In the present work, gelatin type B is modified with highly reactive norbornene functionalities (Gel-NB) following a one-pot synthesis approach to enable subsequent thiol-ene photo-click crosslinking. The modification strategy displays close control over the amount of introduced functionalities. Additionally, Gel-NB exhibits considerably improved processing capabilities in terms of two-photon polymerization when benchmarked to earlier-reported crosslinkable gelatin derivatives (e.g., gelatin-methacrylamide (Gel-MOD) and gelatin-methacrylamide-aminoethylmethacrylate (Gel-MOD-AEMA)). The improvement is especially apparent in terms of minimally required laser power (20 mW vs ≥60 mW (Gel-MOD) vs ≥40 mW (Gel-MOD-AEMA) at 100 mm s scan speed) and processable concentration range (≥5 w/v% vs ≥10 w/v% (Gel-MOD/Gel-MOD-AEMA)). Furthermore, the proposed functionalization scheme maintains the excellent biocompatibility and cell interactivity of gelatin. Additionally, the norbornene functionalities have potential for straightforward postprocessing "thiol-ene" surface grafting of active molecules. As a consequence, a very promising material toward tissue engineering applications and more specifically, biofabrication, is presented.
Stereolithography (SLA) is a widely used technique for the fabrication of prototypes and small series products. The main advantage of SLA and related solid freeform fabrication (SFF) techniques is their capability to fabricate parts with complex shapes with high resolution. Although the spectrum of available materials has been widened in recent years, there is still a lack of materials which can be processed with SLA on a routine basis. In this work, a micro-SLA (μSLA) system is presented which can shape a number of different photopolymers with resolutions down to 5 μm in the xy-plane and 10 μm in the z-direction. The system is capable of processing various specifically tailored photopolymers which are based on acrylate chemistry. The materials processed for this work range from hybrid sol-gel materials (ORMOCER) to photo-crosslinked elastomers and hydrogels. The elastic moduli of these materials can be tuned over several orders of magnitude and range from 0.1 MPa to 8000 MPa. The reactivity of these monomers is sufficient for achieving writing speeds up to 500 mm s −1 which is comparable to commercial SLA resins. Various test structures are presented which show the suitability of the process for fabricating parts required for applications in micro-mechanical systems as well as for applications in biomedical engineering. Using the presented system, internal channels with a diameter of 50 μm and a length of 1500 μm could be fabricated. It was also possible to manufacture a micro-mechanical system consisting of a fixed axe and a free spinning turbine wheel.
In photopolymerization reactions, mostly multifunctional monomers are employed, as they ensure fast reaction times and good final mechanical properties of the cured materials. Drawing conclusions about the influence of the components and curing conditions on the mechanical properties of the subsequently formed insoluble networks is challenging. Therefore, an in situ observation of chemical and mechanical characteristics during the photopolymerization reaction is desired. By coupling of an infrared spectrometer with a photorheometer, a broad spectrum of different photopolymerizable formulations can be analyzed during the curing reaction. The rheological information (i.e., time to gelation, final modulus, shrinkage force) can be derived from a parallel plate rheometer equipped with a UV- and IR-translucent window (glass for NIR and CaF window for MIR). Chemical information (i.e., conversion at the gel point and final conversion) is gained by monitoring the decrease of the corresponding IR-peak for the reactive monomer unit (e.g., C═C double bond peak for (meth)acrylates, H-S thiol and C═C double bond peak in thiol-ene systems, C-O epoxy peak for epoxy resins). Depending on the relative concentration of reactive functional groups in the sample volume and the intensity of the IR signal, the conversion can be monitored in the near-infrared region (e.g., acrylate double bonds, epoxy groups) or the MIR region (e.g., thiol signal). Moreover, an integrated Peltier element and external heating hood enable the characterization of photopolymerization reactions at elevated temperatures, which also widens the window of application to resins that are waxy or solid at ambient conditions. By switching from water to heavy water, the chemical conversion during photopolymerization of hydrogel precursor formulations can also be examined. Moreover, this device could also represent an analytical tool for a variety of thermally and redox initiated systems.
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.
A bis(acyl)phosphane oxide (BAPO) photoinitiator was conveniently synthesized in an efficient one-pot process. It shows excellent dispersibility in water, good storage stability, and high photo-reactivity in 3D printing of hydrogels under visible-light irradiation (460 nm).
Thermoplastic polyurethane (TPU) elastomers with biodegradable chain extenders were synthesized and tested for mechanical performance and biocompatibility. The design of the TPUs was based on structural modification of a mechanically appropriate aromatic isocyanate‐based TPU. As the aromatic isocyanate was substituted with a less toxic but also less “hard” aliphatic isocyanate, the chain extender plays an important role on the mechanical properties. Here, the terephthalate ester chain extender was found to work better than hydroxyl ethyl lactate in providing polymers with mechanical properties similar to commercial aromatic isocyanate‐based TPUs. The polymers were degraded in aqueous solutions at elevated temperatures and compared to polylactic acid (PLA) to partially simulate biodegradation. The lactate‐based TPUs degraded about twice as fast as PLA while the terephthalate‐based TPU degraded much more slowly. The latter material was processed by electrospinning to give a tubular graft approximately the size of a large rat blood vessel. Initial results from implantation of these TPUs into rats are promising and indicate that biodegradation occurs and is likely beneficial to cell proliferation. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011
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 gelatinbased hydrogels show promising potential for future applications in cartilage or bone TE.
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