Poly(Ethylene Furanoate) along Its Life-Cycle from a Polycondensation Approach to High-Performance Yarn and Its Recyclate.

We report on the pilot scale synthesis and melt spinning of poly(ethylene furanoate) (PEF), a promising bio-based fiber polymer that can heave mechanical properties in the range of commercial poly(ethylene terephthalate) (PET) fibers. Catalyst optimization and solid state polycondensation (SSP) allowed for intrinsic viscosities of PEF of up to 0.85 dL·g-1. Melt-spun multifilament yarns reached a tensile strength of up to 65 cN·tex-1 with an elongation of 6% and a modulus of 1370 cN·tex-1. The crystallization behavior of PEF was investigated by differential scanning calorimetry (DSC) and XRD after each process step, i.e., after polymerization, SSP, melt spinning, drawing, and recycling. After SSP, the previously amorphous polymer showed a crystallinity of 47%, which was in accordance with literature. The corresponding XRD diffractograms showed signals attributable to α-PEF. Additional, clearly assignable signals at 2θ > 30° are discussed. A completely amorphous structure was observed by XRD for as-spun yarns, while a crystalline phase was detected on drawn yarns; however, it was less pronounced than for the granules and independent of the winding speed.

Melt-Spun Fibers for Textile Applications.

Textiles have a very long history, but they are far from becoming outdated. They gain new importance in technical applications, and man-made fibers are at the center of this ongoing innovation. The development of high-tech textiles relies on enhancements of fiber raw materials and processing techniques. Today, melt spinning of polymers is the most commonly used method for manufacturing commercial fibers, due to the simplicity of the production line, high spinning velocities, low production cost and environmental friendliness. Topics covered in this review are established and novel polymers, additives and processes used in melt spinning. In addition, fundamental questions regarding fiber morphologies, structure-property relationships, as well as flow and draw instabilities are addressed. Multicomponent melt-spinning, where several functionalities can be combined in one fiber, is also discussed. Finally, textile applications and melt-spun fiber specialties are presented, which emphasize how ongoing research efforts keep the high value of fibers and textiles alive.

Out-of-plane auxetic nonwoven as a designer meta-biomaterial.

Biomaterials are porous and three-dimensional (3D) templates, which are used as biological substitutes in tissue engineering. Targeting the optimal design of biomaterials requires a synergy between mechanical, porous, mass transport, and biological properties. To address this challenge, we propose a non-periodic meta-biomaterial in the form of an out-of-plane auxetic nonwoven scaffold that possesses a 3D interconnected highly porous structure with remarkable mechanical properties corresponding to conventional nonwoven material. A design strategy of utilizing larger fiber diameters to enhance the porosity and permeability characteristics successfully devised the nonwoven scaffold with an extraordinary out-of-plane auxetic effect. In situ tensile-X-ray microcomputed tomography (microCT) analysis has been carried out to monitor the variation in the morphological characteristics.

Co-culture Model for Cutaneous Wound Healing to Assess a Porous Fiber-Based Drug Delivery System.

In vitro tissue-engineered cell culture models are an essential instrument to investigate physiological and pathophysiological wound healing mechanisms and to evaluate new beneficial wound dressing materials and therapeutics to identify possible drug targets and to improve regeneration processes in nonhealing and chronic wounds. In this study, the authors established an in vitro model for cutaneous wound healing, based on primary human dermal microvascular endothelial cells (HDMEC) and primary human dermal fibroblasts (HDF) to study wound healing-associated processes. Co-cultivation of HDMEC and HDF results in the formation of microvessel-like structures in long-term co-cultures. The proposed in vitro co-culture model can be easily modified by adding macrophages to simulate the process of inflammation, thus allowing in vitro investigation of pathophysiological wound healing processes present in nonhealing wounds. Furthermore, the beneficial in vitro wound healing model was used to evaluate a porous fiber-based drug delivery dressing material consisting of melt-spun porous fibers that were filled with a hydrogel carrier (gellan gum) containing vascular endothelial growth factor (VEGF). Angiogenic capability was chosen as functional parameter for improved wound healing, and release of deposited VEGF from the dressing material was evaluated up to 7 days of cultivation. The experiments demonstrated that the porous fiber-based drug delivery dressing material for dermal wound healing with incorporated VEGF strongly enhances the process of angiogenesis in the in vitro co-culture model through a release of VEGF over 7 days of cultivation. In conclusion, tissue-engineered human skin equivalents could contribute significantly to the understanding and improvement of drug releasing dressing materials in the context of treating chronic wounds.

Meniscus-shaped cell-free polyglycolic acid scaffold for meniscal repair in a sheep model.

Since loss of meniscus is correlated with an increasing risk for osteoarthritis, meniscal scaffolds are proposed as new strategies. Development of a suitable scaffold has to take into account differing meniscus thickness, exposure to compressive and tensile forces combined with high porosity and biocompatibility of the material. After physical testing of three flat scaffolds composed of different modified polyglycolic acid (PGA) fibers, a three-dimensional meniscus-shaped PGA-hyaluronan implant was generated. Micro-computed tomography showed 90% porosity in the outer area with 50% in the inner area of the implant. Biocompatibility and expression of meniscus typical cartilaginous genes were shown for human meniscus cells cultivated in the implant with 10% human serum or 5% platelet-rich plasma for 14 days in vitro. The proof-of-concept study in sheep demonstrated proteoglycan- and collagen type I-rich repair tissue formation in partial meniscectomy combined with a meniscus-shaped PGA-hyaluronan implant after 6 months. In contrast, the control showed nearly no repair tissue formation. Thus, meniscus-shaped PGA-hyaluronan implants might be a suitable therapeutic approach to support repair tissue formation in partial meniscectomy.

Scavenging Bacterial Siderophores with Engineered Lipocalin Proteins as an Alternative Antimicrobial Strategy.

Iron acquisition mediated by siderophores, high-affinity chelators for which bacteria have evolved specific synthesis and uptake mechanisms, plays a crucial role in microbiology and in host-pathogen interactions. In the ongoing fight against bacterial infections, this area has attracted biomedical interest. Beyond several approaches to interfere with siderophore-mediated iron uptake from medicinal and immunochemistry, the development of high-affinity protein scavengers that tightly complex the siderophores produced by pathogenic bacteria has appeared as a novel strategy. Such binding proteins have been engineered based on siderocalin-also known as lipocalin 2-an endogenous human scavenger of enterobactin and bacillibactin that controls the systemic spreading of commensal bacteria such as Escherichia coli. By using combinatorial protein design, siderocalin was reshaped to bind several siderophores from Pseudomonas aeruginosa and, in particular, petrobactin from Bacillus anthracis, none of which is recognized by the natural protein. Such engineered versions of siderocalin effectively suppress the growth of corresponding pathogenic bacteria by depriving them of their iron supply and offer the potential to complement antibiotic therapy in situations of acute or persistent infection.

A feasibility study of a multimodal stimulation bioreactor for the conditioning of stem cell seeded cardiac patches via electrical impulses and pulsatile perfusion.

BACKGROUND/OBJECTIVE: Ischemic heart disease is a major cause of mortality worldwide. Myocardial tissue engineering aims to create transplantable units of myocardium for the treatment of myocardial necrosis caused by ischemic heart disease - bioreactors are used to condition these bioartificial tissues before application. METHODS: Our group developed a multimodal bioreactor consisting of a linear drive motor for pulsatile flow generation (500 ml/min) and an external pacemaker for electrical stimulation (10 mA, 3 V at 60 Hz) using LinMot-Talk Software to synchronize these modes of stimulation. Polyurethane scaffolds were seeded with 0.750 × 106 mesenchymal stem cells from umbilical cord tissue per cm2 and stimulated in our system for 72 h, then evaluated. RESULTS: After conditioning histology showed that the patches consisted of a cell multilayer surviving stimulation without major damage by the multimodal stimulation, scanning electron microscopy showed a confluent cell layer with no cell-cell interspaces visible. No cell viability issues could be identified via Syto9-Propidium Iodide staining. CONCLUSIONS: This bioreactor allows mechanical stimulation via pulsatile flow and electrical stimulation through a pacemaker. Our stem cell-polyurethane constructs displayed survival after conditioning. This system shows feasibility in preliminary tests.

An engineered lipocalin that tightly complexes the plant poison colchicine for use as antidote and in bioanalytical applications.

Colchicine is a toxic alkaloid prevalent in autumn crocus (Colchicum autumnale) that binds to tubulin and inhibits polymerization of microtubules. Using combinatorial and rational protein design, we have developed an artificial binding protein based on the human lipocalin 2 that binds colchicine with a dissociation constant of 120 pm, i.e. 10000-fold stronger than tubulin. Crystallographic analysis of the engineered lipocalin, dubbed Colchicalin, revealed major structural changes in the flexible loop region that forms the ligand pocket at the open end of the eight-stranded ß-barrel, resulting in a lid-like structure over the deeply buried colchicine. A cis-peptide bond between residues Phe71 and Pro72 in loop #2 constitutes a peculiar feature and allows intimate contact with the tricyclic ligand. Using directed evolution, we achieved an extraordinary dissociation half-life of more than 9 h for the Colchicalin-colchicine complex. Together with the chemical robustness of colchicine and availability of activated derivatives, this also opens applications as a general-purpose affinity reagent, including facile quantification of colchicine in biological samples. Given that engineered lipocalins, also known as Anticalin® proteins, represent a class of clinically validated biopharmaceuticals, Colchicalin may offer a therapeutic antidote to scavenge colchicine and reverse its poisoning effect in situations of acute intoxication.

Reprogramming Human Siderocalin To Neutralize Petrobactin, the Essential Iron Scavenger of Anthrax Bacillus.

Bacillus anthracis owes its pronounced virulence-apart from specific toxins-to a twofold import mechanism for FeIII ions. This pathogenic bacterium secretes the siderophores bacillibactin (BB) and petrobactin (PB), of which only BB is neutralized by human siderocalin, an abundant lipocalin in plasma. We describe its reshaping via combinatorial protein design to bind PB⋅FeIII instead of BB⋅FeIII , and with even higher affinity (KD ≈20 pm). X-ray crystallographic analysis of the resulting "petrocalin" in complex with PB⋅GaIII reveals a positively charged ligand pocket while the extended butterfly-like conformation of the bound PB provides a rationale for the missing recognition by the natural siderocalin. In microbiological studies, a combination of petrocalin and siderocalin effectively suppressed the growth of a BB+ /PB+ strain of Bacillus cereus under iron-limiting culture conditions. Thus, our reprogrammed lipocalin may offer novel treatment options for devastating infections caused by B. anthracis.

Synthetic Glycosphingolipids for Live-Cell Labeling.

Glycosphingolipids are an important component of cell membranes that are involved in many biological processes. Fluorescently labeled glycosphingolipids are frequently used to gain insight into their localization. However, the attachment of a fluorophore to the glycan part or-more commonly-to the lipid part of glycosphingolipids is known to alter the biophysical properties and can perturb the biological function of the probe. Presented here is the synthesis of novel glycosphingolipid probes with mono- and disaccharide head groups and ceramide moieties containing fatty acids of varying chain length (C4 to C20). These glycosphingolipids bear an azide or an alkyne group as chemical reporter to which a fluorophore can be attached through a bioorthogonal ligation reaction. The fluorescent tag and any linker connected to it can be chosen in a flexible manner. We demonstrate the suitability of the probes by selective visualization of the plasma membrane of living cells by confocal microscopy techniques. Whereas the derivatives with the shorter fatty acids can be directly applied to HEK 293T cells, the hydrophobic glycosphingolipids with longer fatty acids can be delivered to cells using fusogenic liposomes.

Optimisation of a system for the co-translational incorporation of a keto amino acid and its application to a tumour-specific Anticalin.

The bioorthogonal keto group has attracted interest for the site-specific chemical conjugation of recombinant proteins under mild conditions, e.g. with aminooxy-functionalised fluorescent probes, radiometal chelates, toxins or polymers. However, the cotranslational incorporation of the corresponding non-canonical amino acid p-acetyl-L-phenylalanine (Apa) into proteins expressed in Escherichia coli by means of amber suppression using a previously described system with a mutated tRNA and an engineered tyrosyl-tRNA synthetase from Methanococcus jannaschii shows limited efficiency and considerable promiscuity towards endogenous amino acids. Employing a one-plasmid system that encodes all three components required for selection, i.e. the modified aminoacyl-tRNA synthetase (aaRS), the cognate amber suppressor tRNA and the enhanced green fluorescent protein equipped with an amber stop codon and serving as reporter, we have generated an Apa-specific aaRS&tRNA pair with considerably improved efficiency (17-fold increased expression) and also fidelity (6-fold). To this end, both the aaRS and the tRNA were subjected to doped random mutagenesis and selection in altogether four evolutionary cycles using fluorescence-activated bacterial cell sorting as well as automated screening of microcultures. The resulting aaRS&tRNA pair was applied to the functionalisation of an Anticalin with specificity towards oncofetal fibronectin by introducing a keto group at a permissible site for subsequent conjugation with a fluorescent dye, thus allowing visualisation of this tumour target under the microscope.

Hyaluronic acid/chitosan multilayer coatings on neuronal implants for localized delivery of siRNA nanoplexes.

Binding, stabilizing and promoting cellular uptake of siRNA are all critical efforts in creating matrices for the localized delivery of siRNA molecules to target cells. In this study, we describe the generation of chitosan imidazole/siRNA nanoplexes (NPs) embedded in nano scope polyelectrolyte multilayers (PEMs) composed of hyaluronic acid and chitosan for sustained and localized drug delivery. Regular PEM build-up, successful integration of NPs and controlled release under physiological conditions were shown. Biological efficacy was evaluated in neuronal cell culture concerning cell adhesion, viability, NPs uptake and gene silencing. The additionally shown biological functionalization of neuronal implants possesses potential for future applications in the field of regenerative medicine and treatment of spinal cord injuries.

Noninvasive analysis of synthetic and decellularized scaffolds for heart valve tissue engineering.

Microcomputed tomography (µ-CT) is a nondestructive, high-resolution, three-dimensional method of analyzing objects. The aim of this study was to evaluate the feasibility of using µ-CT as a noninvasive method of evaluation for tissue-engineering applications. The polyurethane aortic heart valve scaffold was produced using a spraying technique. Cryopreserved/thawed homograft and biological heart valve were decellularized using a detergent mixture. Human endothelial cells and fibroblasts were derived from saphenous vein segments and were verified by immunocytochemistry. Heart valves were initially seeded with fibroblasts followed by colonization with endothelial cells. Scaffolds were scanned by a µ-CT scanner before and after decellularization as well as after cell seeding. Successful colonization was additionally determined by scanning electron microscopy (SEM) and immunohistochemistry (IHC). Microcomputed tomography accurately visualized the complex geometry of heart valves. Moreover, an increase in the total volume and wall thickness as well as a decrease in total surface was demonstrated after seeding. A confluent cell distribution on the heart valves after seeding was confirmed by SEM and IHC. We conclude that µ-CT is a new promising noninvasive method for qualitative and quantitative analysis of tissue-engineering processes.

Use of a special bioreactor for the cultivation of a new flexible polyurethane scaffold for aortic valve tissue engineering.

BACKGROUND: Tissue engineering represents a promising new method for treating heart valve diseases. The aim of this study was evaluate the importance of conditioning procedures of tissue engineered polyurethane heart valve prostheses by the comparison of static and dynamic cultivation methods. METHODS: Human vascular endothelial cells (ECs) and fibroblasts (FBs) were obtained from saphenous vein segments. Polyurethane scaffolds (n = 10) were primarily seeded with FBs and subsequently with ECs, followed by different cultivation methods of cell layers (A: static, B: dynamic). Group A was statically cultivated for 6 days. Group B was exposed to low flow conditions (t1=3 days at 750 ml/min, t2=2 days at 1100 ml/min) in a newly developed conditioning bioreactor. Samples were taken after static and dynamic cultivation and were analyzed by scanning electron microscopy (SEM), immunohistochemistry (IHC), and real time polymerase chain reaction (RT-PCR). RESULTS: SEM results showed a high density of adherent cells on the surface valves from both groups. However, better cell distribution and cell behavior was detected in Group B. IHC staining against CD31 and TE-7 revealed a positive reaction in both groups. Higher expression of extracellular matrix (ICAM, Collagen IV) was observed in Group B. RT- PCR demonstrated a higher expression of inflammatory Cytokines in Group B. CONCLUSION: While conventional cultivation method can be used for the development of tissue engineered heart valves. Better results can be obtained by performing a conditioning step that may improve the tolerance of cells to shear stress. The novel pulsatile bioreactor offers an adequate tool for in vitro improvement of mechanical properties of tissue engineered cardiovascular prostheses.

Enzymatic synthesis of peptides on a solid support.

We have previously shown that dipeptides can be synthesised in high yields from amino acids using protease catalysis in aqueous media, if the amino component is immobilised on porous PEGA resin (a copolymer of polyethylene glycol and polyacrylamide). Here we explore the scope of this methodology for using protected and glycosylated amino acids as well as the synthesis of longer peptides on resin and show that such a method can also be applied on non-porous surfaces, in particular on gold.

Chondrogenesis of adipose-derived adult stem cells in a poly-lactide-co-glycolide scaffold.

Adult adipose-derived stem cells (ASCs) are considered to be an alternative cell source for cell-based cartilage repair because of their multiple differentiation potentials. This article addresses the chondrogenic differentiation of ASCs seeded into poly-lactide-co-glycolide (PLGA) scaffolds after implantation in a subcutaneous pocket of nude mice. Human ASCs were seeded into PLGA (polylactic acid:polyglycolic acid = 90:10) scaffolds and cultured in transforming growth factor beta 1 (TGF-beta1)-containing medium for 3 weeks in vitro. Then specimens were implanted into a subcutaneous pocket of severe combined immunodeficiency mice and harvested after 8 weeks. Chondrospecific messenger RNA (mRNA) expression was analyzed using reverse transcriptase polymerase chain reaction. Corresponding extracellular matrix (ECM) synthesis was demonstrated using immunohistochemical staining. Chondrospecific marker molecules such as collagen type II and type X, cartilage oligomeric matrix protein, and aggrecan subsequently increased during the 3 weeks period in vitro. After a further 8 weeks, in vivo samples pretreated with TGF-beta1 continued expressing collagen type II and aggrecan mRNA, and collagen type II was found within the ECM using immunohistochemistry. Chondrospecific mRNA was not detected in control samples. ASC-seeded PLGA scaffolds express a stable chondrogenic phenotype in a heterotopic model of cartilage transplantation and represent a suitable tool for tissue engineering of cartilage.

Interactive effects of growth factors and three-dimensional scaffolds on multipotent mesenchymal stromal cells.

The creation of tissue-engineered constructs with autologous cells is a central goal in regenerative medicine. With respect to ligament replacement, we have evaluated the influences of matrix and growth factors on hMSCs (human mesenchymal stromal cells). hMSCs were seeded in two different 3D (three-dimensional) systems consisting of either a collagen type I gel or a synthetic PLA [poly-(L-lactic acid)] scaffold. After cultivation for 14 days with rhTGFbeta1 (recombinant human transforming growth factor beta1), rhPDGF-BB (recombinant human platelet-derived growth factor homodimer of B-chain) or rhBMP13 (recombinant human bone morphogenetic protein 13), we assessed the proliferation potential, mRNA expression and protein expression of various matrix-interacting and matrix-degrading molecules by quantitative real-time RT (reverse transcriptase)-PCR, immunohistochemistry and gelatin zymography in comparison with unstimulated cells. Cellular reactions to the type of scaffold or soluble factors could be found in the expression of tenascin-C as well as integrin subunits alpha1, alpha3 and beta1. Collagen type X expression was induced by 3D culture and stimulated by rhTGFbeta1 on PLA. The expression of MMP-1 (matrix metalloproteinase 1) tended to increase, and MMP-13 was induced in the collagen culture system. The activation of MMP-2 was stimulated by the cultivation of MSCs within the collagenous matrix. These results demonstrated that various interactive effects of growth factors and scaffolds influence the cell-biological behaviour of MSCs. It is important to take these complex interactions, which partly differ from differentiated cells, into account in further tissue-engineering approaches.

Chondrogenic differentiation of human articular chondrocytes differs in biodegradable PGA/PLA scaffolds.

Cartilage tissue engineering is applied clinically to cover and regenerate articular cartilage defects. Two bioresorbable nonwoven scaffolds, polyglycolic acid (PGA) and poly(lactic-co-glycolic acid) (PLGA) (90/10 copolymer of L-lactide and glycolide), were seeded with human chondrocytes after initial progeny in a monolayer with a serum-free medium. Two subgroups of nontreated and plasma-treated (using low-pressure plasma technique) scaffolds were investigated. The constructs were cultivated after seeding in six-well plates with serum-free medium for 7 days and implanted subcutaneously into nude mice for 6 and 12 weeks. Chondrogenic differentiations were investigated using immunhistology and reverse transcriptase-polymerase chain reaction. Cell adhesion only differed from 50% to 65% without a significant difference between the groups. During further cultivation for 7 days, the aggrecan synthesis of the seeded constructs was always higher in the PGA groups (p < 0.05). The mRNA gene expression for collagen type II was significantly higher in the PGA groups after 6 and 12 weeks (p < 0.05). A decrease in the expression of collagen type I was investigated in all groups. The expression for collagen type X and cartilage oligomeric matrix protein (COMP) increased in all groups over time. After cell proliferation in serum-free medium, the long-term chondrogenic differentiation in PGA scaffolds in vitro is cartilage specific and may be utilized in cartilage tissue engineering applications.

Human mesenchymal progenitor cell responses to a novel textured poly(L-lactide) scaffold for ligament tissue engineering.

Biocompatibility and cell seeding capability of a new cell scaffold made of textured polylactic acid (PLA) fibers was investigated as a new material for tissue engineering of anterior cruciate ligaments (ACL). Adhesion and proliferation of human mesenchymal progenitor cells (MPC) was investigated after 15 days by scanning electron microscopy and standard histology. Expression of collagen type I and III, fibronectin, tenascin C, decorin, smooth muscle actin, and the matrix metalloproteinases MMP-1 and MMP-2, as well as their tissue inhibitors TIMP-1 and TIMP-2 was analyzed using real-time PCR. Protein expression of collagen I and III, tenascin C, and proliferating nuclear antigen (PCNA) was determined by immunohistology. Apoptosis was analyzed by detection of p53 expression and TUNEL staining. MPC seeded the scaffold homogeneously and showed good cell growth and no increased rate of apoptosis. After 15 days, the matrix forming genes collagen type I, tenascin C, and decorin were upregulated, indicating the formation of a ligament-like matrix. MMP-1 and TIMP-1 were also significantly increased, suggesting initial matrix remodeling. It was concluded that the new porous PLA scaffold allowed homogeneous cell seeding, a fibroblastic phenotype and the production of a ligament-like matrix and, therefore, might be a suitable cell carrier for ACL tissue engineering.

Biological response to a new composite polymer augmentation device used for cruciate ligament reconstruction.

A resorbable composite augmentation cord braided of poly(L-lactide) and poly(L-lactide-co-glycolide) fibers was designed for the temporary protection of repaired cruciate ligaments. This study examined the biocompatibility of the new device and the influence of augmentation duration on ligament healing in a sheep model. The anterior cruciate ligament (ACL) was cut close to the femoral insertion and reinserted with sutures. The repaired ACLs were augmented with the slowly degrading new composite cord and alternatively with a faster degrading polydioxanone cord (PDS). A tendon graft group (gold standard) served as control. Histological evaluation and biomechanical testing were performed after 6 months. The composite cord showed no signs of degradation, whereas the PDS was intra-articularly resorbed. Both devices showed only minor foreign body reactions, proving their good biocompatibility. However, 9 of 11 composite cords had ruptured too early because of fatigue at the bone tunnel entrances. All operated knees were less stable than the nonoperated collateral joints. Knees equipped with the composite cord showed the largest anterior instabilities, whereas the PDS-augmented group exhibited in some cases knee instabilities comparable with that of the tendon group. A positive effect of a longer mechanical protection by a slowly degrading augmentation could not yet be shown. The fatigue strength of the device still needs improvement.