Bioengineering of living renal membranes consisting of hierarchical, bioactive supramolecular meshes and human tubular cells
Introduction
In epithelial organs such as the pancreas, lungs, salivary glands, and kidneys the appropriate signals for differentiation, maintenance of epithelial cell polarity, and epithelial function are substantially provided by the basement membrane and the underlying matrix [1]. The major protein components of the basement membrane are collagen type IV, and laminin [1], [2]. Both molecules self-assemble into large supramolecular structures forming a hierarchically organized network of nano-fibers anchored together by nidogen/entactin and perlecan [1], [3], [4]. Minor components of the basement membrane include fibronectin, osteonectin, fibulin, and agrin. The underlying extracellular matrix (ECM) consists mainly of fibrous proteins such as different collagen types, and proteoglycans [4]. Besides providing physical support, the constituents of the basement membrane interact with cell-surface receptors such as integrins, syndecans and glycosaminoglycan receptors, and invoke intracellular signalling [5].
Bioengineering of epithelial tissues through reconstruction of the ECM’s hierarchical fiber-like structure from nano-meter to micro-meter scale [2], [4], as well as the incorporation of complex bioactive cues of the natural basement membrane, is a major challenge. The heterogeneity of basement membranes proves that the composition of the ECM is critical for epithelial function. Hence, different mixtures of several natural ECM proteins might fulfil the need of regulating cell specific responses. Nevertheless, their use is limited due to poor availability and processability, and there is little control of their composition and stability. Thus, for epithelial tissue engineering one would prefer to use materials that show no batch-to-batch differences, are conveniently processable, and defined in composition [6]. Additionally, they have to be free-standing and stable ex vivo in the presence of cells and media. Therefore synthetic materials might be the solution. It has been shown that nano-fibrillar scaffolds produced by electro-spinning are beneficial in maintaining and regulating specific cells in vitro. For example, self-renewal of embryonic stem cells was promoted by synthetic meshes [7], and also in-vivo-like organizations were found when different kinds of cells were cultured on these synthetic meshes [8]. However, the cells did not receive specific bioactive signals, but were only influenced by the topology provided by the fiber-like structure. In addition, combinations of natural polymers and synthetic bioactive peptides as membrane materials have been used to promote cell growth and differentiation [9]. These synthetic peptides regulated specific cell responses; nevertheless the membranes were made by freeze-drying and therefore lack the fiber-like structure. Besides these covalent polymeric structures, also supramolecular nano-fibers formed by peptide amphiphiles might be applied as basement membrane and ECM mimics [10]. These nano-fibers display high amounts of bioactive signals which render them applicable to instruct cells. Another advantage is their dynamic nature at a molecular level, which is common in a natural micro-environment [11], [12]. However, these nano-fibers formed hydrogels with low mechanical strength, which makes them not directly suitable for application as free-standing, stable membranes.
Therefore, we propose here a synthetic, self-assembly approach to stable hierarchical, fibrous bioactive membranes by means of directed supramolecular interactions between LMW hydrogen-bonded polymers and bioactive peptides (Fig. 1A, B). It has been shown in a modular approach that mixing of supramolecular polymers [13], [14] and peptides [15] with the dimerizing four-fold hydrogen bonding ureido-pyrimidinone (UPy) unit [16] lead to bioactive supramolecular biomaterials [17]. Furthermore, different polymers modified with a UPy-moiety via a urea (U) functionality have been shown to form nano-fibers in lateral direction via additional hydrogen bonding between the urea groups and π–π interactions between the UPy-U-dimers [18], [19], [20]. Using electro-spinning as a convenient processing technique, we show here a bottom-up approach in which we combine bioactive biomaterials construction in a modular fashion with the introduction of different ECM-derived bioactive peptides in the UPy-U-nano-fibers in a stable manner (Fig. 1).
In order to show the bioactivity of our supramolecular membranes, we aimed at bioengineering of a living kidney membrane based on our bioactive mesh and human renal primary tubular epithelial cells (PTEC) (Fig. 1C). Therefore, the ECM-derived bioactive peptides used in this study, were selected for their presence in kidney basement membranes and in the underlying ECM. Coatings of laminin and collagen IV have been shown to be beneficial for the maintenance of differentiated monolayers of human proximal tubular epithelial cells [21]. Accordingly, we selected laminin, collagens I and IV, and fibronectin as main cell-binding ECM-components for instructing renal epithelial cells. To show the proof-of-principle we used a mixture of the cell-binding motifs present in these ECM-components i.e. the ECM-derived peptides [22]: GRGDS (Gly-Arg-Gly-Asp-Ser) [22], [23] present in laminin, collagen I and IV, and fibronectin; PHSRN (Pro-His-Ser-Arg-Asn) [24], [25] derived from fibronectin; YIGSR (Tyr-Ile-Gly-Ser-Arg) [26], [27] from laminin, and DGEA (Asp-Gly-Glu-Ala) [28] present in collagen I and IV were employed in our investigations. These sequences are able to bind and activate several integrins [29], [30] expressed on renal tubular epithelial cells [31], [32], except for YIGSR which is a ligand for 67LR (67 kDa laminin) receptor [33].
Our bioactive, supramolecular polymers were assembled in a hierarchical fashion and processed into fibrous, bioactive membranes, and as proof-of-principle their long-term biological performance was read-out by studying the behaviour of human primary tubular epithelial cells with respect to their capacity to form monolayers, their brush border enzyme activity and gene expression profile. The living renal membranes were assessed at organotypical culture conditions in a double chamber perfusion bioreactor.
Section snippets
Synthesis of UPy-U-prepolymer and UPy-U-peptides
These compounds were synthesized in a similar manner as reported in the supplementary information and in the references [15], [34].
Preparation of electro-spun membranes
A home-built electro-spinning set-up equipped with a KD Scientific syringe pump and a high voltage source was used. The membranes were collected on a glass plate covering the ground plate. PCLdi(U-UPy) meshes were prepared from solutions varying between 25 and 30 w/w% PCLdi(U-UPy) in 5 w/w% water in tetrahydrofuran (THF) by slow addition of the prepolymer to the
Hierarchical, bioactive supramolecular membranes
The electro-spinning technique has been studied and used extensively for processing of many natural and synthetic polymers [40], [41]. However, electro-spinning of low molecular weight (LMW) compounds has not been explored; difficulties arise in the production of the fibers and/or the fibers formed are not stable. Here, we propose that using a mixture of molecules that form supramolecular polymers by self-assembly induce fiber formation upon arrival at the collecting plate. Supramolecular
Discussion
The creation of a bio-artificial matrix that resembles the natural basement membrane and ECM as the foundation for engineered tissues has been the subject of extensive research in regenerative medicine and tissue engineering sciences [45], [46]. In this study, we provide the proof-of-principle that such a bio-artificial matrix can be created from ECM peptide-bearing supramolecular polymers electro-spun into membranes. The great benefits of using supramolecular polymers particularly lie in the
Conclusions
We have successfully developed living renal membranes composed of bioactive, free-standing supramolecular membranes and human primary tubular epithelial cells. Low molecular weight supramolecular prepolymers could be conveniently processed into micro-fiber structures which are proposed to be composed of supramolecular nano-fibers. Bioactivity was introduced by supramolecular intercalation of UPy-modified ECM-derived peptides, designed for stable binding, into these nano-fibers. This resulted in
Acknowledgements
We thank Jolanda Spiering for synthesizing the UPy-modified oligocaprolactone, Jan Kolijn for electro-spinning the membranes, Jan van der Wijk for supplying the tissues, Jasper Koerts, Marja Brinker, Jelleke Dokter, Björne Mollet, and Saskia de Rond for technical assistance, and Guido Krenning, Diana Ploeger, Ruud Bank and Eliane Popa for useful discussions. This work is supported by SupraPolix, the Council for Chemical Sciences of the Netherlands Organization for Scientific Research (CW-NWO),
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P.Y.W.D. and J.M.B. contributed equally to this work.