Design of Plasma Surface‐Activated,Electrospun Polylactide Non‐Wovens with Improved Cell Acceptance

Electrospinning is a versatile technique to generate tissue engineering matrices possessing structural features similar to the extracellular matrix. Biodegradable polylactides are well suited for processing by this technique, but their innate hydrophobicity impairs initial protein adsorption and cell adhesion. In this work, therefore, electrospun poly(L ‐lactide‐co‐D,L ‐lactide) (70/30) non‐wovens are modified with an ultrathin plasma‐polymerized allylamine (PPAAm) coating. Using scanning electron microsocopy (SEM), it is shown that the fiber structure of the non‐woven is not affected by the plasma treatment. X‐ray photoelectron spectroscopy (XPS) and contact angle measurements of PPAAm‐coated non‐wovens confirm the presence of nitrogen and oxygen‐functional groups in the coating and a hydrophilic nature of the coated non‐woven surface. Cell experiments in vitro demonstrate that the PPAAm‐coated surface promotes occupancy of the non‐woven by human MG‐63 osteoblasts accompanied by improved initial cell spreading and filopodia formation along and between the electrospun polylactide fibers. Overall, plasma‐assisted incorporation of amino groups into electrospun polylactone non‐wovens represents a promising approach to tissue engineering scaffolds with improved cell–material interfaces.     

A Simple Modification Method to Obtain Anisotropic and Porous 3D Microfibrillar Scaffolds for Surgical and Biomedical Applications

In native tissues, cellular organization is predominantly anisotropic. Yet, it remains a challenge to engineer anisotropic scaffolds that promote anisotropic cellular organization at macroscopic length scales. To overcome this challenge, an innovative, cheap and easy method to align clinically approved non‐woven surgical microfibrillar scaffolds is presented. The method involves a three‐step process of coating, unidirectional stretching of scaffolds after heating them above glass transition temperature, and cooling back to room temperature. Briefly, a polymer coating is applied to a non‐woven mesh that results in a partial welding of randomly oriented microfibers at their intersection points. The coated scaffold is then heated above the glass transition temperature of the coating and the scaffold polymer. Subsequently, the coated scaffold is stretched to produce aligned and three dimentional (3D) porous fibrillar scaffolds. In a proof of concept study, a polyglycolic acid (PGA) micro‐fibrillar scaffold was coated with poly(4‐hydroxybutirate) (P4HB) acid and subsequently aligned. Fibroblasts were cultured in vitro within the scaffold and results showed an increase in cellular alignment along the direction of the PGA fibers. This method can be scaled up easily for industrial production of polymeric meshes or directly applied to small pieces of scaffolds at the point of care.     

Evolution of PVA gels prepared without crosslinking agents as a cell adhesive surface

Physical parameters (such as crosslinking density, crystallinity and mechanical properties) have been found to largely affect cellular behavior on polymer scaffolds. This study demonstrated that transparent pure Poly (vinyl alcohol) hydrogels prepared via a freeze–thaw method can be made to support cell adhesion by controlling physical parameters such as concentration and the number of freeze–thaw cycles. For a given number of freeze–thaw cycles, (specifically 45), polymer concentration dependent structural and mechanical properties (such as tensile strength and stiffness) were correlated with cell adhesion. The maximum cell attachment occurred on the hydrogels with the greatest mechanical properties, crystallinity and crosslinking density. The hydrogel surfaces were more favorable to human dermal fibroblasts than human lens epithelial cells and retained their transparency as well as dimensional stability with only a small degree of swelling. Fibroblast laden hydrogels showed extensive alkaline phosphatase activity which confirmed their healthy proliferation and function. In this manner, this study suggests that transparent Poly (vinyl alcohol) hydrogels prepared by the freeze thaw method described here should be further studied for numerous tissue engineering applications.     

Structuring of Functional Spider Silk Wires,Coatings, and Sheets by Self‐Assembly on Superhydrophobic Pillar Surfaces

Spider silk has recently become a material of high interest for a large number of biomedical applications. Previous work on structuring of silk has resulted in particles (0D), fibers (1D), films (2D), and foams, gels, capsules, or microspheres (3D). However, the manufacturing process of these structures is complex and involves posttreatment of chemicals unsuitable for biological applications. In this work, the self‐assembly of recombinant spider silk on micropatterned superhydrophobic surfaces is studied. For the first time, structuring of recombinant spider silk is achieved using superhydrophobic surfaces under conditions that retain the bioactivity of the functionalized silk. By tuning the superhydrophobic surface geometry and the silk solution handling parameters, this approach allows controlled generation of silk coatings, nanowires, and sheets. The underlying mechanisms and governing parameters are discussed. It is believed that the results of this work pave the way for fabrication of silk formations for applications including vehicles for drug delivery, optical sensing, antimicrobial coatings, and cell culture scaffolds.     

Enhanced mechanical properties of thermosensitive chitosan hydrogel by silk fibers for cartilage tissue engineering

Articular cartilage has limited repair capability following traumatic injuries and current methods of treatment remain inefficient. Reconstructing cartilage provides a new way for cartilage repair and natural polymers are often used as scaffold because of their biocompatibility and biofunctionality. In this study, we added degummed chopped silk fibers and electrospun silk fibers to the thermosensitive chitosan/glycerophosphate hydrogels to reinforce two hydrogel constructs which were used as scaffold for hyaline cartilage regeneration. The gelation temperature and gelation time of hydrogel were analyzed by the rheometer and vial tilting method. Mechanical characterization was measured by uniaxial compression, indentation and dynamic mechanical analysis assay. Chondrocytes were then harvested from the knee joint of the New Zealand white rabbits and cultured in constructs. The cell proliferation, viability, production of glycosaminoglycans and collagen type II were assessed. The results showed that mechanical properties of the hydrogel were significantly enhanced when a hybrid with two layers of electrospun silk fibers was made. The results of GAG and collagen type II in cell-seeded scaffolds indicate support of the chondrogenic phenotype for chondrocytes with a significant increase in degummed silk fiber–hydrogel composite for GAG content and in two-layer electrospun fiber–hydrogel composite for Col II. It was concluded that these two modified scaffolds could be employed for cartilage tissue engineering.     

Biofunctionalized poly(ethylene glycol)-<Emphasis Type="Italic">block</Emphasis>-poly(ε-caprolactone) nanofibers for tissue engineering

Electrospun fibers with contrasting cell adhesion properties provided non-woven substrates with enhanced in vitro acceptance and controllable cell interactions. Poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-b-PCL) block copolymers with varying segment lengths were synthesized in two steps and characterized by NMR and GPC. A cell adhesive peptide sequence, GRGDS, was covalently coupled to the PEG segment of the copolymer in an additional step. The suitability of polymers with molecular weights ranging from 10 to 30 kDa for electrospinning and the influences of molecular weight, solvent, and concentration on the resulting morphologies were investigated. Generally, electrospun fibers were obtained by electrospinning polymers with molecular weight larger than 25 kDa and concentrations of 10 wt%. Methanol/chloroform (25/75, v/v) mixtures proved to be good solvent systems for electrospinning the PEG-b-PCL and resulted in hydrophilic, non-woven fiber meshes (contact angle 30°). The mesh without cell adhesive GRGDS ligands showed no attachment of human dermal fibroblasts after 24 h cell culture demonstrating that the particular combination of the material and electrospinnig conditions yielded protein and cell repellent properties. The GRGDS immobilized mesh showed excellent cellular attachment with fibroblasts viable after 24 h with spread morphology. Electrospun nanofibers based on block copolymers have been produced which are capable of specifically targeting cell receptor binding and are a promising material for tissue engineering and controlling cell material interactions.     

Tuning of Cell–Biomaterial Anchorage for Tissue Regeneration

Which mechanisms mediate cell attachment to biomaterials? What role does the surface charge or wettability play on cell–material anchorage? What are the currently investigated strategies to modify cell–matrix adherence spatiotemporally? Considering the development of scaffolds made of biocompatible materials to temporarily replace the structure and/or function of the extracellular matrix, focus is given to the analysis of the specific (i.e., cell adhesive peptide sequences) and unspecific (i.e., surface charge, wettability) mechanisms mediating cell‐matrix interactions. Furthermore, because natural tissue regeneration is characterized by the dynamic attachment/detachment of different cell populations, the design of advanced scaffolds for tissue engineering, based in the spatiotemporal tuning of cell–matrix anchorage is discussed.     

Fabrication and Fibroblast Attachment Property of Regenerated Silk Fibroin/Tetramethoxysilane Nanofibrous Biocomposites

In this study, regenerated silk fibroin (RSF)/tetramethoxysilane (TMOS) composite electrospun fibers with improved hydrophilicity were successfully prepared by electrospinning method, which was superior for fibroblast attachment. The electrospinning process caused adjacent fibers to “weld” at contact points, as confirmed by scanning electron microscope (SEM) analysis. Fourier transform infrared spectroscopy (FTIR) showed that TMOS has been well incorporated into the silk fibroin electrospun fibers. Thermogravimetric analysis (TGA) was carried out to quantify the water content in RSF/TMOS composite electrospun fibers. The cytotoxicity and L929 adhesion of three‐dimensional RSF/TMOS fibrous biocompoaites were investigated and compared to pure RSF membrane. The water contact angle of RSF/TMOS nanofibrous composites showed a sharp decrease compared to the pure RSF electrospun fibers, which has a great effect on the early stage of cell attachment behavior due to an relatively enhanced hydrophilicity.     

Combinatorial On‐Chip Study of Miniaturized 3D Porous Scaffolds Using a Patterned Superhydrophobic Platform

One of the main challenges in tissue engineering (TE) is to obtain optimized products, combining biomaterials, cells and soluble factors able to stimulate tissue regeneration. Multiple combinations may be considered by changing the conditions among these three factors. The unpredictable response of each combination requires time‐consuming tests. High‐throughput methodologies have been proposed to master such complex analyses in TE. Usually, these tests are performed using cells cultured into 2D biomaterials or by dispensing arrays of cell‐loaded hydrogels. For the first time an on‐chip combinatorial study of 3D miniaturized porous scaffolds is proposed, using a patterned bioinspired superhydrophobic platform. Arrays of biomaterials are dispensed and processed in situ as porous scaffolds with distinct composition, surface characteristics, porosity/pore size, and mechanical properties. On‐chip porosity, pore size, and mechanical properties of scaffolds based on chitosan and alginate are assessed by adapting microcomputed tomography equipment and a dynamic mechanical analyzer, as well as cell response after 24 hours. The interactions between cell types of two distinct origins—osteoblast‐like and fibroblasts—and the scaffolds modified with fibronectin are studied and validated by comparison with conventional destructive methods (dsDNA quantification and MTS tests). Physical and biological on‐chip analyses are coherent with the conventional measures, and conclusions about the most favorable conditions for each cell type are taken.     

Silk RGD融合蛋白修饰羟基磷灰石/丝素蛋白支架对成骨细胞生长的影响

通过浸渍吸附的方法, 用桑蚕丝素-RGD融合蛋白(简称Silk-RGD)对多孔状磷灰石/丝素蛋白(HA/SF)复合支架材料进行表面修饰, 研究了复合支架材料在不同浓度Silk-RGD蛋白溶液中浸渍后对两种不同成骨细胞MG-63和MC3T3-E1黏附、增殖和分化的影响。结果表明, Silk-RGD融合蛋白修饰的复合支架材料的细胞黏附性能显著高于未经修饰的对照组, 且其促黏附性能具有Silk-RGD浓度依赖性; 体外培养7天时, 细胞增殖能力较对照组更显著,当Silk-RGD的吸附量为11 μg/mg时, MG-63的增殖率较对照样提高了21%, MC3T3-E1提高了50%; 而碱性磷酸酶活性检测结果显示, 复合支架经Silk-RGD表面修饰后对MC3T3-E1细胞的分化有一定的促进作用, 但对MG-63细胞的影响不明显。      

Synergetic Material and Structure Optimization Yields Robust Spider Web Anchorages

Millions of years of evolution have adapted spider webs to achieve a range of properties, including the well‐known capture of prey, with efficient use of materials. One feature that remains poorly understood is the attachment disc, a network of silk fibers that mechanically anchors a web to its environment. Experimental observations suggest that one possible attachment disc adheres to a substrate through multiple symmetrically branched structures composed of sub‐micrometer scale silk fibers. Here, a theoretical model is used to explore the adaptation of the strength of attachment of such an anchorage, and complementary mesoscale simulations are applied to demonstrate a novel mechanism of synergetic material and structural optimization, such that the maximum anchorage strength can be achieved regardless of the initial anchor placement or material type. The optimal delamination (peeling) angle is facilitated by the inherent extensibility of silk, and is attained automatically during the process of delamination. This concept of self‐optimizing peeling angle suggests that attachment discs do not require precise placement by the spider, irrespective of adhesion strength. Additional hierarchical branching of the anchorage increases efficiency, where both the delamination force and toughness modulus increase with a splitting of the cross‐sectional area.     

Silkworm and spider silk scaffolds for chondrocyte support

Objective To create scaffolds with silkworm cocoon, spider egg sac and spider dragline silk fibres and examine their use for chondrocyte attachment and support. Methods Three different kinds of scaffolds were developed with Bombyx mori cocoon, Araneus diadematus egg sac and dragline silk fibres. The attachment of human articular cartilage cells were investigated on these bioprotein matrices. The chondrocytes produced an extracellular matrix which was studied by immunostaining. Moreover, the compression behaviour in relation to the porosity was studied. Results The compression modulus of a silkworm silk scaffold was related to its porosity. Chondrocytes were able to attach and to grow on the different fibres and in the scaffolds for several weeks while producing extracellular matrix products. Conclusion Porous scaffolds can be made out of silkworm and spider silk for cartilage regeneration. Mechanical properties are related to porosity and pore size of the construct. Cell spreading and cell expression depended on the porosity and pore-size.     

A smart bilayer scaffold of elastin-like recombinamer and collagen for soft tissue engineering

Elastin-like recombinamers (ELRs) are smart, protein-based polymers designed with desired peptide sequences using recombinant DNA technology. The aim of the present study was to produce improved tissue engineering scaffolds from collagen and an elastin-like protein tailored to contain the cell adhesion peptide RGD, and to investigate the structural and mechanical capacities of the resulting scaffolds (foams, fibers and foam-fiber bilayer scaffolds). The results of the scanning electron microscopy, mercury porosimetry and mechanical testing indicated that incorporation of ELR into the scaffolds improved the uniformity and continuity of the pore network, decreased the pore size (from 200 to 20 μm) and the fiber diameter (from 1.179 μm to 306 nm), broadened the pore size distribution (from 70–200 to 4–200 μm) and increased their flexibility (from 0.007 to 0.011 kPa⁻¹). Culture of human fibroblasts and epithelial cells in ELR-collagen scaffolds showed the positive contribution of ELR on proliferation of both types of cells.     

Assessment of the Cytocompatibility of Poly‐(N‐hexylvinylpyridinium) Used as an Antibacterial Implant Coating

Medical implants made of titanium have a wide variety of applications, ranging from replacement of a single tooth to extraoral maxillofacial prosthetic rehabilitation or hip endoprosthesis. The long‐term success of such osseointegrated titanium implants is endangered by inflammation of periimplant hard or soft tissues caused by a bacterial infection. Therefore, implants should ideally inhibit bacterial adhesion and growth, but allows strong attachment of connective tissues or epithelium at the same time. Antimicrobial polymers like poly(vinyl‐N‐hexylpyridinium bromide) (hexyl‐PVP) are a promising approach as implant coatings to inhibit bacterial adhesion, but little is known about the biocompatibility of these polymers. The aim of the present study was to develop a method for evaluation of the cell acceptance of hexyl‐PVP or copolymers of vinyl‐N‐hexylpyridinium bromide and (4‐vinylbenzyl)phosphonic acid diethylester (poly((hexyl‐VP)‐co‐VBP)) as coating on titanium disks. Primary human gingival fibroblasts were used and biocompatibility was assessed by cell adhesion and proliferation. The cell morphology of the fibroblasts on these surfaces was analyzed by scanning electron microscopy (SEM) and was used as additional criterion. The results indicate no significant differences in adhesion or proliferation rate between primary human gingival fibroblasts seeded on polymer‐coated titanium disks and uncoated titanium disks as a control. Although SEM micrographs displayed moderate differences in cell morphology between the two groups, application of hexyl‐PVP or the corresponding copolymers as antibacterial coatings for medical implants or devices appears to be promising.     

Surface Modification of Electrospun Polycaprolactone Nanofiber Meshes by Plasma Treatment to Enhance Biological Performance

A critical aspect in the development of biomaterials is the optimization of their surface properties to achieve an adequate cell response. In the present work, electrospun polycaprolactone nanofiber meshes (NFMs) are treated by radio‐frequency (RF) plasma using different gases (Ar or O₂), power (20 or 30 W), and exposure time (5 or 10 min). Morphological and roughness analysis show topographical changes on the plasma‐treated NFMs. X‐ray photoelectron spectroscopy (XPS) results indicate an increment of the oxygen‐containing groups, mainly ? OH and ? C?O, at the plasma‐treated surfaces. Accordingly, the glycerol contact angle results demonstrate a decrease in the hydrophobicity of plasma‐treated meshes, particularly in the O₂‐treated ones. Three model cell lines (fibroblasts, chondrocytes, and osteoblasts) are used to study the effect of plasma treatments over the morphology, cell adhesion, and proliferation. A plasma treatment with O₂ and one with Ar are found to be the most successful for all the studied cell types. The influence of hydrophilicity and roughness of those NFMs on their biological performance is discussed. Despite the often claimed morphological similarity of NFMs to natural extracellular matrixes, their surface properties contribute substantially to the cellular performance and therefore those should be optimized.     

Upregulation of matrix and adhesion molecules induced by controlled topography

Electrostatic spinning is receiving increasing attention in the field of tissue engineering, due to its ability to produce 3-dimensional, multidirectional, microfibrous scaffolds. These structures are capable of supporting a wide range of cell growth; however, there is little knowledge relating material substrates with specific cellular interactions and responses. The aim of this research was to investigate if electrostatically spun scaffolds, with controlled topographical features, would affect the adhesion mechanisms of contacting cells. A range of electrostatically spun Tecoflex SG-80A polyurethane scaffolds was characterized in terms of inter-fibre separation, fibre diameter, surface roughness, void fraction and fibre orientation. Human embryonic lung fibroblasts and human vein endothelial cells were cultured on these scaffolds for 7, 14, 28 days, and analysed for their expression of extracellular matrix and adhesion molecules using image analysis and laser scanning confocal microscopy. There were significant differences in adhesion mechanisms between scaffolds, cell types and culture periods. Fibroblast-scaffolds were stimulated and oriented to a greater degree, and at earlier cultures, by the controlled topographical features than the endothelial cells. These conclusions confirm that cellular behaviour can be influenced by the induced scaffold topography at both molecular and cellular levels, with implications for optimum application specific tissue engineering constructs.     

Engineered spider silk-based 2D and 3D materials prevent microbial infestation

Biofilm formation, especially of antimicrobiotic-resistant microbial strains, are a major problem in health care. Therefore, there is great interest in developing advanced materials that are selectively inhibiting microbial adhesion to surfaces, but at the same time promoting mammalian cell growth. In nature, some spider silks have evolved to repel microbes, a feature that could be used in biomaterials. To unravel how microbe repellence can be achieved in engineered spider silk, different recombinant spider silk proteins based on the consensus sequences of Araneus diadematus dragline silk proteins (fibroin 3 and 4) were processed into 2D-patterned films and 3D-hydrogels. Strikingly, protein structure characteristics on the nanoscale are the basis for the detected microbe-repellence. Designed spider silk materials promoted mammalian cell attachment and proliferation while inhibiting microbial infestation, demonstrating the great potential of these engineered spider silk-based materials as bio-selective microbial-resistant coatings in biomedical as well as technical applications.     

Osteoblast attachment to a textured surface in the absence of exogenous adhesion proteins

The present study investigated whether osteoblasts could attach to a culture substratum through a surface texture-dependent mechanism. Four test groups were used: (A) untextured, and three texture groups with maximum feature sizes of (B) <0.5 /spl mu/m, (C) 2 /spl mu/m, and (D) 4 /spl mu/m, respectively. All surfaces were coated with the nonadhesive protein bovine serum albumin (BSA). Osteoblasts were allowed to adhere in serum-free medium for either 1 or 4 h, at which time nonadherent cells were removed. At 4 h, untextured surface A exhibited no cell attachment, while textured surfaces B, C, and D exhibited 9%, 32%, and 16% cell adhesion, respectively. At 16 h of incubation, adherent osteoblasts on textured surface C exhibited focal adhesion contacts and microfilament stress-fiber bundles. These results indicate that microtextured surfaces in the absence of exogenous adhesive proteins can facilitate osteoblast adhesion.     

Cell adhesion and accelerated detachment on the surface of temperature-sensitive chitosan and poly(N-isopropylacrylamide) hydrogels

A series of temperature-sensitive poly(NIPAAm-co-CSA) hydrogels were synthesized by the copolymerization of acrylic acid-derivatized Chitosan (CSA) and N-isopropylacrylamide (NIPAAm) in aqueous solution. Their swelling properties and L929 cell adhesion and detachment behaviors were studied. It was found that poly(NIPAAm-co-CSA) hydrogels were temperature-sensitive associated with the roles of the component PNIPAAm. Most significantly, poly(NIPAAm-co-CSA) hydrogels exhibited simultaneously good swelling properties. The investigation of L929 cell adhesion and detachment of poly(NIPAAm-co-CSA) hydrogels indicated the cell adhesion and spreading was higher on the surface of poly(NIPAAm-co-CSA) hydrogels than that of PNIPAAm hydrogel at 37 degrees C due to the incorporation of CS, which having excellent cell affinity. Poly(NIPAAm-co-CSA) hydrogels showed more rapid detachment of cell sheet compared to PNIPAAm hydrogel because of the highly hydrophilic and hygroscopic nature of CS chains when reducing the culture temperature from 37 degrees C to 20 degrees C.     

Amino Acids and Peptide‐Based Supramolecular Hydrogels for Three‐Dimensional Cell Culture

Supramolecular hydrogels assembled from amino acids and peptide‐derived hydrogelators have shown great potential as biomimetic three‐dimensional (3D) extracellular matrices because of their merits over conventional polymeric hydrogels, such as non‐covalent or physical interactions, controllable self‐assembly, and biocompatibility. These merits enable hydrogels to be made not only by using external stimuli, but also under physiological conditions by rationally designing gelator structures, as well as in situ encapsulation of cells into hydrogels for 3D culture. This review will assess current progress in the preparation of amino acids and peptide‐based hydrogels under various kinds of external stimuli, and in situ encapsulation of cells into the hydrogels, with a focus on understanding the associations between their structures, properties, and functions during cell culture, and the remaining challenges in this field. The amino acids and peptide‐based hydrogelators with rationally designed structures have promising applications in the fields of regenerative medicine, tissue engineering, and pre‐clinical evaluation.