its pathological consequences.     

Effects of polycationic peptides on mucin release from airway goblet cells: relationship between polymer size and activity

OBJECTIVES AND DESIGN: Various sizes of poly-L-lysine (PLL) and poly-L-arginine (PLA) were tested for their possible effects on airway goblet cell mucin release using primary hamster tracheal surface epithelial (HTSE) cells in an attempt to identify the smallest size of the polycationic peptide to suppress mucin release without cytotoxicity. MATERIALS AND METHODS: HTSE cells were metabolically labeled using 3H-glucosamine and chased in the presence of varying concentrations of various sizes of the polycationic peptides. The amount of 3H-mucin in the spent media was measured by Sepharose CL-4B gel-filtration column chromatography. Possible cytotoxicity of the peptides was assessed by measuring the release of lactic dehydrogenase (LDH) during the treatment period. RESULTS: (1) PLL (MW 78,000) inhibited whereas PLA (MW 92,000) stimulated mucin release. However, these peptides were cytotoxic at the effective concentrations; (2) Both PLL (MW 9,600) and PLA (MW 8,900) could inhibit mucin release in a dose dependent manner without cytotoxicity; (3) Both PLL and PLA were effective in suppressing mucin release in 20-mer but not in either 10-mer or 5-mer; (4) 14-mers of both PLL and PLA also inhibited mucin release without cytotoxicity; (5) PLL and poly-D-lysine (PDL) of 14-mer were equipotent in its ability to suppress mucin release. CONCLUSION: Both PLL and PLA are cytotoxic at 'high' molecular weights, but have an ability to suppress mucin release without cytotoxicity at 'low' molecular weights. 14-mer seems to be the small, effective size, if not the smallest, for both PLL and PLA to suppress mucin release without cytotoxicity. The inhibitory effect of these polycationic peptides seems to be determined by the presence and the absolute number of positive charges and also to be independent of optical isomerism.     

Preparation and Characterization of a Porous Scaffold Based on Poly(D,L-Lactide) and N-Hydroxyapatite by Phase Separation

In this study, a series of porous scaffolds were prepared from poly(D,L-lactide) (PLA) and nanohydroxyapatite (HA) using the phase separation method. HA/PLA composite membranes and PLA membranes with a microporous structure (pore size around 10–20 μm) were observed by scanning electron microscopy and these micropores were well distributed throughout the PLA membranes. The surface morphology of HA/PLA composite membranes was significantly improved compared to pure PLA membrane. Also, the mechanical property and contact angle of composite membranes were different from that of pure PLA films. The immortalized rat osteoblastic ROS 17/2.8 cell line was used in this research to study the cell adhesion and proliferation behavior, and the results indicated that composite membranes had great cell affinity and good biocompatibility.     

Preparation and characterization of a porous scaffold based on poly(D,L-lactide) and N-hydroxyapatite by phase separation

In this study, a series of porous scaffolds were prepared from poly(D,L-lactide) (PLA) and nanohydroxyapatite (HA) using the phase separation method. HA/PLA composite membranes and PLA membranes with a microporous structure (pore size around 10-20 μm) were observed by scanning electron microscopy and these micropores were well distributed throughout the PLA membranes. The surface morphology of HA/PLA composite membranes was significantly improved compared to pure PLA membrane. Also, the mechanical property and contact angle of composite membranes were different from that of pure PLA films. The immortalized rat osteoblastic ROS 17/2.8 cell line was used in this research to study the cell adhesion and proliferation behavior, and the results indicated that composite membranes had great cell affinity and good biocompatibility.     

Poly(D,L-lactic acid)-poly(ethylene glycol)-monomethyl ether diblock copolymers control adhesion and osteoblastic differentiation of marrow stromal cells

Biodegradable polymers, such as poly(lactic acid) (PLA) and poly(lactic-coglycolic acid) (PLGA), are attractive materials for tissue engineering because of their degradative and mechanical properties, which permit scaffolds to be tailored to the individual requirements of different tissues. Although these materials support tissue development, their chemical properties offer no control of cell adhesion or function because their surfaces become immediately masked by adsorbing serum proteins when the materials come into contact with body fluids. Furthermore, adhesion proteins undergo conformational changes and a decrease in bioactivity when adsorbed to hydrophobic materials, such as PLA. To overcome these limitations, we modified the properties of PLA by synthesizing a diblock copolymer with poly(ethylene glycol) (PEG), which is known to reduce the amount of adsorbed proteins and to modify their conformation. By altering the PEG content of these diblock copolymers we were able to control the adsorption of adhesion proteins and, because cell adhesion takes place only in the presence of serum proteins, to control cell adhesion and cell shape. Marrow stromal cell differentiation to the osteoblastic phenotype was strongly improved on PEG-PLA compared with PLA, PLGA and tissue culture polystyrene and led to a 2-fold increase in alkaline phosphatase activity and mineralization.     

Biodegradable thermogelling poly(ester urethane)s consisting of poly(lactic acid)--thermodynamics of micellization and hydrolytic degradation

Multiblock poly(ether ester urethane)s comprising of poly(lactic acid) (PLA), poly(ethylene glycol) (PEG), and poly(propylene glycol) (PPG) segments were synthesized, and their aqueous solutions exhibited thermogelling behavior at critical gelation concentrations (CGC) ranging from 7 to 9 wt%. The chemical structures and molecular characteristics of the copolymers were studied by GPC, 1H NMR, 13C NMR and FTIR. The thermal stability of the poly(PEG/PPG/PLA urethane)s was studied by thermogravimetry analysis (TGA), and the PLA contents were calculated based on the thermal degradation profile. The results were in good agreement with those obtained from the 1H NMR measurements. The critical micellization concentration (CMC) of these water-soluble poly(ether ester urethane)s was determined at different temperatures using a dye solubilization method. The thermodynamic parameters for micelle formation were calculated, indicating that the process is largely entropy-driven. Interestingly, it appears that there exists a requirement for the system to possess a minimum gain in entropy before the thermogelling effect can be observed. Dilute copolymer solutions showed a lower critical solution temperature (LCST) behavior similar to pNIPAM dissolved in aqueous solutions. The thermogels hydrolytically degraded to polymer fragments corresponding to the constituent segment blocks within 3 months.     

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…     

Microdomain structure in polylactide-block-poly(ethylene oxide) copolymer films

Structured surface is an important property of polymer biomaterials for tissue engineering, for its capacity to expose domains with different surface energy and functional groups. For this purpose, amphiphilic A-B-A block copolymers with polylactide (PLA) as A blocks and poly(ethylene oxide) (PEO 3, Mn = 3090; PEO6, Mn = 6110) as B block were synthesized by ring-opening polymerization of either L-lactide (L-LA) or DL-lactide (DL-LA), using poly(ethylene glycol)s as macroinitiators and tin(II) octanoate (Sn(Oct)2) as a catalyst. Differential scanning calorimetry (DSC) and electron microscopy were used to study the phase separation of the hydrophobic (PLA) and hydrophilic (PEO) segments in films made of the copolymers and their blends with high-molecular-weight PLA homopolymers. Hydrophilic (PEO) and hydrophobic (PLA) domains were formed at the polymer film surface due to the separation of phases. The phase separation was affected by the copolymer composition and the stereoregularity of PLA blocks in the copolymers.     

Preparation of poly(D,L-lactide) nanoparticles assisted by amphiphilic poly(methyl methacrylate-co-methacrylic acid) copolymers.

When co-precipitated with amphiphilic copolymers from DMSO, poly(D,L-lactide) (PLA) can be readily converted into stable sub-200 nm nanoparticles by addition of an aqueous phase, free of any polymeric stabilizers such as poly(vinyl alcohol) or Poloxamer. In this work, the ability of random poly(methyl methacrylate-co-methacrylic acid) copolymers (PMMA-co-MA) to stabilize PLA nanoparticles was demonstrated, and the properties of PLA/PMMA-co-MA nanoparticles were investigated. When co-precipitated with PMMA-co-MA, PLA was totally converted into nanoparticles using a polymer concentration in DMSO (Cp) below 17.6 mg ml(-1), and a PMMA-co-MA proportion above a critical value depending on the content of MA repeating units (X). For instance, the lowest PMMA-co-MA proportion required was 0.9 mg mg(-1) PLA for X = 12%, and 0.5 mg mg(-1) PLA for X = 25% (for C(PLA) = 16 mg ml(-1) DMSO). The nanoparticle diameter was essentially independent of X, the proportion of PMMA-co-MA, and the PLA molecular weight, except for oligomers for which the nanoparticle diameter was smaller. It decreased when the organic phase was diluted (126 +/- 13 nm for Cp = 17.6 mg ml(-1), and 81 +/- 5 nm for C(P) = 5.6 mg ml(-1)). The time-dependence of the stability and the degradation of PLA/PMMA-co-MA nanoparticles was discussed. One of the main advantages of this technique is the ability to control surface properties and to bring functional groups to otherwise non-functionalized PLA nanoparticles. To illustrate this, a conjugate of PMMA-co-MA25 and biotin was synthesized, and used to prepare biotinylated nanoparticles that could be detected by fluorescence and transmission electron microscopy after infiltration into ligatured rat small intestine.     

Effect of RGD functionalization and stiffness modulation of polyelectrolyte multilayer films on muscle cell differentiation

Skeletal muscle tissue engineering holds promise for the replacement of muscle damaged by injury and for the treatment of muscle diseases. Although arginylglycylaspartic acid (RGD) substrates have been widely explored in tissue engineering, there have been no studies aimed at investigating the combined effects of RGD nanoscale presentation and matrix stiffness on myogenesis. In the present work we use polyelectrolyte multilayer films made of poly(l-lysine) (PLL) and poly(l-glutamic) acid (PGA) as substrates of tunable stiffness that can be functionalized by a RGD adhesive peptide to investigate important events in myogenesis, including adhesion, migration, proliferation and differentiation. C2C12 myoblasts were used as cellular models. RGD presentation on soft films and increasing film stiffness could both induce cell adhesion, but the integrins involved in adhesion were different in the case of soft and stiff films. Soft films with RGD peptide appeared to be the most appropriate substrate for myogenic differentiation, while the stiff PLL/PGA films induced significant cell migration and proliferation and inhibited myogenic differentiation. ROCK kinase was found to be involved in the myoblast response to the different films. Indeed, its inhibition was sufficient to rescue differentiation on stiff films, but no significant changes were observed on stiff films with the RGD peptide. These results suggest that different signaling pathways may be activated depending on the mechanical and biochemical properties of multilayer films. This study emphasizes the advantage of soft PLL/PGA films presenting the RGD peptide in terms of myogenic differentiation. This soft RGD-presenting film may be further used as a coating of various polymeric scaffolds for muscle tissue engineering.     

Stable modification of poly(lactic acid) surface with neurite outgrowth-promoting peptides via hydrophobic collagen-like sequence

Surface modification of poly(dl-lactic acid) (PLA) scaffolds has been performed using a biofunctional small peptide composed of collagen-like repetitive sequence and laminin-derived sequence (AG73-G₃-(PPG)₅) via hydrophobic interaction. The results of surface analysis suggest that AG73-G₃-(PPG)₅ can be stably adsorbed onto PLA films via hydrophobic interaction at the (PPG)₅ region, and form an extracellular matrix-like layer composed of both structural and biosignalling sequences. In addition, neurite outgrowth of PC12 cells was observed on the AG73-G₃-(PPG)₅-adsorbed PLA film. These results indicate that AG73-G₃-(PPG)₅ very effectively enhances neurite outgrowth activity on PLA films. The hydrophobic adsorption of collagen-like peptide bound to biosignalling molecules may be widely applied as a surface modifier of PLA films for tissue engineering.     

Poly(D,L-lactic acid)-block-(ligand-tethered poly(ethylene glycol)) copolymers as surface additives for promoting chondrocyte attachment and growth

The poly(D,L-lactic acid)-block-(ligand-tethered poly(ethylene glycol)) copolymer was explored to engineer poly(D,L-lactic acid) (PLA) material to promote chondrocyte attachment and growth. The poly(D,L-lactic acid)-block-poly(ethylene glycol) copolymer (PLE) was synthesized by a coupling reaction between PLA and poly(ethylene glycol) (PEG) (M(n) 1000, 2000, and 4000 respectively), with the use of 4,4'-methylenediphenyl diisocyanate (MDI). Then the PLE was activated by methyl sulfonyl chloride and the amino acids or arginine-glycine-aspartic acid tripeptide (RGD) was attached, which was verified by the ninhydrin-UV method. The modified PLA films were simply prepared by blending PLA with PLE derivatives. ATR-FTIR, XPS, contact angle, and AFM results clearly showed that the PEG chain stably enriched on the surface of PLE-modified PLA films. The chondrocyte cytocompatibility test showed the modified PLA films could significantly improve chondrocyte attachment and proliferation.     

Synthesis and characterization of poly(L-lysine)-g-poly(D,L-lactic-co-glycolic acid) biodegradable micelles

A series of amphipathic graft copolymers composed of poly(L-lysine) (PLL) as the cationic polymer backbone and biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA) as the grafting chains were synthesized and characterized. The terminal group of PLGA was activated and chemically conjugated to the primary epsilon-amine groups of PLL to produce PLL-g-PLGA copolymers. PLL-g-PLGA formed a self-assembling micelle structure in aqueous solution. The micelle size ranged from 69.4 to 149.6 nm in diameter, depending on the grafting percentage of PLGA. Upon increasing the number of PLGA chains grafted onto the PLL backbone, the size of the micelles gradually decreased, at the same time lowering their critical micelle concentration. The micelles were individually separated and had a spherical geometry, as observed by atomic force microscopy (AFM). These PLL-g-PLGA copolymers can be applied as cell adhesive surface coating materials for biodegradable tissue engineering scaffolds and can be used as non-viral DNA carriers for gene therapy.     

Interaction between poly(L-lysine) and sulfated poly(vinyl alcohol)

Ionic polymer-polymer interaction was studied in aqueous solution for poly(L -lysine) (PLL) and sulfated poly(vinyl alcohol) (PVS) as functions of pH, the degree of sulfation, the functional unit mole ratio of the two polymers and temperature by means of circular dichroism and viscosity measurements. In all the cases studied, strong inter-polymer complexes were formed at the functional unit mole ratio (VS)/(LL) higher than 1. Although PLL itself is well known to take the α-helical conformation at such a high pH as 11, the PLL conformation in the PLL/PVS complexes did not depend on pH but on the degree of sulfation: at room temperature, PLL took random coil conformation in PLL/PVS-25 (25: degree of sulfation in mole-%) and PLL/PVS-30, and the α-helical conformation (helicity of 70%) in PLL/PVS-46 and PLL/PVS-95. Models for the complex structures are postulated. Methanesulfonic acid did not influence the conformational transition of PLL, supporting that a polymer effect took place in the complex formation between PLL and PVS. Thermal effect on the PLL conformation in the complex is also discussed.     

Patterned poly(chlorotrifluoroethylene) guides primary nerve cell adhesion and neurite outgrowth

Central nervous system (CNS) neurons, unlike those of the peripheral nervous system, do not spontaneously regenerate following injury. Recently it has been shown that in the developing CNS, a combination of cell-adhesive and cell-repulsive cues guide growing axons to their targets. We hypothesized that by mimicking these guidance signals, we could guide nerve cell adhesion and neurite outgrowth in vitro. Our objective was to direct primary nerve cell adhesion and neurite outgrowth on poly(chlorotrifluoroethylene) (PCTFE) surfaces by incorporating alternating patterns of cell-adhesive (peptide) and nonadhesive (polyethylene glycol; PEG) regions. PCTFE was surface-modified with lithium PEG-alkoxide, demonstrating the first report of metal-halogen exchange with an alkoxide and PCTFE. Titanium and then gold were sputtered onto PEG-modified films, using a shadow-masking technique that creates alternating patterns on the micrometer scale. PCTFE-Au regions then were modified with one of two cysteine-terminated laminin-derived peptides, C-GYIGSR or C-SIKVAV. Hippocampal neuron cell-surface interactions on homogeneously modified surfaces showed that neuron adhesion was decreased significantly on PEG-modified surfaces and was increased significantly on peptide-modified surfaces. Cell adhesion was greatest on CGYIGSR surfaces while neurite length was greatest on CSIKVAV surfaces and PLL/laminin positive controls, indicating the promise of peptides for enhanced cellular interactions. On patterned surfaces, hippocampal neurons adhered and extended neurites preferentially on peptide regions. By incorporating PEG and peptide molecules on the surface, we were able to simultaneously mimic cell-repulsive and cell-adhesive cues, respectively, and maintain the biopatterning of primary CNS neurons for over 1 week in culture.