Multiphoton lithography of 3D hydrogel structures within microfluidic chips

Multiphoton lithography (MPL) is a 3D printing approach based on localized polymerization of materials induced by femtosecond laser pulses. This technology can produce complex 3D structures with remarkably high spatial resolution, down to submicron range. Furthermore, in contrast to other 3D printing technologies, MPL produces structures within the volume of the sample, without the necessity to deposit the material layer-by-layer. Being an optical technology closely related to microscopy, MPL is also well compatible with microfluidic technology. We have used this capacity of MPL to produce 3D hydrogel structures directly within the microfluidic chips. The photosensitive hydrogel formulations can be injected into the microfluidic channels, by this way enabling MPL within already assembled chips. In the subsequent step the microfluidic chip is perfused with PBS or cell culture medium in order to remove the unpolymerized material. This approach allows to test different materials, construct geometries and cell types with the same set of microfluidic chips without changing their initial design or fabrication process. Furthermore, by producing 3D cell traps, it is possible to position the cells at the desired location within the microfluidic channel. Our results demonstrate the general practicability of MPL for producing complex cell-containing 3D constructs within the microfluidic chips. This approach opens exciting perspectives towards realization of 3D tissue models and organ-on-a-chip devices

Bioink properties before, during and after 3D bioprinting

Bioprinting is a process based on additive manufacturing from materials containing living cells. These materials, often referred to as bioink, are based on cytocompatible hydrogel precursor formulations, which gel in a manner compatible with different bioprinting approaches. The bioink properties before, during and after gelation are essential for its printability, comprising such features as achievable structural resolution, shape fidelity and cell survival. However, it is the final properties of the matured bioprinted tissue construct that are crucial for the end application. During tissue formation these properties are influenced by the amount of cells present in the construct, their proliferation, migration and interaction with the material. A calibrated computational framework is able to predict the tissue development and maturation and to optimize the bioprinting input parameters such as the starting material, the initial cell loading and the construct geometry. In this contribution relevant bioink properties are reviewed and discussed on the example of most popular bioprinting approaches. The effect of cells on hydrogel processing and vice versa is highlighted. Furthermore, numerical approaches were reviewed and implemented for depicting the cellular mechanics within the hydrogel as well as for prediction of mechanical properties to achieve the desired hydrogel construct considering cell density, distribution and material–cell interaction

Biocompatible micropatterning of o‐nitrobenzyl crosslinked hydrogels by sensitized two-photon cleavage

Hydrogels play a major role as biomaterials for 3D-cell encapsulation. Dependent on the intended biomedical application of a hydrogel platform degradability is a crucial characteristic. By integrating photolabile junctions into the backbone or linker of a hydrated polymer network, photodegradable hydrogels can be created. Such systems, when used for cell encapsulation, allow for targeted dynamical and localized manipulation of the cell surrounding matrix by the non-invasive use of light. Photolabile o-nitrobenzyl (oNB) derivatives are frequently utilized linkages here, as they permit photoscission either by one-photon irradiation using UV light or by a two-photon process applying pulsed IR-laser light [1]. The later mode of excitation is of particular interest, if high resolution 3D micropatterning in presence of cells is desired. Since the two-photon absorption cross-sections a of oNB functionalities are usually rather low,[2] relatively high laser powers and long irradiation times are required for photoscission. However, at such parameters encapsulated cells can be harmed. To improve the two-photon induced process, we report a modular system permitting the sensitization of the oNB photoscission. By adding a small molecule exhibiting high two-photon absorption, we demonstrate that the efficiency of the oNB photocleavage can be effectively promoted in a concentration dependent manner and demonstrate the efficacy of this method in the presence of cells. The model hydrogel used in this study is based on hyaluronic acid (HA) and poly(ethylene glycol) (PEG) and assembled by a biocompatible Michael-type thiol-ene click reaction. ____ [1] C. A. DeForest, K. S. Anseth, Nat. Chem. 2011, 3, 925-931. [2] I. Aujard, C. Benbrahim, M. Gouget, O. Ruel, J.-B. Baudin, P. Neveu, L. Jullien, Chem. – Eur. J. 2006, 12, 6865-687

On-chip high-definition bioprinting of microvascular structures

'Organ-on-chip' devices which integrate three-dimensional (3D) cell culture techniques with microfluidic approaches have the capacity to overcome the limitations of classical 2D platforms. Although several different strategies have been developed to improve the angiogenesis within hydrogels, one of the main challenges in tissue engineering remains the lack of vascularization in the fabricated 3D models. The present work focuses on the high-definition (HD) bioprinting of microvascular structures directly on-chip using two-photon polymerization (2PP). 2PP is a nonlinear process, where the near-infrared laser irradiation will only lead to the polymerization of a very small volume pixel (voxel), allowing the fabrication of channels in the microvascular range (10-30 mu m in diameter). Additionally, 2PP not only enables the fabrication of sub-micrometer resolution scaffolds but also allows the direct embedding of cells within the produced structure. The accuracy of the 2PP printing parameters were optimized in order to achieve high-throughput and HD production of microfluidic vessel-on-chip platforms. The spherical aberrations stemming from the refractive index mismatch and the focusing depth inside the sample were simulated and the effect of the voxel compensation as well as different printing modes were demonstrated. Different layer spacings and their dependency on the applied laser power were compared both in terms of accuracy and required printing time resulting in a 10-fold decrease in structuring time while yielding well-defined channels of small diameters. Finally, the capacity of 2PP to create vascular structures within a microfluidic chip was tested with two different settings, by direct embedding of a co-culture of endothelial- and supporting cells during the printing process and by creating a supporting, cell-containing vascular scaffold barrier where the endothelial cell spheroids can be seeded afterwards. The functionality of the formed vessels was demonstrated with immunostaining of vascular endothelial cadherin (VE-Cadherin) endothelial adhesion molecules in both static and perfused culture

Thiol-gelatin-norbornene bioink for laser‐based high‐definition bioprinting

Two-photon polymerization (2PP) is a lithography-based 3D printing method allowing the fabrication of 3D structures with sub-micrometer resolution. This work focuses on the characterization of gelatin-norbornene (Gel-NB) bioinks which enables the embedding of cells via 2PP. The high reactivity of the thiol-ene system allows 2PP processing of cell-containing materials at remarkably high scanning speeds (1000 mm s(-1)) placing this technology in the domain of bioprinting. Atomic force microscopy results demonstrate that the indentation moduli of the produced hydrogel constructs can be adjusted in the 0.2-0.7 kPa range by controlling the 2PP processing parameters. Using this approach gradient 3D constructs are produced and the morphology of the embedded cells is observed in the course of 3 weeks. Furthermore, it is possible to tune the enzymatic degradation of the crosslinked bioink by varying the applied laser power. The 3D printed Gel-NB hydrogel constructs show exceptional biocompatibility, supported cell adhesion, and migration. Furthermore, cells maintain their proliferation capacity demonstrated by Ki-67 immunostaining. Moreover, the results demonstrate that direct embedding of cells provides uniform distribution and high cell loading independently of the pore size of the scaffold. The investigated photosensitive bioink enables high-definition bioprinting of well-defined constructs for long-term cell culture studies

Impact of hydrogel stiffness on differentiation of human adipose-derived stem cell microspheroids

Hydrogels represent an attractive material platform for realization of three-dimensional (3D) tissue-engineered constructs, as they have tunable mechanical properties, are compatible with different types of cells, and resemble elements found in natural extracellular matrices. So far, numerous hydrogel-cartilage/bone tissue engineering (TE)-related studies were performed by utilizing a single cell encapsulation approach. Although multicellular spheroid cultures exhibit advantageous properties for cartilage or bone TE, the chondrogenic or osteogenic differentiation potential of stem cell microspheroids within hydrogels has not been investigated much. This study explores, for the first time, how stiffness of gelatin-based hydrogels (having a storage modulus of 538, 3584, or 7263 Pa) affects proliferation and differentiation of microspheroids formed from telomerase-immortalized human adipose-derived stem cells (hASC/hTERT). Confocal microscopy indicates that all tested hydrogels supported cell viability during their 3- to 5-week culture period in the control, chondrogenic, or osteogenic medium. Although in the softer hydrogels cells from neighboring microspheroids started outgrowing and interconnecting within a few days, their protrusion was slower or limited in stiffer hydrogels or those cultured in chondrogenic medium, respectively. High expressions of chondrogenic markers (SOX9, ACAN, COL2A1), detected in all tested hydrogels, proved that the chondrogenic differentiation of hASC/hTERT microspheroids was very successful, especially in the two softer hydrogels, where superior cartilage-specific properties were confirmed by Alcian blue staining. These chondrogenically induced samples also expressed COL10A1, a marker of chondrocyte hypertrophy. Interestingly, the hydrogel itself (with no differentiation medium) showed a slight chondrogenic induction. Regardless of the hydrogel stiffness, in the samples stimulated with osteogenic medium, the expression of selected markers RUNX2, BGLAP, ALPL, and COL1A1 was not conclusive. Nevertheless, the von Kossa staining confirmed the presence of calcium deposits in osteogenically stimulated samples in the two softer hydrogels, suggesting that these also favor osteogenesis. This observation was also confirmed by Alizarin red quantification assay, with which higher amounts of calcium were detected in the osteogenically induced hydrogels than in their controls. The presented data indicate that the encapsulation of adipose-derived stem cell microspheroids in gelatin-based hydrogels show promising potential for future applications in cartilage or bone TE. Impact Statement Osteochondral defects represent one of the leading causes of disability in the world. Although numerous tissue engineering (TE) approaches have shown success in cartilage and bone tissue regeneration, achieving native-like characteristics of these tissues remains challenging. This study demonstrates that in the presence of a corresponding differentiation medium, gelatin-based hydrogels support moderate osteogenic and excellent chondrogenic differentiation of photo-encapsulated human adipose-derived stem cell microspheroids, the extent of which depends on hydrogel stiffness. Because photosensitive hydrogels are a convenient material platform for creating stiffness gradients in three dimensions, the presented microspheroid-hydrogel encapsulation strategy holds promise for future strategies of cartilage or bone TE

Synthesis of Fast Curing, Water-Resistant and Photopolymerizable Glass for Recording of Holographic Structures by One- and Two-Photon Lithography

Advancements in hybrid sol-gel technology have provided a new class of holographic materials as photopolymerizable glasses. Recently, a number of photosensitive glass compositions with high dynamic range and high spatial resolution have been reported and their excellent capability for volume holography has been demonstrated. Nevertheless, challenges remain, particularly in relation to the processing time and environmental stability of these materials, that strongly affect the performance and durability of the fabricated holograms. State-of-the-art photopolymerizable glasses possess long curing times (few days) required to achieve thick films, thus limiting the industrial implementation of this technology and its commercial viability. This article presents a novel, fast curing, water-resistant, photopolymerizable hybrid sol-gel (PHSG) for holographic applications. Due to introducing an amine-based modifier that increases the condensation ability of the sol-gel network, this PHSG overcomes the problem of long curing time and can readily produce thick (up to a few hundred micrometers) layers without cracking and breaking. In addition, this PHSG exhibits excellent water-resistance, providing stable performance of holographic gratings for up to 400 h of immersion in water. This finding moves photopolymerizable glasses to the next development stage and renders the technology attractive for the mass production of holographic optical elements and their use across a wide number of outdoor applications

Two-photon polymerization of microneedles for transdermal drug delivery

Microneedles are small-scale devices that are finding use for transdermal delivery of protein-based pharmacologic agents and nucleic acid-based pharmacologic agents; however, microneedles prepared using conventional microelectronics-based technologies have several shortcomings, which have limited translation of these devices into widespread clinical use

Cross-linkable gelatins with superior mechanical properties through carboxylic acid modification : increasing the two-photon polymerization potential

The present work reports on the development of photo-cross-linkable gelatins sufficiently versatile to overcome current biopolymer two-photon polymerization (2PP) processing limitations. To this end, both the primary amines as well as the carboxylic acids of gelatin type B were functionalized with photo-cross-linkable moieties (up to 1 mmol/g) resulting in superior and tunable mechanical properties (G' from 5000 to 147000 Pa) enabling efficient 2PP processing. The materials were characterized in depth prior to and after photoinduced cross-linking using fully functionalized gelatin-methacrylamide (gel-MOD) as a benchmark to assess the effect of functionalization on the protein properties, cross-linking efficiency, and mechanical properties. In addition, preliminary experiments on hydrogel films indicated excellent in vitro biocompatibility (close to 100% viability) both in the presence of MC3T3 preosteoblasts and L929 fibroblasts. Moreover, 2PP processing of the novel derivative was superior in terms of applied laser power (>= 40 vs >= 60 mW for gel-MOD at 100 mm/s) as well as post-production swelling (0-20% vs 75-100% for gel-MOD) compared to those of gel-MOD. The reported novel gelatin derivative (gel-MOD-AEMA) proves to be extremely suitable for direct laser writing as both superior mimicry of the applied computer-aided design (CAD) was obtained while maintaining the desired cellular interactivity of the biopolymer. It can be anticipated that the present work will also be applicable to alternative biopolymers mimicking the extra cellular environment such as collagen, elastin, and glycosaminoglycans, thereby expanding current material-related processing limitations in the tissue engineering field

Photo-crosslinkable recombinant collagen mimics for tissue engineering applications

Gelatin is frequently used in various biomedical applications. However, gelatin is generally extracted from an animal source, which can result in issues with reproducibility as well as pathogen transmittance. Therefore, we have investigated the potential of a recombinant peptide based on collagen I (RCPhC1) for tissue engineering applications and more specifically for adipose tissue regeneration. In the current paper, RCPhC1 was functionalized with photo-crosslinkable methacrylamide moieties to enable subsequent UV-induced crosslinking in the presence of a photo-initiator. The resulting biomaterial (RCPhC1-MA) was characterized by evaluating the crosslinking behaviour, the mechanical properties, the gel fraction, the swelling properties and the biocompatibility. The obtained results were compared with the data obtained for methacrylamide-modified gelatin (Gel-MA). The results indicated that the properties of RCPhC1-MA networks are comparable to those of animal-derived Gel-MA. RCPhC1-MA is thus an attractive synthetic alternative for animal-derived Gel-MA and is envisioned to be applicable for a wide range of tissue engineering purposes