Lydia A.M. Bolhuis-Versteeg

A novel approach for blood purification: Mixed-matrix membranes combining diffusion and adsorption in one step

Hemodialysis is a commonly used blood purification technique in patients requiring kidney replacement therapy. Sorbents could increase uremic retention solute removal efficiency but, because of poor biocompatibility, their use is often limited to the treatment of patients with acute poisoning. This paper proposes a novel membrane concept for combining diffusion and adsorption of uremic retention solutes in one step: the so-called mixed-matrix membrane (MMM). In this concept, adsorptive particles are incorporated in a macro-porous membrane layer whereas an extra particle-free membrane layer is introduced on the blood-contacting side of the membrane to improve hemocompatibility and prevent particle release. These dual-layer mixed-matrix membranes have high clean-water permeance and high creatinine adsorption from creatinine model solutions. In human plasma, the removal of creatinine and of the protein-bound solute para-aminohippuric acid (PAH) by single and dual-layer membranes is in agreement with the removal achieved by the activated carbon particles alone, showing that under these experimental conditions the accessibility of the particles in the MMM is excellent. This study proves that the combination of diffusion and adsorption in a single step is possible and paves the way for the development of more efficient blood purification devices, excellently combining the advantages of both techniques.

Hollow fibers of poly(lactide-co-glycolide) and poly(ε-caprolactone) blends for vascular tissue engineering applications.

At present the manufacture of small-diameter blood vessels is one of the main challenges in the field of vascular tissue engineering. Currently available vascular grafts rapidly fail due to development of intimal hyperplasia and thrombus formation. Poly(lactic-co-glycolic acid) (PLGA) hollow fiber (HF) membranes have previously been proposed for this application, but as we show in the present work, they have an inhibiting effect on cell proliferation and rather poor mechanical properties. To overcome this we prepared HF membranes via phase inversion using blends of PLGA with poly(ε-caprolactone) (PCL). The influence of polymer composition on the HF physicochemical properties (topography, water transport and mechanical properties) and cell attachment and proliferation were studied. Our results show that only the ratio PCL/PLGA of 85/15 (PCL/PLGA85/15) yielded a miscible blend after processing. A higher PLGA concentration in the blend led to immiscible PCL/PLGA phase-separated HFs with an inhomogeneous morphology and variation in the cell culture results. In fact, the PCL/PLGA85/15 blend, which had the most homogeneous morphology and suitable pore structure, showed better human adipose stem cell (hASC) attachment and proliferation compared with the homopolymers. This, combined with the good mechanical and transport properties, makes them potentially useful for the development of small-caliber vascular grafts.

A facile method to fabricate poly(l-lactide) nano-fibrous morphologies by phase inversion

Scaffolds with a nano-fibrous morphology are favored for certain tissue engineering applications as this morphology mimics the tissues natural extracellular matrix secreted by the cells, which consists of mainly collagen fibers with diameters ranging from 50 to 400 nm. Porous poly(L-lactide) (PLLA) scaffolds obtained by phase inversion methods generally have a solid-wall pore morphology. In contrast, this work presents a facile method to fabricate highly porous and highly interconnected nano-fibrous scaffold sheets by phase inversion using PLLA of very high molecular weight (5.7x10(5) g mol(-1)). The scaffold sheets consist of nano-fibers within the desired range of 50-500 nm. When applying phase separation micromolding as a fabrication method besides the porous nano-fibrous morphology, an additional topography can be introduced into these sheets. Culturing of C2C12 pre-myoblasts on these nano-fibrous sheets reveals very good cell adhesion, morphology and proliferation. Excellent alignment of the cells is induced by fabrication of 25 microm wide microchannels in these sheets. These results warrant further evaluation of these sheets as tissue engineering scaffolds.

Designing porosity and topography of poly(1,3-trimethylene carbonate) scaffolds

Using phase separation micromolding (PSmicroM) we developed porous micro-patterned sheets from amorphous poly(1,3-trimethylene carbonate) (PTMC). The use of these PTMC sheets can be advantageous in tissue engineering applications requiring highly flexible constructs. Addition of poly(ethylene oxide) (PEO) in various amounts to PTMC casting solutions provides PTMC sheets with tailored porosity and pore sizes in the range 2-20 microm. The pore-forming effect of PEO during the phase separation process is evaluated and glucose transport measurements show that the pores are highly interconnected. Additionally, tailoring the micro-pattern design yields PTMC sheets with various surface topographies. Cell culturing experiments with C2C12 pre-myoblasts revealed that cell attachment and proliferation on these sheets is relatively high and that the micro-pattern topography induces a clearly defined cell organization.

Efficient generation of smooth muscle cells from adipose-derived stromal cells by 3D mechanical stimulation can substitute the use of growth factors in vascular tissue engineering

Occluding artery disease causes a high demand for bioartificial replacement vessels. We investigated the combined use of biodegradable and creep-free poly (1,3-trimethylene carbonate) (PTMC) with smooth muscle cells (SMC) derived by biochemical or mechanical stimulation of adipose tissue-derived stromal cells (ASC) to engineer bioartificial arteries. Biochemical induction of cultured ASC to SMC was done with TGF-β1 for 7d. Phenotype and function were assessed by qRT-PCR, immunodetection and collagen contraction assays. The influence of mechanical stimulation on non-differentiated and pre-differentiated ASC, loaded in porous tubular PTMC scaffolds, was assessed after culturing under pulsatile flow for 14d. Assays included qRT-PCR, production of extracellular matrix and scanning electron microscopy. ASC adhesion and TGF-β1-driven differentiation to contractile SMC on PTMC did not differ from tissue culture polystyrene controls. Mesenchymal and SMC markers were increased compared to controls. Interestingly, pre-differentiated ASC had only marginal higher contractility than controls. Moreover, in 3D PTMC scaffolds, mechanical stimulation yielded well-aligned ASC-derived SMC which deposited ECM. Under the same conditions, pre-differentiated ASC-derived SMC maintained their SMC phenotype. Our results show that mechanical stimulation can replace TGF-β1 pre-stimulation to generate SMC from ASC and that pre-differentiated ASC keep their SMC phenotype with increased expression of SMC markers.

A bioartificial kidney device with polarized secretion of immune modulators

The accumulation of protein‐bound toxins in dialyzed patients is strongly associated with their high morbidity and mortality. The bioartificial kidney device (BAK), containing proximal tubule epithelial cells (PTECs) seeded on functionalized synthetic hollow fibre membranes, may be a powerful solution for the active removal of those metabolites. In an earlier study, we developed an upscaled BAK containing conditionally immortalized human PTEC with functional organic cationic transporter 2. Here, we first extended this development to a BAK device having cells with the organic anionic transporter 1, capable of removing anionic uraemic wastes. We confirmed the quality of the conditionally immortalized human PTEC monolayer by confocal microscopy and paracellular inulin‐fluorescein isothiocyanate leakage, as well as by the active transport of anionic toxin, indoxyl sulphate. Furthermore, we assessed the immune safety of our system by measuring the production of relevant cytokines by the cells after lipopolysaccharide stimulation. Upon lipopolysaccharide treatment, we observed a polarized secretion of proinflammatory cytokines by the cells: 10‐fold higher in the extraluminal space, corresponding to the urine compartment, as compared with the intraluminal space, corresponding to the blood compartment. To the best of our knowledge, our work is the first to show this favourable cell polarization in a BAK upscaled device.