Promoting nerve cell functions on hydrogels grafted with poly(L-lysine)

We present a novel photopolymerizable poly(L-lysine) (PLL) and use it to modify polyethylene glycol diacrylate (PEGDA) hydrogels for creating a better, permissive nerve cell niche. Compared with their neutral counterparts, these PLL-grafted hydrogels greatly enhance pheochromocytoma (PC12) cell survival in encapsulation, proliferation, and neurite growth and also promote neural progenitor cell proliferation and differentiation capacity, represented by percentages of both differentiated neurons and astrocytes. The role of efficiently controlled substrate stiffness in regulating nerve cell behavior is also investigated and a polymerizable cationic small molecule, [2-(methacryloyloxy)ethyl]-trimethylammonium chloride (MTAC), is used to compare with this newly developed PLL. The results indicate that these PLL-grafted hydrogels are promising biomaterials for nerve repair and regeneration.     

Carbon nanotubes promote neuron differentiation from human embryonic stem cells

Human embryonic stem cells (hESCs) hold great promise for regenerative medicine and transplantation therapy due to their self-renewal and pluripotent properties. We report that 2D thin film scaffolds composed of biocompatible polymer grafted carbon nanotubes (CNTs), can selectively differentiate human embryonic stem cells into neuron cells while maintaining excellent cell viability. According to fluorescence image analysis, neuron differentiation efficiency of poly(acrylic acid) grafted CNT thin films is significant greater than that on poly(acrylic acid) thin films. When compared with the conventional poly-l-ornithine surfaces, a standard substratum commonly used for neuron culture, this new type thin film scaffold shows enhanced neuron differentiation. No noticeable cytotoxic effect difference has been detected between these two surfaces. The surface analysis and cell adhesion study have suggested that CNT-based surfaces can enhance protein adsorption and cell attachment. This finding indicates that CNT-based materials are excellent candidates for hESCs’ neuron differentiation.     

RGD-grafted poly-L-lysine-graft-(polyethylene glycol) copolymers block non-specific protein adsorption while promoting cell adhesion

A novel class of surface-active copolymers is described, designed to protect surfaces from nonspecific protein adsorption while still inducing specific cell attachment and spreading. A graft copolymer was synthesized, containing poly-(L-lysine) (PLL) as the backbone and substrate binding and poly(ethylene glycol) (PEG) as protein adsorption-resistant pendant side chains. A fraction of the grafted PEG was pendantly functionalized by covalent conjugation to the peptide motif RGD to induce cell binding. The graft copolymer spontaneously adsorbs from dilute aqueous solution onto negatively charged surfaces. The performance of RGD-modified PLL-g-PEG copolymers was analyzed in protein adsorption and cell culture assays. These coatings efficiently blocked the adsorption of serum proteins to Nb(2)O(5) and tissue culture polystyrene while specifically supporting attachment and spreading of human dermal fibroblasts. This surface functionalization technology is expected to be valuable in both the biomaterial and biosensor fields, because different signals can easily be combined, and sterilization and application are straightforward and cost-effective.     

Synthesis and evaluation of novel biodegradable hydrogels based on poly(ethylene glycol) and sebacic acid as tissue engineering scaffolds

Novel biodegradable poly(ethylene glycol) (PEG) based hydrogels, namely, PEG sebacate diacrylate (PEGSDA) were synthesized, and their properties were evaluated. Chemical structures of these polymers were confirmed by Fourier transform infrared and proton nuclear magnetic resonance (1H NMR) spectroscopy. After photopolymerization, the dynamic shear modulus of the hydrogels was up to 0.2 MPa for 50% PEGSDA hydrogel, significantly higher than conventional hydrogels such as PEG diacrylate (PEGDA). The swelling ratios of these macromers were significantly lower than PEGDA. The in vitro degradation study demonstrated that these hydrogels were biodegradable with weight losses about 66% and 32% for 25% and 50% PEGSDA after 8 weeks of incubation in phosphate-buffered saline at 37 degrees C. In vitro biocompatibility was assessed using cultured rat bone marrow stromal cells (MSCs) in the presence of unreacted monomers or degradation products. Unlike conventional PEGDA hydrogels, PEGSDA hydrogel without RGD peptide modification induced MSC cell adhesion similar to tissue culture polystyrene. Finally, complex three-dimensional structures of PEGSDA hydrogels using solid free form technique were fabricated and their structure integrity was better maintained than PEGDA hydrogels. These hydrogels may find use as scaffolds for tissue engineering applications.     

Synthesis and characterization of novel thiol-reactive poly(ethylene glycol) cross-linkers for extracellular-matrix-mimetic biomaterials

Synthetic extracellular matrix hydrogels can be used for three-dimensional cell culture, wound repair, and tissue engineering. Using the bifunctional electrophile poly(ethylene glycol) diacrylate (PEGDA), thiol-modified glycosaminoglycans and polypeptides can be cross-linked into biocompatible materials in the presence of cells or tissues. However, the rate of in situ cross-linking with PEGDA under physiological conditions may occur too slowly for clinical applications requiring a fast-curing preparation. To explore a wider range of cross-linking time courses, five homo-bifunctional PEG derivatives were synthesized and examined as cross-linking agents for thiol-modified derivatives of hyaluronan (HA). Thiol reaction rate constants were measured over a pH range of 7.4 to 8.6. The order of reactivity for the functional groups used was determined to be maleimide > iodoacetate > bromoacetate > iodoacetamide > acrylate > bromoacetamide, with rates increasing exponentially with increasing pH. The range of gelation times at physiological pH varied from less than 1 min to over 2 h. Addition of the cross-linkers to cell culture medium showed minimal cytotoxicity toward primary human dermal fibroblasts at concentrations anticipated during in situ cross-linking. Moreover, hydrogels prepared from thiol-modified gelatin and thiol-modified HA were biocompatible and supported attachment and proliferation of fibroblasts and hepatocytes.     

Modulation of Huh7.5 Spheroid Formation and Functionality Using Modified PEG-Based Hydrogels of Different Stiffness

Physical cues, such as cell microenvironment stiffness, are known to be important factors in modulating cellular behaviors such as differentiation, viability, and proliferation. Apart from being able to trigger these effects, mechanical stiffness tuning is a very convenient approach that could be implemented readily into smart scaffold designs. In this study, fibrinogen-modified poly(ethylene glycol)-diacrylate (PEG-DA) based hydrogels with tunable mechanical properties were synthesized and applied to control the spheroid formation and liver-like function of encapsulated Huh7.5 cells in an engineered, three-dimensional liver tissue model. By controlling hydrogel stiffness (0.1–6 kPa) as a cue for mechanotransduction representing different stiffness of a normal liver and a diseased cirrhotic liver, spheroids ranging from 50 to 200 μm were formed over a three week time-span. Hydrogels with better compliance (i.e. lower stiffness) promoted formation of larger spheroids. The highest rates of cell proliferation, albumin secretion, and CYP450 expression were all observed for spheroids in less stiff hydrogels like a normal liver in a healthy state. We also identified that the hydrogel modification by incorporation of PEGylated-fibrinogen within the hydrogel matrix enhanced cell survival and functionality possibly owing to more binding of autocrine fibronectin. Taken together, our findings establish guidelines to control the formation of Huh7.5 cell spheroids in modified PEGDA based hydrogels. These spheroids may serve as models for applications such as screening of pharmacological drug candidates.     

Biomimetic poly(ethylene glycol)-based hydrogels as scaffolds for inducing endothelial adhesion and capillary-like network formation

The extracellular matrix (ECM) is an attractive model for designing synthetic scaffolds with a desirable environment for tissue engineering. Here, we report on the synthesis of ECM-mimetic poly(ethylene glycol) (PEG) hydrogels for inducing endothelial cell (EC) adhesion and capillary-like network formation. A collagen type I-derived peptide GPQGIAGQ (GIA)-containing PEGDA (GIA-PEGDA) was synthesized with the collagenase-sensitive GIA sequence attached in the middle of the PEGDA chain, which was then copolymerized with RGD capped-PEG monoacrylate (RGD-PEGMA) to form biomimetic hydrogels. The hydrogels degraded in vitro with the rate dependent on the concentration of collagenase and also supported the adhesion of human umbilical vein ECs (HUVECs). Biomimetic RGD/GIA-PEGDA hydrogels with incorporation of 1% RGD-PEGDA into GIA-PEGDA hydrogels induced capillary-like organization when HUVECs were seeded on the hydrogel surface, while RGD/PEGDA and GIA-PEGDA hydrogels did not. These results indicate that both cell adhesion and biodegradability of scaffolds play important roles in the formation of capillary-like networks.     

Development of a biostable replacement for PEGDA hydrogels

The exceptional tunability of poly(ethylene glycol) (PEG) hydrogel chemical, mechanical, and biological properties enables their successful use in a wide range of biomedical applications. Although PEG diacrylate (PEGDA) hydrogels are often used as nondegradable controls in short-term in vitro studies, it is widely acknowledged that the hydrolytically labile esters formed upon acrylation of the PEG diol make them susceptible to slow degradation in vivo. A PEG hydrogel system that maintains the desirable properties of PEGDA while improving biostability would be valuable in preventing degradation-related failure of gel-based devices in long-term in vivo applications. To this end, PEG diacrylamide (PEGDAA) hydrogels were synthesized and characterized in quantitative comparison to traditional PEGDA hydrogels. It was found that PEGDAA hydrogel modulus and swelling can be tuned over a similar range and to comparable degrees as PEGDA hydrogels with changes in macromer molecular weight and concentration. Additionally, PEGDAA cytocompatibility, low cell adhesion, and capacity for incorporation of bioactivity were analogous to that of PEGDA. In vitro hydrolytic degradation studies showed that the amide-based PEGDAA had significantly increased biostability relative to PEGDA. Overall, these findings indicate that PEGDAA hydrogels are a suitable replacement for PEGDA hydrogels with enhanced hydrolytic resistance. In addition, these studies provide a quantitative measure of the hydrolytic degradation rate of PEGDA hydrogels which was previously lacking in the literature.     

Biomimetic hydrogels with immobilized ephrinA1 for therapeutic angiogenesis

The formation of a microvasculature is regulated in large part by cell-cell interactions. Ephrins and their Eph receptors mediate cell adhesion, repulsion, and migration, all critical processes in angiogenesis. (1) Here we use a covalently immobilized ephrinA1, conjugated to poly(ethylene glycol), to induce vessel formation both in vitro and in vivo in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Human umbilical vein endothelial cell (HUVEC) tubulogenesis in matrix metalloproteinase-sensitive hydrogels was visualized from 6 h to 7 days in response to three different concentrations of PEG-ephrinA1. The deposition of extracellular matrix proteins collagen IV and laminin that stabilize tubule formation were imaged, quantified, and found to be dependent on PEG-ephrinA1 concentration. To confirm the importance of the EphA2-ephrinA1 interaction in tubule formation, soluble EphA2 was used to disrupt the EphA2-ephrinA1 interaction between a coculture of HUVEC and human brain vascular pericyte cells. HUVECs seeded onto PEGDA hydrogels displayed a dose-dependent reduction in tubule formation in response to the soluble EphA2. Finally, hydrogels with releasable platelet-derived growth factor (PDGF), immobilized RGDS, and covalently immobilized PEG-ephrinA1 were implanted into the mouse cornea micropocket. These hydrogels induced a more robust vascular response with an increase in vessel density as compared with hydrogels with releasable PDGF alone. As such, PEG-ephrinA1 may represent a promising molecule to regulate cell adhesion and migration for formation of a microvasculature in tissue-engineered constructs.     

Characterization of PLL-g-PEG-DNA nanoparticles for the delivery of therapeutic DNA

Local and controlled DNA release is a critical issue in current gene therapy. As viral gene delivery systems are associated with severe security problems, nonviral gene delivery vehicles were developed. Here, DNA-nanoparticles using grafted copolymers of PLL and PEG to increase their biocompatibility and stealth properties were systematically studied. Ten different PLL-based polymers with no, low, and high PEG grafting and PEG molecular weights as well as different PLL backbone lengths were complexed with plasmids containing 3200 to 10,100 base pairs. Stable complexes were formed and selected for cytotoxicity and transfection efficiency. Predominantly, PLL-g-PEG-DNA nanoparticles grafted with 4 or 5% PEG moieties of 5 kDa transfected 40% COS-7 cells without reduction of cell viability when formed at N/P ratios between 0.1 and 12.5. The molecular weight of PLL did not significantly affect transfection efficiency or cytotoxicity indicating that a specific cationic charge-density-to-PEG-ratio is important for efficient transfection and low cytotoxicity. The PLL-g-PEG-DNA nanoparticles were spherical with a diameter of approximately 100 nm and did not aggregate over 2 weeks. Moreover, they protected included plasmid DNA against serum components and DNase I digestion. Therefore, such storage stable and versatile PLL-g-PEG-DNA nanoparticles might be useful to deliver differently sized therapeutic DNA for in vivo applications.     

Hydrolytically degradable poly(ethylene glycol) hydrogel scaffolds as a cell delivery vehicle: Characterization of PC12 cell response

The central nervous system (CNS) has a low intrinsic potential for regeneration following injury and disease, yet neural stem/progenitor cell (NPC) transplants show promise to provide a dynamic therapeutic in this complex tissue environment. Moreover, biomaterial scaffolds may improve the success of NPC‐based therapeutics by promoting cell viability and guiding cell response. We hypothesized that a hydrogel scaffold could provide a temporary neurogenic environment that supports cell survival during encapsulation, and degrades completely in a temporally controlled manner to allow progression of dynamic cellular processes such as neurite extension. We utilized PC12 cells as a model cell line with an inducible neuronal phenotype to define key properties of hydrolytically degradable poly(ethylene glycol) hydrogel scaffolds that impact cell viability and differentiation following release from the degraded hydrogel. Adhesive peptide ligands (RGDS, IKVAV, or YIGSR), were required to maintain cell viability during encapsulation; as compared to YIGSR, the RGDS, and IKVAV ligands were associated with a higher percentage of PC12 cells that differentiated to the neuronal phenotype following release from the hydrogel. Moreover, among the hydrogel properties examined (e.g., ligand type, concentration), total polymer density within the hydrogel had the most prominent effect on cell viability, with densities above 15% w/v leading to decreased cell viability likely due to a higher shear modulus. Thus, by identifying key properties of degradable hydrogels that affect cell viability and differentiation following release from the hydrogel, we lay the foundation for application of this system towards future applications of the scaffold as a neural cell delivery vehicle. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1255–1264, 2013     

Rheological characterization of in situ cross-linkable hyaluronan hydrogels

This report investigates the rheological properties of cross-linked, thiol-functionalized HA (HA-DTPH) hydrogels prepared by varying the concentration and molecular weight (MW) of the cross-linker, poly(ethylene glycol) diacrylate (PEGDA). Hydrogels were subsequently cured for either short-term (hours) or long-term (days) and subjected to oscillatory shear rheometry (OSR). OSR allows the evaluation and comparison of the shear storage moduli (G'), an index of the total number of effective cross-links formed in the hydrogels. While the oscillatory time sweep monitored the evolution of G' during in situ gelation, the stress and frequency sweeps measured the G' of preformed and subsequently cured hydrogels. From stress sweeps, we found that, for the hydrogels, G' scaled linearly with PEGDA concentration and was independent of its MW. Upon comparison with the classical Flory's theory of elasticity, stress sweep tests on short-term cured hydrogels revealed the simultaneous, but gradual, formation of spontaneous disulfide cross-links in the hydrogels. Results from time and frequency sweeps suggested that the formation of a stable, three-dimensional network depended strictly on PEGDA concentration. Results from the equilibrium swelling of hydrogels concurred with those obtained from oscillatory stress sweeps. Such a detailed rheological characterization of our HA-DTPH-PEGDA hydrogels will aid in the design of biomaterials targeted for biomedical or pharmaceutical purposes, especially in applications involving functional tissue engineering.     

Development of multilayered cell‐hydrogel composites using an acoustic focusing technique

Multilayered composites, composed of mammalian cells arranged in a hydrogel, have been prepared using an acoustic focusing technique. Acoustic focusing is a simple, nonchemical technique that allows for the fast arrangement of cells in matrices where the control of cell geometry is beneficial. Breast cancer cells (MDA‐MB231) were dispersed in a 30 wt % solution of poly(ethylene glycol) diacrylate (PEGDA) of molecular weight 400 at a density of 5 × 10⁶ cells/mL of PEGDA solution. An ultrasonic field was used to organize the cells before polymerization of PEGDA. Disk‐shaped hydrogel composites, typically 1 cm in diameter and 2‐mm thick were prepared based on a PEGDA solution volume of 130 μL. At an acoustic frequency of 2.32 MHz, composites having cells positioned within concentric cylindrical shells interspersed with zones of cell‐free hydrogel were produced. The cells were located in annuli approximately 80‐μm thick and about 300 μm apart. The structure and viability of the cells within these constructs were studied using a fluorescent LIVE/DEAD assay. The viability of the cells was on the order of 50%. For the conditions used in this study, cell death was primarily attributed to exposure of cells to the PEGDA solution prior to polymerization, rather than adverse effects of polymerization or the sound field itself. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Characterization of photo-cross-linked oligo[poly(ethylene glycol) fumarate] hydrogels for cartilage tissue engineering

Photo-cross-linkable oligo[poly(ethylene glycol) fumarate] (OPF) hydrogels have been developed for use in tissue engineering applications. We demonstrated that compressive modulus of these hydrogels increased with increasing polymer concentration, and hydrogels with different mechanical properties were formed by altering the ratio of cross-linker/polymer in precursor solution. Conversely, swelling of hydrogels decreased with increasing polymer concentration and cross-linker/polymer ratio. These hydrogels are degradable and degradation rates vary with the change in cross-linking level. Chondrocyte attachment was quantified as a method for evaluating adhesion of cells to the hydrogels. These data revealed that cross-linking density affects cell behavior on the hydrogel surfaces. Cell attachment was greater on the samples with increased cross-linking density. Chondrocytes on these samples exhibited spread morphology with distinct actin stress fibers, whereas they maintained their rounded morphology on the samples with lower cross-linking density. Moreover, chondrocytes were photoencapsulated within various hydrogel networks. Our results revealed that cells encapsulated within 2-mm thick OPF hydrogel disks remained viable throughout the 3-week culture period, with no difference in viability across the thickness of hydrogels. Photoencapsulated chondrocytes expressed the mRNA of type II collagen and produced cartilaginous matrix within the hydrogel constructs after three weeks. These findings suggest that photo-cross-linkable OPF hydrogels may be useful for cartilage tissue engineering and cell delivery applications.     

Iron promotes the survival and neurite extension of serum-starved PC12 cells in the presence of NGF by enhancing cell attachment

Delayed death of serum-starved PC12 cells on a poly-L-lysine (PLL) matrix was observed, even in the presence of NGF. NGF blocked the apoptotic death of attached but not detached cells, which suggests that delayed death may be related to cell detachment from the PLL matrix. Iron selectively blocked this anoikis-like death by increasing cell attachment. Interestingly, the addition of > 10 microM FeCl2 to the culture medium generated gelatinous iron precipitates, and the removal of the precipitates abolished the iron effect. Attachment experiments using poly-HEMA supported the role of iron precipitates on cell-to-matrix adhesion. The expression of integrin beta1, neither N-cadherin nor alpha/beta-catenin, was also significantly increased by iron. In addition to its effect on cell viability, iron promoted the outgrowth of neurites. Our results collectively indicate that iron functions as a necessary co-element for NGF by enhancing cell attachment, survival, and neurite extension.     

Stem cell-derived extracellular matrix enables survival and multilineage differentiation within superporous hydrogels

Hydrophilic poly(ethylene glycol) diacrylate (PEGDA) hydrogel surfaces resist protein adsorption and are generally thought to be unsuitable for anchorage-dependent cells to adhere. Intriguingly, our previous findings revealed that PEGDA superporous hydrogel scaffolds (SPHs) allow anchorage of bone marrow derived human mesenchymal stem cells (hMSCs) and support their long-term survival. Therefore, we hypothesized that the physicochemical characteristics of the scaffold impart properties that could foster cellular responses. We examined if hMSCs alter their microenvironment to allow cell attachment by synthesizing their own extracellular matrix (ECM) proteins. Immunofluorescence staining revealed extensive expression of collagen type I, collagen type IV, laminin, and fibronectin within hMSC-seeded SPHs by the end of the third week. Whether cultured in serum-free or serum-supplemented medium, hMSC ECM protein gene expression patterns exhibited no substantial changes. The presence of serum proteins is required for initial anchorage of hMSCs within the SPHs but not for the hMSC survival after 24 h. In contrast to 2D expansion on tissue culture plastic (TCP), hMSCs cultured within SPHs proliferate similarly in the presence or absence of serum. To test whether hMSCs retain their undifferentiated state within the SPHs, cell-seeded constructs were cultured for 3 weeks in stem cell maintenance medium and the expression of hMSC-specific cell surface markers were evaluated by flow cytometry. CD105, CD90, CD73, and CD44 were present to a similar extent in the SPH and in 2D monolayer culture. We further demonstrated multilineage potential of hMSCs grown in the PEGDA SPHs, whereby differentiation into osteoblasts, chondrocytes, and adipocytes could be induced. The present study demonstrates the potential of hMSCs to alter the "blank" PEGDA environment to a milieu conducive to cell growth and multilineage differentiation by secreting adhesive ECM proteins within the porous network of the SPH scaffolds.     

Probing vocal fold fibroblast response to hyaluronan in 3D contexts

A number of treatments are being investigated for vocal fold (VF) scar, including designer implants. The aim of the present study was to validate a 3D model system for probing the effects of various bioactive moieties on VF fibroblast (VFF) behavior toward rational implant design. We selected poly(ethylene glycol) diacrylate (PEGDA) hydrogels as our base‐scaffold due to their broadly tunable material properties. However, since cells encapsulated in PEGDA hydrogels are generally forced to take on rounded/stellate morphologies, validation of PEGDA gels as a 3D VFF model system required that the present work directly parallel previous studies involving more permissive scaffolds. We therefore chose to focus on hyaluronan (HA), a polysaccharide that has been a particular focus of the VF community. Toward this end, porcine VFFs were encapsulated in PEGDA hydrogels containing consistent levels of high M _w HA (${\rm HA}_{{\rm H}{M}_{\rm W} } $ ), intermediate M_w HA (${\rm HA}_{{\rm I}{M}_{\rm W} } $ ), or the control polysaccharide, alginate, and cultured for 7 and 21 days. ${\rm HA}_{{\rm H}{M}_{\rm W} } $ promoted sustained increases in active ERK1/2 relative to ${\rm HA}_{{\rm I}{M}_{\rm W} } $ . Furthermore, VFFs in ${\rm HA}_{{\rm I}{M}_{\rm W} } $ gels displayed a more myofibroblast‐like phenotype, higher elastin production, and greater protein kinase C (PkC) levels at day 21 than VFFs in ${\rm HA}_{{\rm H}{M}_{\rm W} } $ and alginate gels. The present results are in agreement with a previous 3D study of VFF responses to ${\rm HA}_{{\rm I}{M}_{\rm W} } $ relative to alginate in collagen‐based scaffolds permissive of cell elongation, indicating that PEGDA hydrogels may serve as an effective 3D model system for probing at least certain aspects of VFF behavior. Biotechnol. Bioeng. 2009; 104: 821–831 © 2009 Wiley Periodicals, Inc.     

Adhesion and growth of HUVEC endothelial cells on films of enzymatically functionalized chitosan with phenolic compounds

Human Umbilical Vein Endothelial Cell (HUVEC) growth on chitosan films and its enzymatically functionalized derivatives films with ferulic acid (FA) and ethyl ferulate (EF) was assessed by evaluating cell adhesion, morphology and cell viability. The results indicated that chitosan derivative films improved protein adsorption properties compared to chitosan films. The HUVEC cell morphology showed well attachment and spread phenotype on chitosan derivative films compared to those growing on chitosan films which did not spread and remained round. Evaluation of cell viability revealed improvement of cell adhesion on chitosan derivative films compared to chitosan film depending on the quantity of oxidized phenols grafted on chitosan. In addition, FA-/EF-chitosan films allowed almost similar cell adhesion. Furthermore, cell adhesion was increased with the film thickness. These results suggested that the oxidized phenols grafting on chitosan is a promising process to enhance cell adhesion, growth and creating useful functional biomaterials.     

The effect of titanium with heparin/BMP-2 complex for improving osteoblast activity

The objective of this study was to investigate the enhanced osteoblast activity of MG-63 cells cultured on titanium (Ti) with a heparin/BMP-2 (Hep/BMP-2) complex. The Ti substrates were initially modified by chemical grafting poly-l-lysine (PLL) using condensing agent, followed by immobilizing the heparin/BMP-2 complex to the PLL-grafted Ti substrate via electrostatic interactions. The surface modification of Ti substrates with PLL and/or Hep/BMP-2 complex were confirmed with scanning electron microscopy, contact angle measurements, and X-ray photoelectron spectroscopy. Immobilized BMP-2 was released from the Hep/BMP-2/Ti substrate in a sustained manner. In vitro studies revealed that osteoblasts grown on Hep/BMP-2/Ti substrate increased ALP activity, calcium deposition, ALP and osteocalcin levels as compared to those grown on pristine Ti or PLL-Ti. These results indicated that heparin/BMP-2 complex immobilized Ti substrate can be useful to effectively improve osteoblast activity.     

Filamentous condensation of DNA induced by pegylated poly-L-lysine and transfection efficiency

In this paper we propose a detailed analysis of structural and morphological properties of two poly-L-lysine (PLL)-based transfection formulations, PLL/DNA and pegylated PLL (PLL-g-PEG)/DNA, by means of atomic force microscopy (AFM) and transmission electron microscopy (TEM). Comparing PLL-g-PEG/DNA with PLL/DNA polyplexes, we demonstrate that, due to the presence of PEG, the particles differ not only in size, shape, and crystalline structure, but also in transfection efficiency. While PLL condensates DNA in large agglomerates, PLL grafted with polyethylene glycol 2000 can condensate DNA in long filaments with diameters of some nanometers (6-20 nm). These structures are dependent on the grafting ratio and are more efficient than compacted ones, showing that DNA uptake and processing by cell is directly related to physicochemical properties of the polyplexes.