Retinal pigment epithelial cell adhesion on novel micropatterned surfaces fabricated from synthetic biodegradable polymers

Novel synthetic biodegradable polymer substrates with specific chemical micropatterns were fabricated from poly(DL-lactic-coglycolic acid) (PLGA) and diblock copolymers of poly(ethylene glycol) and poly(DL-lactic acid) (PEG/PLA). Thin films of PLGA and PEG/PLA supported and inhibited, respectively, retinal pigment epithelial (RPE) cell proliferation, with a corresponding cell density of 352,900 and 850 cells/cm2 after 7 days (from an initial seeding density of 15,000 cells/cm2). A microcontact printing technique was used to define arrays of circular (diameter of 50 microm) PLGA domains surrounded and separated by regions (width of 50 microm) of PEG/PLA. Reversed patterns composed of PEG/PLA circular domains surrounded by PLGA regions were also fabricated. Both micropatterned surfaces were shown to affect initial RPE cell attachment, limit cell spreading, and promote the characteristic cuboidal cell morphology during the 8-h period of the experiments. In contrast, RPE cells on plain PLGA (control films) were elongated and appeared fibroblast-like. The reversed patterns had continuous PLGA regions that allowed cell-cell interactions and thus higher cell adhesion. These results demonstrate the feasibility of fabricating micropatterned synthetic biodegradable polymer surfaces to control RPE cell morphology.     

Retinal pigment epithelium engineering using synthetic biodegradable polymers

Retinal pigment epithelium (RPE) plays a key role in the maintenance of the normal functions of the retina, especially photoreceptors. Alteration in RPE structure and function is implicated in a variety of ocular disorders. Tissue engineering strategies using synthetic biodegradable polymers as temporary substrates for RPE cell culture and subsequent transplantation may provide a promising new therapy. In this review article, the manufacture of thin biodegradable poly(DL-lactic-co-glycolic acid) (PLGA) films and their degradation behavior in vitro are discussed. RPE cell proliferation and differentiation on these PLGA films are reviewed. The fabrication of model substrates with desired chemical micropatterns in the micrometer scale is discussed and the effects of surface patterning on RPE morphology and function are assessed. Finally. the preparation of biodegradable micropatterns with adhesive PLGA and non-adhesive poly(ethylene glycol)/PLA domains to modulate RPE cell adhesion is presented.     

Effect of nano- and micro-roughness on adhesion of bioinspired micropatterned surfaces

In this work, the adhesion of biomimetic polydimethylsiloxane (PDMS) pillar arrays with mushroom-shaped tips was studied on nano- and micro-rough surfaces and compared to unpatterned controls. The adhesion strength on nano-rough surfaces invariably decreased with increasing roughness, but pillar arrays retained higher adhesion strengths than unpatterned controls in all cases. The results were analyzed with a model that focuses on the effect on adhesion of depressions in a rough surface. The model fits the data very well, suggesting that the pull-off strength for patterned PDMS is controlled by the deepest dimple-like feature on the rough surface. The lower pull-off strength for unpatterned PDMS may be explained by the initiation of the pull-off process at the edge of the probe, where significant stress concentrates. With micro-rough surfaces, pillar arrays showed maximum adhesion with a certain intermediate roughness, while unpatterned controls did not show any measurable adhesion. This effect can be explained by the inability of micropatterned surfaces to conform to very fine and very large surface asperities.     

Surface tethering of phosphorylcholine groups onto poly(dimethylsiloxane) through swelling--deswelling methods with phospholipids moiety containing ABA-type block copolymers

The surface modification of poly(dimethylsiloxane) (PDMS) substrates by using ABA-type block copolymers comprising poly(2-methacryloyloxyethyl phosphorylcholine (MPC)) (PMPC) and PDMS segments was investigated. The hydrophobic interaction between the swelling-deswelling nature of PDMS and PDMS segments in block copolymers was the main mechanism for surface modification. Block copolymers with various compositions were synthesized by using the atom transfer radical polymerization (ATRP) method. The kinetic plots revealed that polymerization could be initiated by PDMS macroinitiators and it proceeds in a well-controlled manner; therefore, the compositions of the block copolymers were controllable. The obtained block copolymers were dissolved in a chloroform/ethanol mixed solvent. The surface of the PDMS substrate was modified using block copolymers by the swelling-deswelling method. Static contact angle and X-ray photoelectron spectroscopy (XPS) measurements revealed that the hydrophobic surface of the PDMS substrate was converted to a hydrophilic surface because of modification by surface-tethered PMPC segments. Protein adsorption test and L929 cell adhesion test were carried out for evaluating the biocompatibility. As observed, the amount of adsorbed proteins and cell adhesion were drastically reduced as compared to those in the non-treated PDMS substrate. We conclude that this procedure is effective in fabricating biocompatible surfaces on PDMS substrates.     

Synergistic effects of physical and chemical guidance cues on neurite alignment and outgrowth on biodegradable polymer substrates

This article demonstrates that directional outgrowth of neurites is promoted by applying a combination of physical and chemical cues to biodegradable polymer substrates. Films of poly-D,L-lactic acid and poly(lactide-co-glycolide) were micropatterned to form grooves on substrate surfaces, using novel indirect transfer techniques developed specifically for biodegradable polymers that cannot be micropatterned directly. Laminin was selectively adsorbed in the grooves. Whole and dissociated dorsal root ganglia were seeded on the substrates and neurite outgrowth and alignment along the microgrooves were measured. The microgrooves provide physical guidance, whereas laminin provides chemical cues to the neurons. The groove depth and spacing were found to significantly influence neurite alignment. The presence of laminin was found to promote neurite adhesion and outgrowth along the grooves. Using a combination of optimized physical and chemical cues, excellent spatial control of directional neurite outgrowth, with up to 95% alignment of neurites, was obtained. The synergistic effect of physical and chemical guidance cues was found to be more effective than individual cues in promoting directional outgrowth of neurites.     

Effect of functionalized micropatterned PLGA on guided neurite growth

When coaptation is not possible in the repair of nerve injuries, a bridge of biomaterial scaffold provides a structural support for neuronal cell growth and guides nerve regeneration. Poly(lactide-co-glycolide) (PLGA) scaffolds have been widely investigated for neural tissue engineering applications. In order to investigate guided neurite growth, we have fabricated micropatterns on PLGA films using laser ablation methods. The micropatterned PLGA films were coated with collagen type I or laminin peptide (PPFLMLLKGSTR) to promote axon growth. Micropatterned PLGA films provide a guidance effect on both early stage neurite outgrowth and elongation. Small (5 microm) grooves showed more statistically significant parallel neurite growth compared with larger size grooves (10 microm). Micropatterned PLGA films coated with laminin peptide showed more parallel neurite growth compared with those coated with collagen type I. Primary neurite number and total neurite length per cell decreased on micropatterned PLGA films compared with the controls. Neurites showed a preference for growth in the microgrooves rather than on the spaces. This study indicates that surface micropatterned structures with conjugated functional molecules can be used to guide neurite growth.     

载TK基因聚丙交酯乙交酯纳米粒的制备及有关性质研究

我们构建了含AFP启动子、TK基因和EGFP真核表达载体重组的质粒，并以可生物降解、生物相容的高分子载体材料PLGA包埋制成载自杀基因的纳米粒。结果表明纳米粒形态圆整，大小均匀，平均粒径68nm，包封率可达80%，该纳米粒能显著提高质粒DNA抵抗核酸酶降解和超声波剪切的能力。     

Cornea engineering on polyester carriers

In this study, biodegradable polyester based carriers were designed for tissue engineering of the epithelial and the stromal layers of the cornea, and the final construct was tested in vitro. In the construction of the epithelial layer, micropatterned films were prepared from blends of biodegradable and biocompatible polyesters of natural (PHBV) and synthetic (P(L/DL)LA) origin, and these films were seeded with D407 (retinal pigment epithelial) cells. To improve cell adhesion and growth, the films were coated with fibronectin. To serve as the stromal layer of the cornea, highly porous foams of P(L/DL)LA-PHBV blends were seeded with 3T3 fibroblasts. Cell numbers on the polyester carriers were significantly higher than those on the tissue culture polystyrene control. The cells and the carriers were characterized scanning electron micrographs showed that the foam was highly porous and the pores were interconnected. 3T3 Fibroblasts were distributed quite homogeneously at the seeding site, but probably because of the high thickness of the carrier ( approximately 6 mm); they could not sufficiently populate the core (central parts of the foam) during the test duration. The D407 cells formed multilayers on the micropatterned polyester film. Immunohistochemical studies showed that the cells retained their phenotype during culturing; D407 cells formed tight junctions characteristic of epithelial cells, and 3T3 cells deposited collagen type I into the foams. On the basis of these results, we concluded that the micropatterned films and the foams made of P(L/DL)LA-PHBV blends have a serious potential as tissue engineering carriers for the reconstruction of the epithelial and stromal layers of the cornea.     

Modification of Polydimethylsiloxane Surfaces Using Benzophenone

New biocompatible materials have been obtained by different modifications of polydimethylsiloxane (PDMS) surfaces. PDMS is of great interest for several biomedical applications. For some applications the native silicone does not provide an optimal performance. PDMS attracts proteins and salts. To reduce protein adhesion and salt deposition selected monomers were grafted by radical polymerization on the silicone surface. The conditions for surface modifications of PDMS using benzophenone as UV initiator were optimized. The modified surfaces were characterized properly using different methods. The effect of surface modifications on the albumin, as model protein, deposition was tested in an in vitro model.     

Poly(dimethylsiloxane) elastomers with tethered peptide ligands for cell adhesion studies

Poly(dimethylsiloxane) (PDMS) is the choice of material for a wide range of biological and non-biological applications because of its chemical inertness, non-toxicity, ease of handling and commercial availability. However, PDMS exhibits uncontrolled protein adsorption and cell adhesion and it has proved difficult to functionalize to present bioactive ligands. We present a facile strategy for functional surface modification of PDMS using commercial reagents to engineer polymer brushes of oligo(ethylene glycol) methacrylate that prevent cell adhesion and can be functionalized to display bioadhesive ligands. The polymer brushes resist biofouling and prevent cell adhesion and bioadhesive peptides can be tethered either uniformly or constrained to micropatterned domains using standard peptide chemistry approaches. This approach is relevant to various biomedical and biotechnological applications.     

Oriented astroglial cell growth on micropatterned polystyrene substrates

In an effort to develop a permissive environment for neural stem cell differentiation, directional growth of astrocytes has been achieved on polymer substrates in vitro. Manipulating a combination of physical and chemical cues, astrocyte adhesion and alignment in vitro were examined. To provide physical guidance, micropatterned polymer substrates of polystyrene (PS) were fabricated. Laminin was selectively adsorbed onto the grooves of the patterned surface. Rat type-1 astrocytes were seeded onto the micropatterned PS substrates, and the effects of substrate topography and the adsorption of laminin to the PS substrates on the behavior and morphology of the astrocytes were explored. The astrocytes were found to align parallel to the micropatterned grooves at initial seeding densities of approximately 7500, 13,000, and 20,000 cells/cm(2) due to the effects of the physical and chemical guidance mechanisms. Adsorbing laminin in the microgrooves of the micropatterned PS substrates improved cell adhesion and spreading of cytoskeletal filaments significantly. At these initial seeding densities, over 85% astrocyte alignment in the direction of the grooves was achieved on the micropatterned PS substrates with laminin adsorbed in the grooves. This combination of guidance cues has the potential to provide a permissive substrate for in vivo regeneration within the central nervous system.     

Integrating polyurethane culture substrates into poly(dimethylsiloxane) microdevices

Poly(dimethylsiloxane) (PDMS)-based microdevices have enabled rapid, high-throughput assessment of cellular response to precisely controlled microenvironmental stimuli, including chemical, matrix and mechanical factors. However, the use of PDMS as a culture substrate precludes long-term culture and may significantly impact cell response. Here we describe a method to integrate polyurethane (PU), a well-studied and clinically relevant biomaterial, into the PDMS multilayer microfabrication process, enabling the exploration of long-term cellular response on alternative substrates in microdevices. To demonstrate the utility of these hybrid microdevices for cell culture, we compared initial cell adhesion, cell spreading, and maintenance of protein patterns on PU and PDMS substrates. Initial cell adhesion and cell spreading after three days were comparable between collagen-coated PDMS and PU substrates (with or without collagen coating), but significantly lower on native PDMS substrates. However, for longer culture durations (≥6 days), cell spreading and protein adhesion on PU substrates was significantly better than that on PDMS substrates, and comparable to that on tissue culture-treated polystyrene. Thus, the use of a generic polyurethane substrate in microdevices enables longer-term cell culture than is possible with PDMS substrates. More generally, this technique can improve the impact and applicability of microdevice-based research by facilitating the use of alternate, relevant biomaterials while maintaining the advantages of using PDMS for microdevice fabrication.     

乳酸-乙醇酸共聚物载药复合纤维制备及其释药行为研究

乳酸-乙醇酸共聚物(PLGA)复合纤维具有良好的生物相容性和生物可降解性,且降解速率可由相对分子质量和共聚物组成来调控.采用同轴静电纺丝法制备以PLGA为壳层材料、聚乙烯基吡咯烷酮(PVP)为内芯材料的复合纤维,研究两种模型药物(5-氟尿嘧啶和硝苯地平)在同轴复合纤维载体中的药物释放行为,并用扫描、透射电镜观察复合纤维的形貌与结构,用紫外分光光度计测量载药量和累积释放率.实验结果表明,通过改变芯、壳纺丝液浓度、PLGA相对分子质量以及共聚物中LA和GA的组成比,可制得具有芯-壳结构且直径大小不同的复合纤维.采用相同电纺丝条件,可以将不同药物以相同载药量包覆于复合纤维中,但药物的释放行为不相同.     

携载rhBMP-2微球的新型复合人工骨的制备及生物相容性研究

目的制备一种具有良好生物相容性、降解性和成骨活性、可注射的自凝固新型骨修复材料。方法采用复乳溶剂挥发方法制备携载rhBMP-2的聚乳酸与聚乙醇酸共聚物（PLGA）微球，并将其与rhBMP-2／磷酸钙骨水泥（CPC）复合，制备出rhBMP-2／PLGA微球／CPC复合人工骨。探讨材料特性包括形貌和体外rhBMP-2释放速度，采用体外细胞培养的方法测定复合材料的细胞黏附能力及其浸提液对于人骨髓基质干细胞（MSCs）增殖和成骨分化的影响。结果与单纯rhBMP-2／CPC材料相比较，复合材料rhBMP-2体外释药明显提高。材料与MSCs可良好黏附并使其增殖。体外培养时材料不同时间的浸提液对MSCs细胞的增殖具有促进作用，对于细胞成骨分化的影响与单纯CPC无明显差别。结论rhBMP-2／PLGA微球／磷酸钙骨水泥新型复合人工骨具有良好的生物相容性和活性因子缓释功能，是一种有良好应用前景的骨修复材料。     

Surface studies of coated polymer microspheres and protein release from tissue-engineered scaffolds

The controlled release of growth factors from porous, polymer scaffolds is being studied for potential use as tissue-engineered scaffolds. Biodegradable polymer microspheres were coated with a biocompatible polymer membrane to permit the incorporation of the microspheres into tissue-engineered scaffolds. Surface studies with poly(D,L-lactic-co-glycolic acid) [PLGA], and poly(vinyl alcohol) [PVA] were conducted. Polymer films were dip-coated onto glass slides and water contact angles were measured. The contact angles revealed an initially hydrophobic PLGA film, which became hydrophilic after PVA coating. After immersion in water, the PVA coating was removed and a hydrophobic PLGA film remained. Following optimization using these 2D contact angle studies, biodegradable PLGA microspheres were prepared, characterized, and coated with PVA. X-ray photoelectron spectroscopy was used to further characterize coated slides and microspheres. The release of the model protein bovine serum albumin from PVA-coated PLGA microspheres was studied over 8 days. The release of BSA from PVA-coated PLGA microspheres embedded in porous PLGA scaffolds over 24 days was also examined. Coating of the PLGA microspheres with PVA permitted their incorporation into tissue-engineered scaffolds and resulted in a controlled release of BSA.     

Preparation and characterization of cationic PLGA nanospheres as DNA carriers

Nanoparticles formulated from biodegradable polymers such as poly(lactic acid) (PLA) and poly(lactide-co-glycolide) (PLGA) are being extensively investigated as non-viral gene delivery systems due to their controlled release characteristics and biocompatibility. PLGA nanoparticles for DNA delivery are mainly formulated by an emulsion-solvent evaporation technique using PVA as a stabilizer generating negatively charged particles and heterogeneous size distribution. The objective of the present study was to formulate cationically modified PLGA nanoparticles with defined size and shape that can efficiently bind DNA. An Emulsion-diffusion-evaporation technique to make cationic nanospheres composed of biodegradable and biocompatible co-polyester PLGA has been developed. PVA-chitosan blend was used to stabilize the PLGA nanospheres. The nanospheres were characterized by atomic force microscopy (AFM), photon-correlation spectroscopy (PCS), and Fourier transform infrared spectroscopy (FTIR). Zeta potential and gel electrophoresis studies were also performed to understand the surface properties of nanospheres and their ability to condense negatively charged DNA. The designed nanospheres have a zeta potential of 10mV at pH 7.4 and size under 200nm. From the gel electrophoresis studies we found that the charge on the nanospheres is sufficient to efficiently bind the negatively charged DNA electrostatically. These cationic PLGA nanospheres could serve as potential alternatives of the existing negatively charged nanoparticles.     

Adhesion contact dynamics of 3T3 fibroblasts on poly (lactide-co-glycolide acid) surface modified by photochemical immobilization of biomacromolecules

A simple and effective method of biomacromolecule immobilization on biomaterial surface for direct tuning of biophysical parameters such as the initial cell deformation rate, degree of cell spreading and adhesion kinetics is important for tissue engineering. The photochemical immobilization of azide-chitosan (Az-CS) on poly (lactide-co-glycolide) acid (PLGA) is applied here. Chitosan immobilization on PLGA through the photoactive azide group further facilitates subsequent grafting of other biocompatible biomacromolecules like gelatin (Gel) through the active amine groups on CS. This study quantitatively compares the 3T3 fibroblast adhesion dynamics on three PLGA surfaces (Gel-CS-PLGA, CS-PLGA and unmodified PLGA surfaces) using Confocal-Reflectance Interference Contrast Microscopy (C-RICM) together with phase contrast imaging. CS-PLGA and Gel-CS-PLGA surfaces developed were confirmed by X-ray photoelectron spectroscopy, atomic force microscopy and water contact angle and cell adhesion contact dynamics measurements. The cell adhesion was strongest on the Gel-CS-PLGA surface and lowest on unmodified PLGA. The steady state adhesion energy attained by the cells on gelatin modified PLGA surface is determined as 4.0 x 10(-8) J/m(2), which is about 400 times higher than that on PLGA surface (1.1 x 10(-10) J/m(2)). Significantly increased cell adhesion with Gel-CS-PLGA is postulated to result in increased cell spreading. Our integrated biophysical method can quantify the transient contact dynamics and is sufficiently accurate to discriminate even between Gel and CS modified surfaces.     

The potential of polymeric nanoparticles to increase the immunogenicity of the hapten clenbuterol

Micro‐ and nanoparticles prepared from the biodegradable and biocompatible polymers poly(lactide‐co‐glycolide) (PLGA) and polymethylmethacrylate (PMMA) have been successfully used as immunopotentiating antigen delivery systems. In our study, this approach was used to improve polyclonal antibody production to clenbuterol (CBL), a model hapten. PLGA and PMMA nanoparticles were loaded with either CBL alone or with a clenbuterol‐transferrin conjugate (CBL—Tfn) and administered subcutaneously to mice. PLGA nanoparticles were administered with or without the saponin adjuvant Quil A. The anti‐CBL titres present in experimental sera were determined by an enzyme immunoassay (ELISA). CBL‐Tfn‐loaded PLGA nanoparticles co‐administered with Quil A had obvious advantages immunologically over the currently used method of raising antibodies to CBL (the positive control). The combined adjuvanticity of Quil A and PLGA nanoparticles resulted in a positive response in all four of the mice tested and in higher antibody titres than were seen in the positive control group. Furthermore, the sustained release of immunogen from the nanoparticles permitted a reduction in immunizing frequency over the 15‐week study period.     

Surface studies of coated polymer microspheres and protein release from tissue-engineered scaffolds

The controlled release of growth factors from porous, polymer scaffolds is being studied for potential use as tissue-engineered scaffolds. Biodegradable polymer microspheres were coated with a biocompatible polymer membrane to permit the incorporation of the microspheres into tissueengineered scaffolds. Surface studies with poly(D,L-lactic-co-glycolic acid) [PLGA], and poly(vinyl alcohol) [PVA] were conducted. Polymer films were dip-coated onto glass slides and water contact angles were measured. The contact angles revealed an initially hydrophobic PLGA film, which became hydrophilic after PVA coating. After immersion in water, the PVA coating was removed and a hydrophobic PLGA film remained. Following optimization using these 2D contact angle studies, biodegradable PLGA microspheres were prepared, characterized, and coated with PVA. X-ray photoelectron spectroscopy was used to further characterize coated slides and microspheres. The release of the model protein bovine serum albumin from PVA-coated PLGA microspheres was studied over 8 days. The release of BSA from PVA-coated PLGA microspheres embedded in porous PLGA scaffolds over 24 days was also examined. Coating of the PLGA microspheres with PVA permitted their incorporation into tissue-engineered scaffolds and resulted in a controlled release of BSA.     

In vitro characterization of micropatterned PLGA-PHBV8 blend films as temporary scaffolds for photoreceptor cells

In developed countries the aging population faces increasing risks of blinding retinal diseases, for which there are few effective treatments available. Photoreceptor transplantation represents one approach, but generally results have been disappointing. We hypothesize that micropatterned biodegradable poly(L-lactic acid-co-glycolic acid)/poly(hydroxybutyrate-co-hydroxyvaleric acid) (PLGA-PHBV8) blend films could deliver photoreceptor cells in a more organized manner than bolus injections. Blending of PLGA and PHBV8 was used to optimize the degradation rate of the temporary template. At the end of 8 weeks, for both thin and thick films of PLGA-PHBV8 a 50% decrease of their initial weight with increasing water uptake was observed. When photoreceptor cells were seeded onto micropatterned PLGA-PHBV8 films with parallel grooves (21- and 42-microm-wide grooves and 20 microm ridge width and depth), the cells preferred laminin-deposited grooves to ridges and expressed rod- and cone-specific markers such as rhodopsin and arrestin. A loss in photoreceptor viability of 50% was observed after 7 days in culture. The effects of either retinal pigment epithelium (RPE)-derived or Muller glial cell-derived conditioned media or bFGF on the survival of photoreceptor cells seeded on PLGA-PHBV8 films were investigated. Addition of either RPE- and Muller-conditioned media increased statistically (p < 0.01) the viability of photoreceptor cells after 7 days of incubation. Our results suggest that such biodegradable micropatterned PLGA-PHBV8 blend films have a potential to deliver photoreceptor cells to the subretinal space and ensure laminar organization and maintenance of differentiation, and that incorporation of intrinsic factors within the scaffold would enhance the survival rate of transplanted cells.