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Porous three-dimensional (3D) scaffolds are promising treatment options in regenerative medicine. Supercritical and dense-phase fluid technologies provide an attractive alternative to solvent-based scaffold fabrication methods. In this work, we report on the fabrication of poly-etheresterurethane (PPDO-PCL) based porous scaffolds with tailorable pore size, porosity, and pore interconnectivity by using supercritical CO2(scCO(2)) fluid-foaming. The influence of the processing parameters such as soaking time, soaking temperature and depressurization on porosity, pore size, and interconnectivity of the foams were investigated. The average pore diameter could be varied between 100-800 mu m along with a porosity in the range from (19 +/- 3 to 61 +/- 6)% and interconnectivity of up to 82%. To demonstrate their applicability as scaffold materials, selected foams were sterilized via ethylene oxide sterilization. They showed negligible cytotoxicity in tests according to DIN EN ISO 10993-5 and 10993-12 using L929 cells. The study demonstrated that the pore size, porosity and the interconnectivity of this multi-phase semicrystalline polymer could be tailored by careful control of the processing parameters during the scCO(2)foaming process. In this way, PPDO-PCL scaffolds with high porosity and interconnectivity are potential candidate materials for regenerative treatment options.
Silicones are widely used as biomaterials for medical devices such as extracorporeal equipments. However, there is often conflicting evidence about their supposed cell-and histocompatibility. Macrophages could mediate silicone-induced adverse responses such as foreign body reaction and fibrous encapsulation. The polarization behaviour of macrophages could determine the clinical outcome after implantation of biomaterials. Induction of classically activated macrophages (CAM) may induce and support uncontrolled inflammatory responses and undesired material degradation. In contrast, polarization into alternatively activated macrophages (AAM) is assumed to support healing processes and implant integration.
This study compared the interaction of non-polarized macrophages (M0), CAM, and AAM with commercially available tissue culture polystyrene (TCP) and a medical grade silicone-based biomaterial, regarding the secretion of inflammatory mediators such as cytokines and chemokines. Firstly, by using the Limulus amoebocyte lysate (LAL) test the silicone films were shown to be free of soluble endotoxins, which is the prerequisite to investigate their interaction with primary immune cells. Primary human monocyte-derived macrophages (M0) were polarized into CAM and AAM by addition of suitable differentiation factors. These macrophage subsets were incubated on the materials for 24 hours and their viability and cytokine secretion was assessed. In comparison to TCP, cell adhesion was lower on silicone after 24 hours for all three macrophage subsets. However, compared to TCP, silicone induced higher levels of certain inflammatory and chemotactic cytokines in M0, CAM, and AAM macrophage subsets.
Conclusively, it was shown that silicone has the ability to induce a pro-inflammatory state to different magnitudes dependent on the macrophage subsets. This priming of the macrophage phenotype by silicone could explain the incidence of severe foreign body complications observed in vivo.
Glycoproteins adsorbing on an implant upon contact with body fluids can affect the biological response in vitro and in vivo, depending on the type and conformation of the adsorbed biomacromolecules. However, this process is poorly characterized and so far not controllable. Here, protein monolayers of high molecular cohesion with defined density are transferred onto polymeric substrates by the Langmuir-Schaefer (LS) technique and were compared with solution deposition (SO) method. It is hypothesized that on polydimethylsiloxane (PDMS), a substrate with poor cell adhesion capacity, the fibronectin (FN) layers generated by the LS and SO methods will differ in their organization, subsequently facilitating differential stem cell adhesion behavior. Indeed, atomic force microscopy visualization and immunofluorescence images indicated that organization of the FN layer immobilized on PDMS was uniform and homogeneous. In contrast, FN deposited by SO method was rather heterogeneous with appearance of structures resembling protein aggregates. Human mesenchymal stem cells showed reduced absolute numbers of adherent cells, and the vinculin expression seemed to be higher and more homogenously distributed after seeding on PDMS equipped with FN by LS in comparison with PDMS equipped with FN by SO. These divergent responses could be attributed to differences in the availability of adhesion molecule ligands such as the Arg-Gly-Asp (RGD) peptide sequence presented at the interface. The LS method allows to control the protein layer characteristics, including the thickness and the protein orientation or conformation, which can be harnessed to direct stem cell responses to defined outcomes, including migration and differentiation. Copyright (c) 2016 John Wiley & Sons, Ltd.
Gelatin-based hydrogels offer various biochemical cues that support encapsulated cells and are therefore suitable as cell delivery vehicles in regenerative medicine. However, besides the biochemical signals, biomechanical cues are crucial to ensure an optimal support of encapsulated cells. Hence, we aimed to correlate the cellular response of encapsulated cells to macroscopic and microscopic elastic properties of glycidylmethacrylate (GMA)-functionalized gelatin-based hydrogels. To ensure that different observations in cellular behavior could be attributed to differences in elastic properties, an identical concentration as well as degree of functionalization of biopolymers was utilized to form covalently crosslinked hydrogels. Elastic properties were merely altered by varying the average gelatin-chain length. Hydrogels exhibited an increased degree of swelling and a decreased bulk elastic modulus G with prolonged autoclaving of the starting solution. This was accompanied by an increase of hydrogel mesh size and thus by a reduction of crosslinking density. Tougher hydrogels retained the largest amount of cells; however, they also interfered with cell viability. Softer gels contained a lower cell density, but supported cell elongation and viability. Observed differences could be partially attributed to differences in bulk properties, as high crosslinking densities interfere with diffusion and cell spreading and thus can impede cell viability. Interestingly, a microscopic elastic modulus in the range of native soft tissue supported cell viability and elongation best while ensuring a good cell entrapment. In conclusion, gelatin-based hydrogels providing a soft tissue-like microenvironment represent adequate cell delivery vehicles for tissue engineering approaches. Copyright (c) 2016 John Wiley & Sons, Ltd.
While branched polyglycerol (PG)-based molecules are well established as hydrophilic particles, the capacity of utilizing PG in bulk materials and opportunities arising by their further surface functionalization have only recently been considered. Here we investigated how the mold used in PG network synthesis may affect surface composition and how the permeability of substances through PG can be controlled by altering network structure, i.e. introducing 20mol% oligoethylene glycol (OEG) bifunctional spacer molecules. Overall, PG-based bulk network materials were shown to be tailorable, hydrophilic, low swelling and relatively stiff polyether-based materials, with low impact of salt onto material properties. Based on these features, but also on the principal capacity of free hydroxyl groups to be used for functionalization reactions, these materials may be an interesting platform for medical and technical applications, e.g. as diffusion-rate controlling membrane in aqueous environment. Copyright (c) 2016 John Wiley & Sons, Ltd.
Humanoid robots, prosthetic limbs and exoskeletons require soft actuators to perform their primary function, which is controlled movement. In this wont we explored whether crosslinked poly[ethylene-co-(vinyl acetate)] (cPEVA) fibers, with different vinyl acetate (VA) content can serve as torsional fiber actuators. exhibiting temperature controlled reversible rotational changes. Broad melting transitions ranging from 50 to 90 degrees C for cPEVA18-165 or from 40 to 80 degrees C for cPEVA28-165 fibers in combination with complete crystallization at temperatures around 10 degrees C make them suitable actuating materials with adjustable actuation temperature ranges between 10 and 70 degrees C during repetitive cooling and heating. The obtained fibers exhibited a circular cross section with diameters around 0.4 +/- 0.1 mm, while a length of 4 cm was employed for the investigation of reversible rotational actuation after programming by twist insertion using 30 complete rotations at a temperature above melting transition. Repetitive heating and cooling between 10 to 60 degrees C or 70 degrees C of one-end-tethered programmed fibers revealed reversible rotations and torsional force. During cooling 3 +/- 1 complete rotations (Delta theta(r) = + 1080 +/- 360 degrees) in twisting direction were observed, while 4 +/- 1 turns in the opposite direction (Delta theta(r) = - 1440 +/- 1360 degrees) were found during heating. Such torsional fiber actuators, which are capable of approximately one rotation per cm fiber length, can serve as miniaturized rotary motors to provide rotational actuation in futuristic humanoid robots.
Poly(ether imide) (PEI), which can be chemically functionalized with biologically active ligands, has emerged as a potential biomaterial for medical implants. Electrospun PEI scaffolds have shown advantageous properties, such as enhanced endothelial cell adherence, proliferation and low platelet adhesion in in vitro experiments. In this study, the in vivo behaviour of electrospun PEI scaffolds and PEI films was examined in a murine subcutaneous implantation model. Electrospun PEI scaffolds and films were surgically implanted subcutaneously in the dorsae of mice. The surrounding subcutaneous tissue response was examined via histopathological examination at 7 and 28days after implantation. No serious adverse events were observed for both types of PEI implants. The presence of macrophages or foreign body giant cells in the vicinity of the implants and the formation of a fibrous capsule indicated a normal foreign body reaction towards PEI films and scaffolds. Capsule thickness and inflammatory infiltration cells significantly decreased for PEI scaffolds during days 7-28 while remaining unchanged for PEI films. The infiltration of cells into the implant was observed for PEI scaffolds 7days after implantation and remained stable until 28days of implantation. Additionally some, but not all, PEI scaffold implants induced the formation of functional blood vessels in the vicinity of the implants. Conclusively, this study demonstrates the in vivo biocompatibility of PEI implants, with favourable properties of electrospun PEI scaffolds regarding tissue integration and wound healing.
The multiplication and antibody production of murine hybridoma cells cultured on five different polymer membranes were tested and compared with conventional tissue culture polystyrene (TCPS). Membranes were prepared from polyacrylonitrile (PAN) and acrylonitrile copolymerized with N-vinylpyrrolidone (NVP20, NVP30), Na-methallylsulfonate (NaMAS) and N-(3-amino-propyl-methacrylamide-hydrochloride) (APMA). Cell number and antibody concentration were quantified as criteria for viability and productivity. Adhesion of hybridoma cells was characterized by vital and scanning electron microscopy. The results suggest that a strong adhesion of cells, observed on APMA and TCPS, increased cell growth but reduced monoclonal antibody production. In contrast membranes with lowered adhesivity such as NVP20 provided favourable conditions for monoclonal antibody production. In addition it was shown that this membrane also possessed a minor fouling as indicated by the low decrease of water flux across the membrane after protein adsorption. It was concluded that NVP20 could be a suitable material for the development of hollow fibre membranes for bioreactors.
The influence of coating polystyrene tissue culture plates with different proteins on murine hybridoma cell growth and antibody production was investigated. Fibronectin, collagen I, bovine serum albumin and laminin were used to coat NUNC and COSTAR cell culture plates. Cell number and antibody concentration in culture fluids were quantified as indicators for cell viability, proliferation and productivity. Adhesive behaviour, morphology, expression of surface receptors of hybridoma cells and the presence of tyrosine-phosphorylated proteins in cell lysates were characterized by cell adhesion experiments, microscopy, flow cytometry and Western Blot analysis. It was shown that coatings with fibronectin (0.2 ;g/ml) lead to a substantial improvement of cell growth by 50-70% and an increase of monoclonal antibody production by 100-120%. Collagen I coatings showed an improvement in cell growth by 30-70% and by 60% for the production of monoclonal antibodies. Coatings with BSA and laminin had minor effects on these parameters. It was found that the hybridoma cell lines used in this study did not express the ;2-chain of the ;2;1-integrin, which is responsible for binding to collagen and laminin. However, the presence of ;1- integrin on the cell surface was shown, which should enable hybridoma cells to bind fibronectin. We propose, therefore, that fibronectin adsorption to cell culture materials may be a promising approach to enhance the production of monoclonal antibodies by cultivated hybridoma cells.
Highly porous poly(ether imide) (PEI) microparticles prepared by a spraying/coagulation process are discussed as candidate adsorber materials for apheresis applications, i.e. removal of uremic toxins from the blood of renal failure patients. PEI particles obtained by the aforementioned procedure can have a broad size distribution with particle diameters ranging from 20 to 800 mu m. In order to further estimate the adsorption behavior of PEI microparticles packed in application relevant apheresis modules, a quantitative information about the relation between particle size and pore morphology is required. In this study, we explored whether the intraparticle porosity of PEI microparticles varies with altering the diameter of the particulate adsorbers. By an analytical wet sieving procedure, the obtained PEI microparticles were separated into five size fractions, which were analyzed by mercury intrusion porosimetry, nitrogen adsorption, and scanning electron microscopy. Mercury intrusion porosimetry revealed for all size fractions high porosity values in the range from 78% to 84% with pore diameters in the range from 10 to 1000nm. A bimodal pore size distribution was found having a first peak at around 100nm, while a second pronounced peak maximum was found at higher pore sizes that increased with raising particle diameter from 300nm for the smallest particle size fraction (50-100 mu m) to 700nm for particles with a diameter of 200 to 250 mu m. Based on these findings, it can be assumed that the main PEI particle size fraction (200-250 mu m) should exhibit the highest adsorption capacity in an apheresis module. Copyright (c) 2016 John Wiley & Sons, Ltd.