Stable Non‐Covalent Large Area Patterning of Inert Teflon‐AF Surface: A New Approach to Multiscale Cell Guidance

Micro‐ and nano‐patterning of cell adhesion proteins is demonstrated to direct the growth of neural cells, viz. human neuroblastoma SHSY5Y, at precise positions on a strongly antifouling substrate of technolological interest. We adopt a soft‐lithographic approach with oxygen plasma modified PDMS stamps to pattern human laminin on Teflon‐AF films. These patterns are based on the interplay of capillary forces within the stamp and non‐covalent intermolecular and surface interactions. Remarkably, they remain stable for several days upon cell culture conditions. The fabrication of substrates with adjacent antifouling and adhesion‐promoting regions allows us to reach absolute spatial control in the positioning of neuroblastoma cells on the Teflon‐AF films. This patterning approach of a technologically‐relevant substrate can be of interest in tissue engineering and biosensing.     

Zwitterionic sulfobetaine-grafted poly(vinylidene fluoride) membrane with highly effective blood compatibility via atmospheric plasma-induced surface copolymerization

Development of nonfouling membranes to prevent nonspecific protein adsorption and platelet adhesion is critical for many biomedical applications. It is always a challenge to control the surface graft copolymerization of a highly polar monomer from the highly hydrophobic surface of a fluoropolymer membrane. In this work, the blood compatibility of poly(vinylidene fluoride) (PVDF) membranes with surface-grafted electrically neutral zwitterionic poly(sulfobetaine methacrylate) (PSBMA), from atmospheric plasma-induced surface copolymerization, was studied. The effect of surface composition and graft morphology, electrical neutrality, hydrophilicity and hydration capability on blood compatibility of the membranes were determined. Blood compatibility of the zwitterionic PVDF membranes was systematically evaluated by plasma protein adsorption, platelet adhesion, plasma-clotting time, and blood cell hemolysis. It was found that the nonfouling nature and hydration capability of grafted PSBMA polymers can be effectively controlled by regulating the grafting coverage and charge balance of the PSBMA layer on the PVDF membrane surface. Even a slight charge bias in the grafted zwitterionic PSBMA layer can induce electrostatic interactions between proteins and the membrane surfaces, leading to surface protein adsorption, platelet activation, plasma clotting and blood cell hemolysis. Thus, the optimized PSBMA surface graft layer in overall charge neutrality has a high hydration capability and the best antifouling, anticoagulant, and antihemolytic activities when comes into contact with human blood.     

Functionalization of the surface of electrospun poly(epsilon-caprolactone) mats using zwitterionic poly(carboxybetaine methacrylate) and cell-specific peptide for endothelial progenitor cells capture

A novel approach for vascular grafts to achieve rapid endothelialization is to attract endothelial progenitor cells (EPCs) from peripheral blood onto grafts via EPC-specific antibodies, aptamer, or peptides that specifically bind to EPCs. Inspired by this idea, the electrospun poly(epsilon-caprolactone) (PCL) mats were modified with zwitterionic poly(carboxybetaine methacrylate) (PCBMA) and phage display-selected-EPC-specific peptide, TPSLEQRTVYAK (TPS). We tested the physical and chemical properties, cyto-compatibility, and platelet adhesion of the modified material, and investigated the specificity of the functionalized surface for capturing EPCs. The results indicated that PCL modified with zwitterionic PCBMA and TPS peptide showed improved hydrophilicity without morphology change and damage of the mats. Furthermore, the modified material supported adherence and growth of vascular cells and resisted platelets adhesion. The surfaces also specifically captured EPCs rather than bone marrow mesenchymal stem cells and human umbilical vein endothelial cells. This surface-functionalized PCL graft may offer new opportunities for designing new vascular grafts.     

Ultralow‐Fouling,Functionalizable, and Hydrolyzable Zwitterionic Materials and Their Derivatives for Biological Applications

In recent years, zwitterionic materials such as poly(carboxybetaine) (pCB) and poly(sulfobetaine) (pSB) have been applied to a broad range of biomedical and engineering materials. Due to electrostatically induced hydration, surfaces coated with zwitterionic groups are highly resistant to nonspecific protein adsorption, bacterial adhesion, and biofilm formation. Among zwitterionic materials, pCB is unique due to its abundant functional groups for the convenient immobilization of biomolecules. pCB can also be prepared in a hydrolyzable form as cationic pCB esters, which can kill bacteria or condense DNA. The hydrolysis of cationic pCB esters into nonfouling zwitterionic groups will lead to the release of killed microbes or the irreversible unpackaging of DNA. Furthermore, mixed‐charge materials have been shown to be equivalent to zwitterionic materials in resisting nonspecific protein adsorption when they are uniformly mixed at the molecular scale.     

Molecular Design of Antifouling Polymer Brushes Using Sequence‐Specific Peptoids

Material systems that can be used to flexibly and precisely define the chemical nature and molecular arrangement of a surface would be invaluable for the control of complex biointerfacial interactions. For example, progress in antifouling polymer biointerfaces that prevents nonspecific protein adsorption and cell attachment, which can significantly improve the performance of an array of biomedical and industrial applications, is hampered by a lack of chemical models to identify the molecular features conferring their properties. Poly(N‐substituted glycine) “peptoids” are peptidomimetic polymers that can be conveniently synthesized with specific monomer sequences and chain lengths, and are presented as a versatile platform for investigating the molecular design of antifouling polymer brushes. Zwitterionic antifouling polymer brushes have captured significant recent attention, and a targeted library of zwitterionic peptoid brushes with different charge densities, hydration, separations between charged groups, chain lengths, and grafted chain densities, is quantitatively evaluated for their antifouling properties through a range of protein adsorption and cell attachment assays. Specific zwitterionic brush designs are found to give rise to distinct but subtle differences in properties. The results also point to the dominant roles of the grafted chain density and chain length in determining the performance of antifouling polymer brushes.     

Robotic dispensing of composite scaffolds and in vitro responses of bone marrow stromal cells

The development of bioactive scaffolds with a designed pore configuration is of particular importance in bone tissue engineering. In this study, bone scaffolds with a controlled pore structure and a bioactive composition were produced using a robotic dispensing technique. A poly(ε-caprolactone) (PCL) and hydroxyapatite (HA) composite solution (PCL/HA = 1) was constructed into a 3-dimensional (3D) porous scaffold by fiber deposition and layer-by-layer assembly using a computer-aided robocasting machine. The in vitro tissue cell compatibility was examined using rat bone marrow stromal cells (rBMSCs). The adhesion and growth of cells onto the robotic dispensed scaffolds were observed to be limited by applying the conventional cell seeding technique. However, the initially adhered cells were viable on the scaffold surface. The alkaline phosphatase activity of the cells was significantly higher on the HA–PCL than on the PCL and control culture dish, suggesting that the robotic dispensed HA–PCL scaffold should stimulate the osteogenic differentiation of rBMSCs. Moreover, the expression of a series of bone-associated genes, including alkaline phosphatase and collagen type I, was highly up-regulated on the HA–PCL scaffold as compared to that on the pure PCL scaffold. Overall, the robotic dispensed HA–PCL is considered to find potential use as a bioactive 3D scaffold for bone tissue engineering. Seok-Jung Hong and Ishik Jeong contributed equally.     

Surface modified electrospun nanofibrous scaffolds for nerve tissue engineering

The development of biodegradable polymeric scaffolds with surface properties that dominate interactions between the material and biological environment is of great interest in biomedical applications. In this regard, poly-ε-caprolactone (PCL) nanofibrous scaffolds were fabricated by an electrospinning process and surface modified by a simple plasma treatment process for enhancing the Schwann cell adhesion, proliferation and interactions with nanofibers necessary for nerve tissue formation. The hydrophilicity of surface modified PCL nanofibrous scaffolds (p-PCL) was evaluated by contact angle and x-ray photoelectron spectroscopy studies. Naturally derived polymers such as collagen are frequently used for the fabrication of biocomposite PCL/collagen scaffolds, though the feasibility of procuring large amounts of natural materials for clinical applications remains a concern, along with their cost and mechanical stability. The proliferation of Schwann cells on p-PCL nanofibrous scaffolds showed a 17% increase in cell proliferation compared to those on PCL/collagen nanofibrous scaffolds after 8 days of cell culture. Schwann cells were found to attach and proliferate on surface modified PCL nanofibrous scaffolds expressing bipolar elongations, retaining their normal morphology. The results of our study showed that plasma treated PCL nanofibrous scaffolds are a cost-effective material compared to PCL/collagen scaffolds, and can potentially serve as an ideal tissue engineered scaffold, especially for peripheral nerve regeneration.     

Polymer Powder Processing of Cryomilled Polycaprolactone for Solvent‐Free Generation of Homogeneous Bioactive Tissue Engineering Scaffolds

Synthetic polymers used in tissue engineering require functionalization with bioactive molecules to elicit specific physiological reactions. These additives must be homogeneously dispersed in order to achieve enhanced composite mechanical performance and uniform cellular response. This work demonstrates the use of a solvent‐free powder processing technique to form osteoinductive scaffolds from cryomilled polycaprolactone (PCL) and tricalcium phosphate (TCP). Cryomilling is performed to achieve micrometer‐sized distribution of PCL and reduce melt viscosity, thus improving TCP distribution and improving structural integrity. A breakthrough is achieved in the successful fabrication of 70 weight percentage of TCP into a continuous film structure. Following compaction and melting, PCL/TCP composite scaffolds are found to display uniform distribution of TCP throughout the PCL matrix regardless of composition. Homogeneous spatial distribution is also achieved in fabricated 3D scaffolds. When seeded onto powder‐processed PCL/TCP films, mesenchymal stem cells are found to undergo robust and uniform osteogenic differentiation, indicating the potential application of this approach to biofunctionalize scaffolds for tissue engineering applications.     

Surface-mineralized polymeric nanofiber for the population and osteogenic stimulation of rat bone-marrow stromal cells

Nanofibrous biomaterials made of degradable polymers, including poly(?-caprolactone) (PCL), are considered as a potential substrate for populating and differentiating tissue cells. The surface modification of biomaterials is of utmost importance in regulating cell functions. This study examined the effects of an apatite-mineralization of the PCL nanofiber surface on the growth behavior and osteogenic differentiation of rat bone-marrow stromal cells (rBMSCs). BMSCs isolated from adult rats were seeded on the PCL nanofiber with mineralization (PCLnf-M) and without it as a control (PCLnf), and cultured for up to 28 days. Initially (for 1 h), the cells adhered better on the PCLnf-M than on the control, showing favorable cell affinity to the mineralized nanofibrous surface. The secretion of collagen by the cells was shown to increase with culturing time on both types of the nanofiber. The cell viability was similar at day 7 but was higher on the control at a prolonged period of day 14. However, the alkaline phosphatase (AP) activity was noticed to significantly higher level on the PCLnf-M than on the control at days 14 and 28. Overall, the surface-mineralized PCL nanofibrous substrate was shown to support the adhesion and growth of BMSCs and to stimulate the differentiation into an osteogenic lineage. This type of nanofibrous sheet is considered to be useful as a matrix for the regeneration and engineering of skeletal tissues.     

Design of novel three-phase PCL/TZ–HA biomaterials for use in bone regeneration applications

The design of bioactive scaffold materials able to guide cellular processes involved in new-tissue genesis is key determinant in bone tissue engineering. The aim of this study was the design and characterization of novel multi-phase biomaterials to be processed for the fabrication of 3D porous scaffolds able to provide a temporary biocompatible substrate for mesenchymal stem cells (MSCs) adhesion, proliferation and osteogenic differentiation. The biomaterials were prepared by blending poly(ε-caprolactone) (PCL) with thermoplastic zein (TZ), a thermoplastic material obtained by de novo thermoplasticization of zein. Furthermore, to bioactivate the scaffolds, microparticles of osteoconductive hydroxyapatite (HA) were dispersed within the organic phases. Results demonstrated that materials and formulations strongly affected the micro-structural properties and hydrophilicity of the scaffolds and, therefore, had a pivotal role in guiding cell/scaffold interaction. In particular, if compared to neat PCL, PCL–HA composite and PCL/TZ blend, the three-phase PCL/TZ–HA showed improved MSCs adhesion, proliferation and osteogenic differentiation capability, thus demonstrating potential for bone regeneration.     

Electrospun PCL/PLA/HA based nanofibers as scaffold for osteoblast-like cells

Polycaprolactone (PCL), poly (lactic acid) (PLA) and hydroxyapatite (HA) are frequently used as materials for tissue engineering. In this study, PCL/PLA/HA nanofiber mats with different weight ratio were prepared using electrospinning. Their structure and morphology were studied by FTIR and FESEM. FTIR results demonstrated that the HA particles were successfully incorporated into the PCL/PLA nanofibers. The FESEM images showed that the surface of fibers became coarser with the introduction of HA nanoparticles into PCL/PLA system. Furthermore, the addition of HA led to the decreasing of fiber diameter. The average diameters of PCL/PLA/HA nanofiber were in the range of 300-600 nm, while that of PCL/PLA was 776 +/- 15.4 nm. The effect of nanofiber composition on the osteoblast-like MC3T3-E1 cell adhesion and proliferation were investigated as the preliminary biological evaluation of the scaffold. The MC3T3-E1 cell could be attached actively on all the scaffolds. The MTT assay revealed that PCL/PLA/HA scaffold shows significantly higher cell proliferation than PCL/PLA scaffolds. After 15 days of culture, mineral particles on the surface of the cells was appeared on PCL/PLA/HA nanofibers while normal cell spreading morphology on PCL/PLA nanofibers. These results manifested that electrospun PCL/PLA/HA scaffolds could enhance bone regeneration, showing their marvelous prospect as scaffolds for bone tissue engineering.     

Bio-functionalized PCL nanofibrous scaffolds for nerve tissue engineering

Surface properties of scaffolds such as hydrophilicity and the presence of functional groups on the surface of scaffolds play a key role in cell adhesion, proliferation and migration. Different modification methods for hydrophilicity improvement and introduction of functional groups on the surface of scaffolds have been carried out on synthetic biodegradable polymers, for tissue engineering applications. In this study, alkaline hydrolysis of poly (ε-caprolactone) (PCL) nanofibrous scaffolds was carried out for different time periods (1 h, 4 h and 12 h) to increase the hydrophilicity of the scaffolds. The formation of reactive groups resulting from alkaline hydrolysis provides opportunities for further surface functionalization of PCL nanofibrous scaffolds. Matrigel was attached covalently on the surface of an optimized 4 h hydrolyzed PCL nanofibrous scaffolds and additionally the fabrication of blended PCL/matrigel nanofibrous scaffolds was carried out. Chemical and mechanical characterization of nanofibrous scaffolds were evaluated using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, contact angle, scanning electron microscopy (SEM) and tensile measurement. In vitro cell adhesion and proliferation study was carried out after seeding nerve precursor cells (NPCs) on different scaffolds. Results of cell proliferation assay and SEM studies showed that the covalently functionalized PCL/matrigel nanofibrous scaffolds promote the proliferation and neurite outgrowth of NPCs compared to PCL and hydrolyzed PCL nanofibrous scaffolds, providing suitable substrates for nerve tissue engineering.     

Nanofibrous modification on ultra-thin poly(e-caprolactone) membrane via electrospinning

Poly(e-caprolactone) (PCL) is a favorable material for tissue engineering. PCL was successfully fabricated into less than 10 μm thin membranes using a 2-roll-heated-mill and biaxial stretching process. However, PCL is known for its poor cellular adhesion and surface modifications are needed for any tissue engineering applications. This paper reports on a novel surface modification technique of the PCL membrane by coating with electrospun nanofibers. The purpose was to mimic the architecture of the natural extracellular matrix and create nanotopography for enhanced cellular attachment. The surfaces were characterized by scanning electron microscopy (SEM), water contact angle and atomic force microscopy. The results showed that uniform nanofibrous topology were successfully achieved on the surface of the PCL membrane, with increased roughness (more than 17 times) and surface area. This nanofibrous topology induced capillary effects after sodium hydroxide (NaOH) treatment, causing the water contact angle to drop to almost zero. Scratch tests revealed a strong interaction of PCL nanofiber coating on the PCL membrane. AlamarBlue assay indicated that 3T3 fibroblast cells proliferated well on the nanofibrous membrane. Confocal Laser Scanning Microscope revealed better cell attachment onto the nanofibrous membranes than the untreated membranes. Results from SEM showed that the cells' spindle-shaped morphology on the NaOH-treated fibrous surface was evident while they remained in isolated spherical shaped entities in the non-treated fibrous surfaces.     

Type I Collagen Immobilized Poly(caprolactone) Nanofibers: Characterization of Surface Modification and Growth of Fibroblasts

Poly(caprolactone) (PCL) electrospun nanofibers were modified by aminolysis and collagen was immobilized on the aminolysed PCL nanofibers. Considering low immunogenic response collagen elicits, immobilization of the same is anticipated to enhance the tissue engineering application of the PCL nanofibers. Amino groups were introduced into PCL nanofibers through aminolysis process. Aminolysis of PCL nanofibers was confirmed by electron dispersive X‐ray analysis (EDX). Collagen was immobilized on aminolysed PCL nanofibers using glutaraldehyde as crosslinker. The collagen crosslinking on to PCL nanofibers was established by attenuated total reflectance‐Fourier transform infrared (ATR‐FTIR) spectroscopy. The fiber morphologies of PCL nanofibers at different stages were characterized by scanning electron microscopy (SEM). The change in hydrophobicity of PCL nanofibers due to aminolysis and collagen immobilization was determined by water contact angle measurements. Aminolysis followed by collagen immobilization had reduced the intrinsic hydrophobicity of PCL nanofibers. NIH 3T3 fibroblasts were cultured for 2 days on PCL nanofibers, aminolysed PCL nanofibers, and aminolysed PCL nanofibers crosslinked with collagen. Cell attachment and growth were observed by MTT assay in each case. Collagen immobilization improved the biocompatibility of the PCL nanofibers. Thus the modified PCL nanofibers can be used as suitable broad spectrum scaffold for skin, cartilage, bone, cardiac constructs for efficient tissue engineering applications.     

The effects of types of degradable polymers on porcine chondrocyte adhesion, proliferation and gene expression

Understanding how a specific biomaterial may influence chondrocyte adhesion, proliferation and gene expression is important in cartilage tissue engineering. In this study several biodegradable polymers that are commonly used in tissue engineering were evaluated with respect to their influence on chondrocyte attachment, proliferation and gene expression. Primary cultures of porcine chondrocytes were performed in films made of poly-L-lactic acid (PLLA), poly-D,L-lactic acid (PDLLA), poly-(lactide-co-glycolide) (PLGA), or polycaprolactone (PCL). Chondrocytes adhered to PDLLA or PLGA after 1-day incubation better than to PLLA or PCL. After 7 or 14 day culture, the cell numbers on PDLLA or PLGA was still higher than PLLA or PCL. The results suggested that cell attachment and growth might depend on degradation rate of biodegradable polymers. Along with the fact that PDLLA or PLGA supported expression of chondrocyte specific genes more than PLLA or PCL, the former two materials seemed to be more suitable for cartilage tissue engineering than the latter ones. Besides, we found that chondrocyte phenotype prior to seeding was important in the expression of ECM proteins.     

Superdurable Coating Fabricated from a Double‐Sided Tape with Long Term “Zero” Bacterial Adhesion

There is no coating technology currently available to prevent the notorious biofilm formation issue. Here, a potential solution to fully address this tough issue is reported by developing a super‐antifouling coating. The use of zwitterionic hydrogel (a double‐sided tape) and commercial superglue is combined and a durable and ultrarobust antifouling zwitterionic (DURA‐Z) coating is created that can be easily and universally applied on common substrates. Commercial superglue mostly for binding hydrophobic materials is used to strongly immobilize the superhydrophilic DURA‐Z coating through interpenetration. DURA‐Z coating effectively solves several key challenges preventing the current antifouling coatings from practical use, including difficult fabrication, low efficacy, poor toughness, and durability. The fabricated DURA‐Z coating retains antifouling property after 90 d of immersion in water, 50 d of buffer shearing, and 30 d of water flushing, and after repeated knife scratch and sandpaper abrasion under 570 kPa. The DURA‐Z coating achieves a rarely reported long‐term biofilm resistance to both Gram‐positive and Gram‐negative bacteria and fungi: it remains almost “zero” microbe adhesion after continuously challenged by more than 10⁹ cells mL^?1 culture medium for 30 d.     

Amyloid-Like Protein Aggregates: A New Class of Bioinspired Materials Merging an Interfacial Anchor with Antifouling

Surfaces that resist nonspecific protein adsorption in a complex biological milieu are required for a variety of applications. However, few strategies can achieve a robust antifouling coating on a surface in an easy and reliable way, regardless of material type, morphology, and shape. Herein, the preparation of an antifouling coating by one-step aqueous supramolecular assembly of bovine serum albumin (BSA) is reported. Based on fast amyloid-like protein aggregation through the rapid reduction of the intramolecular disulfide bonds of BSA by tris(2-carboxyethyl)phosphine, a dense proteinaceous nanofilm with controllable thickness (≈130 nm) can be covered on virtually arbitrary material surfaces in tens of minutes by a simple dipping or spraying. The nanofilm shows strong stability and adhesion with the underlying substrate, exhibiting excellent resistance to the nonspecific adsorption of a broad-spectrum of contaminants including proteins, serum, cell lysate, cells, and microbes, etc. In vitro and in vivo experiments show that the nanofilm can prevent the adhesion of microorganisms and the formation of biofilm. Compared with native BSA, the proteinaceous nanofilm coating exposes a variety of functional groups on the surface, which have more-stable adhesion with the surface and can maintain the antifouling in harsh conditions including under ultrasound, surfactants, organic solvents, and enzymatic digestion.     

Effect of testosterone incorporation on cell proliferation and differentiation for polymer–bioceramic composites

In the current study, we characterized the polycaprolactone (PCL), poly(lactic acid-co-glycolic acid) (PLGA), and biphasic calcium phosphate (BCP) composites coated with testosterone propionate (T) using Fourier transform infrared spectroscopy (FTIR) and powder X-ray diffraction (XRD). Osteoblastic cells were seeded with PCL/BCP, PCL/BCP/T, PLGA/PCL/BCP and PLGA/PCL/BCP/T scaffolds, and cell viability, proliferation, differentiation and adhesion were analyzed. The results of physic-chemical experiments showed no displacements or suppression of bands in the FTIR spectra of scaffolds. The XRD patterns of the scaffolds showed an amorphous profile. The osteoblastic cells viability and proliferation increased in the presence of composites with testosterone over 72?h, and were significantly greater when PLGA/PCL/BCP/T scaffold was tested against PCL/BCP/T. Furthermore alkaline phosphatase production was significantly greater in the same group. In conclusion, the PLGA/PCL/BCP scaffold with testosterone could be a promising option for bone tissue applications due to its biocompatibility and its stimulatory effect on cell proliferation.     

不同结构两性离子聚酰亚胺超滤膜的性能研究

利用相转化工艺制备了两类化学结构不同的两性离子聚酰亚胺超滤膜,并分别使用扫描电子显微镜和接触角测量仪表征了两种膜的断面形貌和表面亲疏水性。通过多次循环超滤实验显示,这两类两性离子聚酰亚胺超滤膜的抗污染能力明显优于参比的聚酰亚胺超滤膜,特别是不可逆污染明显减少,实现了超滤膜的稳定运行。     

Surface modification of starch based blends using potassium permanganate-nitric acid system and its effect on the adhesion and proliferation of osteoblast-like cells

The surface modification of three starch based polymeric biomaterials, using a KMnO₄/NHO₃ oxidizing system, and the effect of that modification on the osteoblastic cell adhesion has been investigated. The rationale of this work is as follows—starch based polymers have been proposed for use as tissue engineering scaffolds in several publications. It is known that in biodegradable systems it is quite difficult to have both cell adhesion and proliferation. Starch based polymers have shown to perform better than poly-lactic acid based materials but there is still room for improvement. This particular work is aimed at enhancing cell adhesion and proliferation on the surface of several starch based polymer blends that are being proposed as tissue engineering scaffolds.The surface of the polymeric biomaterials was chemically modified using a KMnO₄/HNO₃ system. This treatment resulted in more hydrophilic surfaces, which was confirmed by contact angle measurements. The effect of the treatment on the bioactivity of the surface modified biomaterials was also studied. The bioactivity tests, performed in simulated body fluid after biomimetic coating, showed that a dense film of calcium phosphate was formed after 30 days. Finally, human osteoblast-like cells were cultured on unmodified (control) and modified materials in order to observe the effect of the presence of higher numbers of polar groups on the adhesion and proliferation of those cells. Two of the modified polymers presented changes in the adhesion behavior and a significant increase in the proliferation rate kinetics when compared to the unmodified controls.