Synthetic building block for hierarchical tissue engineering

We focus on designing biomaterial systems for hierarchical tissue engineering. Polymeric molecular and nano- to micron-scale building blocks are assembled into soft 3D biomimetic constructs, which allow studying and controlling cell/material interactions that are difficult or impossible to investigate with current material systems.[1] These biomaterial platforms can then be applied to build ex vivo tissue models for drug testing or investigating pathologies, to grow tissue or organs ex vivo for transplantation in vivo, or for in vivo tissue regenerative therapies. The hybrid artificial biomaterial matrices consist of a polymeric crosslinked network and colloids to create macroscopic structures with new properties. Microgels and fibers are produced by adapted technologies based on fiber spinning, microfluidics, and in-mold polymerization. To arrange the building blocks in a spatially controlled manner, we rely on self-assembly mechanisms and assembly by external fields (e.g., magnetic). A better understanding of cellular processes in contact with synthetic biomaterials will supply information about the parameters, which are most important to make viable and functional regenerative materials for clinical use in a modular manner. The different methods are applied to embed and grow cells in micro-containers to form mini-tissues for transplantation or load specific biological molecules to control temporal release [2], and produce structural magnetic elements that can be aligned for cell guidance. Due to their size, the micro-objects can be injected, with or without a surrounding hydrogel. Their internal structure and degradation properties enable temporal control of tissue formation. To obtain anisotropic matrices after injection, rod-shaped elements are rendered magneto-responsive by the incorporation of superparamagnetic iron oxide nanoparticles. Due to their anisometric shape, the elements align parallel to a low external mTesla magnetic field, after which a surrounding hydrogel can crosslink to fix their unidirectional orientation after removal of the magnetic field. Fibroblasts and nerve cells sense the mechanical anisotropy, induced by a minimal amount of oriented structures, resulting in directed cell growth inside 3D hydrogels.[3] Modification of the guiding elements with adhesive peptides enhances the overall cell alignment, reduces ECM production, and increases nuclear shuttling of mechanosensitive proteins, such as YAP/TAZ.[4] Neurons inside the Anisogel show spontaneous electrical activity proving neuronal functionality and importantly, electrical signals propagate along the anisotropy axis of the material.[5] The developed hybrid hydrogel can be applied as a low invasive, injectable material to repair complex, sensitive tissues, such as the spinal cord. [1] Rose J, De Laporte L. Hierarchical design of tissue regenerative constructs. Review paper. Advanced Healthcare Materials. Published Online January 2018. [2] Guerzoni LPB, De Laporte L et al. Microfluidic fabrication of polyethylene glycol microgel capsules with tailored properties for the delivery of biomolecules. Biomaterials Science. 2017 Aug; 5(8): 1549-57. [3] Rose J, De Laporte L et al. Nerve cells decide to orient inside an injectable hydrogel with minimal structural guidance. Nano Letters, 2017 Jun; 17(6): 3782-91. [4] Rose JC, Gehlen DB, Haraszti T, Köhler J, Licht CJ, De Laporte L. Biofunctionalized aligned microgels to provide 3D cell guidance to mimic complex tissue matrices. Biomaterials, Published Online February 2018 [5] Omidinia-Anarkoli A, De Laporte L et al. An injectable hybrid hydrogel with oriented short fibers induces unidirectional growth of functional nerve cells. Small, 2017 Sept; 13(36

Digitally Fabricated and Naturally Augmented In Vitro Tissues

Human in vitro tissues are extracorporeal 3D cultures of human cells embedded in biomaterials, commonly hydrogels, which recapitulate the heterogeneous, multiscale, and architectural environment of the human body. Contemporary strategies used in 3D tissue and organ engineering integrate the use of automated digital manufacturing methods, such as 3D printing, bioprinting, and biofabrication. Human tissues and organs, and their intra- and interphysiological interplay, are particularly intricate. For this reason, attentiveness is rising to intersect materials science, medicine, and biology with arts and informatics. This report presents advances in computational modeling of bioink polymerization and its compatibility with bioprinting, the use of digital design and fabrication in the development of fluidic culture devices, and the employment of generative algorithms for modeling the natural and biological augmentation of in vitro tissues. As a future direction, the use of serially linked in vitro tissues as human body-mimicking systems and their application in drug pharmacokinetics and metabolism, disease modeling, and diagnostics are discussed. © 2020 The Authors. Advanced Healthcare Materials published by Wiley-VCH Gmb

Controlling Structure with Injectable Biomaterials to Better Mimic Tissue Heterogeneity and Anisotropy

Tissue regeneration of sensitive tissues calls for injectable scaffolds, which are minimally invasive and offer minimal damage to the native tissues. However, most of these systems are inherently isotropic and do not mimic the complex hierarchically ordered nature of the native extracellular matrices. This review focuses on the different approaches developed in the past decade to bring in some form of anisotropy to the conventional injectable tissue regenerative matrices. These approaches include introduction of macroporosity, in vivo pattering to present biomolecules in a spatially and temporally controlled manner, availability of aligned domains by means of self-assembly or oriented injectable components, and in vivo bioprinting to obtain structures with features of high resolution that resembles native tissues. Toward the end of the review, different techniques to produce building blocks for the fabrication of heterogeneous injectable scaffolds are discussed. The advantages and shortcomings of each approach are discussed in detail with ideas to improve the functionality and versatility of the building blocks. © 2021 The Authors. Advanced Healthcare Materials published by Wiley-VCH Gmb

Anisometric Microstructures to Determine Minimal Critical Physical Cues Required for Neurite Alignment

In nerve regeneration, scaffolds play an important role in providing an artificial extracellular matrix with architectural, mechanical, and biochemical cues to bridge the site of injury. Directed nerve growth is a crucial aspect of nerve repair, often introduced by engineered scaffolds imparting linear tracks. The influence of physical cues, determined by well-defined architectures, has been mainly studied for implantable scaffolds and is usually limited to continuous guiding features. In this report, the potential of short anisometric microelements in inducing aligned neurite extension, their dimensions, and the role of vertical and horizontal distances between them, is investigated. This provides crucial information to create efficient injectable 3D materials with discontinuous, in situ magnetically oriented microstructures, like the Anisogel. By designing and fabricating periodic, anisometric, discreet guidance cues in a high-throughput 2D in vitro platform using two-photon lithography techniques, the authors are able to decipher the minimal guidance cues required for directed nerve growth along the major axis of the microelements. These features determine whether axons grow unidirectionally or cross paths via the open spaces between the elements, which is vital for the design of injectable Anisogels for enhanced nerve repair. © 2021 The Authors. Advanced Healthcare Materials published by Wiley-VCH Gmb

Bicyclic RGD peptides enhance nerve growth in synthetic PEG-based Anisogels

Nerve regeneration scaffolds often consist of soft hydrogels modified with extracellular matrix (ECM) proteins or fragments, as well as linear and cyclic peptides. One of the commonly used integrin-mediated cell adhesive peptide sequences is Arg-Gly-Asp (RGD). Despite its straightforward coupling mechanisms to artificial extracellular matrix (aECM) constructs, linear RGD peptides suffer from low stability towards degradation and lack integrin selectivity. Cyclization of RGD improves the affinity towards integrin subtypes but lacks selectivity. In this study, a new class of short bicyclic peptides with RGD in a cyclic loop and 'random screened' tri-amino acid peptide sequences in the second loop is investigated as a biochemical cue for cell growth inside three-dimensional (3D) synthetic poly(ethylene glycol) (PEG)-based Anisogels. These peptides impart high integrin affinity and selectivity towards either αvβ3 or α5β1 integrin subunits. Enzymatic conjugation of such bicyclic peptides to the PEG backbone enables the formulation of an aECM hydrogel that supports nerve growth. Furthermore, different proteolytic cleavable moieties are incorporated and compared to promote cell migration and proliferation, resulting in enhanced cell growth with different degradable peptide crosslinkers. Mouse fibroblasts and primary nerve cells from embryonic chick dorsal root ganglions (DRGs) show superior growth in bicyclic RGD peptide conjugated gels selective towards αvβ3 or α5β1, compared to monocyclic or linear RGD peptides, with a slight preference to αvβ3 selective bicyclic peptides in the case of nerve growth. Synthetic Anisogels, modified with bicyclic RGD peptides and containing short aligned, magneto-responsive fibers, show oriented DRG outgrowth parallel to the fibers. This report shows the potential of PEG hydrogels coupled with bicyclic RGD peptides as an aECM model and paves the way for a new class of integrin selective biomolecules for cell growth and nerve regeneration

Tenascin C Promiscuously Binds Growth Factors via Its Fifth Fibronectin Type III-Like Domain

Tenascin C (TNC) is an extracellular matrix protein that is upregulated during development as well as tissue remodeling. TNC is comprised of multiple independent folding domains, including 15 fibronectin type III-like (TNCIII) domains. The fifth TNCIII domain (TNCIII5) has previously been shown to bind heparin. Our group has shown that the heparin-binding fibronectin type III domains of fibronectin (FNIII), specifically FNIII12-14, possess affinity towards a large number of growth factors. Here, we show that TNCIII5 binds growth factors promiscuously and with high affinity. We produced recombinant fragments of TNC representing the first five TNCIII repeats (TNCIII1-5), as well as subdomains, including TNCIII5, to study interactions with various growth factors. Multiple growth factors of the platelet-derived growth factor (PDGF) family, the fibroblast growth factor (FGF) family, the transforming growth factor beta (TGF-beta) superfamily, the insulin-like growth factor binding proteins (IGF-BPs), and neurotrophins were found to bind with high affinity to this region of TNC, specifically to TNCIII5. Surface plasmon resonance was performed to analyze the kinetics of binding of TNCIII1-5 with TGF-beta 1, PDGF-BB, NT-3, and FGF-2. The promiscuous yet high affinity of TNC for a wide array of growth factors, mediated mainly by TNCIII5, may play a role in multiple physiological and pathological processes involving TNC

Compartmentalized Jet Polymerization as a High-Resolution Process to Continuously Produce Anisometric Microgel Rods with Adjustable Size and Stiffness

In the past decade, anisometric rod-shaped microgels have attracted growing interest in the materials-design and tissue-engineering communities. Rod-shaped microgels exhibit outstanding potential as versatile building blocks for 3D hydrogels, where they introduce macroscopic anisometry, porosity, or functionality for structural guidance in biomaterials. Various fabrication methods have been established to produce such shape-controlled elements. However, continuous high-throughput production of rod-shaped microgels with simultaneous control over stiffness, size, and aspect ratio still presents a major challenge. A novel microfluidic setup is presented for the continuous production of rod-shaped microgels from microfluidic plug flow and jets. This system overcomes the current limitations of established production methods for rod-shaped microgels. Here, an on-chip gelation setup enables fabrication of soft microgel rods with high aspect ratios, tunable stiffness, and diameters significantly smaller than the channel diameter. This is realized by exposing jets of a microgel precursor to a high intensity light source, operated at specific pulse sequences and frequencies to induce ultra-fast photopolymerization, while a change in flow rates or pulse duration enables variation of the aspect ratio. The microgels can assemble into 3D structures and function as support for cell culture and tissue engineering. © 2019 DWI – Leibniz Institute for Interactive Materials. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinhei

Rapid and Robust Coating Method to Render Polydimethylsiloxane Surfaces Cell-Adhesive

Polydimethylsiloxane (PDMS) is a synthetic material with excellent properties for biomedical applications because of its easy fabrication method, high flexibility, permeability to oxygen, transparency, and potential to produce high-resolution structures in the case of lithography. However, PDMS needs to be modified to support homogeneous cell attachments and spreading. Even though many physical and chemical methods, like plasma treatment or extracellular matrix coatings, have been developed over the last decades to increase cell surface interactions, these methods are still very time-consuming, often not efficient enough, complex, and can require several treatment steps. To overcome these issues, we present a novel, robust, and fast one-step PDMS coating method using engineered anchor peptides fused to the cell-adhesive peptide sequence (glycine-arginine-glycine-aspartate-serine, GRGDS). The anchor peptide attaches to the PDMS surface predominantly by by simply dipping PDMS in a solution containing the anchor peptide, presenting the GRGDS sequence on the surface available for cell adhesion. The binding performance and kinetics of the anchor peptide to PDMS are characterized, and the coatings are optimized for efficient cell attachment of fibroblasts and endothelial cells. Additionally, the applicability is proven using PDMS-based directional nanotopographic gradients, showing a lower threshold of 5 mu m wrinkles for fibroblast alignment

Synthetic 3D PEG-Anisogel Tailored with Fibronectin Fragments Induce Aligned Nerve Extension

An enzymatically cross-linked polyethylene glycol (PEG)-based hydrogel was engineered to promote and align nerve cells in a three-dimensional manner. To render the injectable, otherwise bioinert, PEG-based material supportive for cell growth, its mechanical and biochemical properties were optimized. A recombinant fibronectin fragment (FNIII9*-10/12-14) was coupled to the PEG backbone during gelation to provide cell adhesive and growth factor binding domains in close vicinity. Compared to full-length fibronectin, FNIII9*-10/12-14 supports nerve growth at similar concentrations. In a 3D environment, only the ultrasoft 1 w/v% PEG hydrogels with a storage modulus of ∼10 Pa promoted neuronal growth. This gel was used to establish the first fully synthetic, injectable Anisogel by the addition of magnetically aligned microelements, such as rod-shaped microgels or short fibers. The Anisogel led to linear neurite extension and represents a large step in the direction of clinical translation with the opportunity to treat acute spinal cord injuries

Development of a robust and convenient dual-reporter high-throughput screening assay for SARS-CoV-2 antiviral drug discovery.

Massive efforts on both vaccine development and antiviral research were launched to combat the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We contributed, amongst others, by the development of a high-throughput screening (HTS) antiviral assay against SARS-CoV-2 using a fully automated, high-containment robot system. Here, we describe the development of this novel, convenient and phenotypic dual-reporter virus-cell-based high-content imaging assay using the A549+hACE2+TMPRSS2_mCherry reporter lung carcinoma cell line and an ancestral SARS-CoV-2_Wuhan_mNeonGreen reporter virus. Briefly, by means of clonal selection, a host cell subclone was selected that (i) efficiently supports replication of the reporter virus with high expression, upon infection, of the NeonGreen fluorescent reporter protein, (ii) that is not affected by virus-induced cytopathogenic effects and, (iii) that expresses a strong fluorescent mCherry signal in the nucleus. The selected clone matched these criteria with an infection rate on average of 75% with limited cell death. The average (R)Z'-factors of the assay plates were all >0.8, which indicates a robust assay suitable for HTS purposes. A selection of reference compounds that inhibits SARS-CoV-2 replication in vitro were used to validate this novel dual-reporter assay and confirms the data reported in the literature. This assay is a convenient and powerful tool for HTS of large compound libraries against SARS-CoV-2