Kirsten Borchers

Fabrication of 2D protein microstructures and 3D polymer–protein hybrid microstructures by two-photon polymerization

Two-photon polymerization (TPP) offers the possibility of creating artificial cell scaffolds composed of micro- and nanostructures with spatial resolutions of less than 1 µm. For use in tissue engineering, the identification of a TPP-processable polymer that provides biocompatibility, biofunctionality and appropriate mechanical properties is a difficult task. ECM proteins such as collagen or fibronectin, which could mimic native tissues best, often lack the mechanical stability. Hence, by generating polymer-protein hybrid structures, the beneficial properties of proteins can be combined with the advantageous characteristics of polymers, such as sufficient mechanical stability. This study describes three steps toward facilitated application of TPP for biomaterial generation. (1) The efficiency of a low-cost ps-laser source is compared to a fs-laser source by testing several materials. A novel photoinitiator for polymerization with a ps-laser source is synthesized and proved to enable increased fabrication throughput. (2) The fabrication of 3D-microstructures with both systems and the fabrication of polymer-protein hybrid structures are demonstrated. (3) The tissue engineering capabilities of TPP are demonstrated by creating cross-linked gelatin microstructures, which clearly forced porcine chondrocytes to adapt their cell morphology.

Methacrylated gelatin and mature adipocytes are promising components for adipose tissue engineering.

In vitro engineering of autologous fatty tissue constructs is still a major challenge for the treatment of congenital deformities, tumor resections or high-graded burns. In this study, we evaluated the suitability of photo-crosslinkable methacrylated gelatin (GM) and mature adipocytes as components for the composition of three-dimensional fatty tissue constructs. Cytocompatibility evaluations of the GM and the photoinitiator Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) showed no cytotoxicity in the relevant range of concentrations. Matrix stiffness of cell-laden hydrogels was adjusted to native fatty tissue by tuning the degree of crosslinking and was shown to be comparable to that of native fatty tissue. Mature adipocytes were then cultured for 14 days within the GM resulting in a fatty tissue construct loaded with viable cells expressing cell markers perilipin A and laminin. This work demonstrates that mature adipocytes are a highly valuable cell source for the composition of fatty tissue equivalents in vitro. Photo-crosslinkable methacrylated gelatin is an excellent tissue scaffold and a promising bioink for new printing techniques due to its biocompatibility and tunable properties.

Side chain thiol-functionalized poly(ethylene glycol) by post-polymerization modification of hydroxyl groups: synthesis, crosslinking and inkjet printing

Polymers with a poly(ethylene glycol) backbone and mercaptomethyl side chains were synthesized by post-polymerization modification of hydroxymethyl side chains in three steps. As the starting point of the synthetic route, linear copolymers of ethylene oxide and glycidol with molar contents of glycidol repeating units of approximately 20, 40, 60, 80 and 100% were used. The polymer-bound hydroxyl groups were converted to thiol groups in three steps, comprising tosylation, introduction of a triphenylmethyl protected thiol and thiol deprotection by acid treatment. The degree of thiol-functionalization was controlled by the degree of functionalization of the starting material. The degree of conversion of hydroxyl groups to thiol groups determined by 1H NMR spectroscopy was quantitative for copolymers with approximately 20 and 40% glycidol repeating units and 92, 81 and 87% for copolymers with approximately 60, 80 and 100% glycidol repeating units, respectively. Exemplarily, poly(glycidylthiol) obtained by conversion of poly(glycidol) was crosslinked with poly(ethylene glycol) diacrylate (PEG-DA) to yield hydrogels which supported adhesion and proliferation of human fibroblasts 48 h after cell seeding. Spatially defined and surface attached gel structures were fabricated by subsequent inkjet printing of poly(glycidylthiol) and PEG-DA solutions onto acrylated glass slides.

Blood-Vessel Mimicking Structures by Stereolithographic Fabrication of Small Porous Tubes Using Cytocompatible Polyacrylate Elastomers, Biofunctionalization and Endothelialization

Blood vessel reconstruction is still an elusive goal for the development of in vitro models as well as artificial vascular grafts. In this study, we used a novel photo-curable cytocompatible polyacrylate material (PA) for freeform generation of synthetic vessels. We applied stereolithography for the fabrication of arbitrary 3D tubular structures with total dimensions in the centimeter range, 300 µm wall thickness, inner diameters of 1 to 2 mm and defined pores with a constant diameter of approximately 100 µm or 200 µm. We established a rinsing protocol to remove remaining cytotoxic substances from the photo-cured PA and applied thio-modified heparin and RGDC-peptides to functionalize the PA surface for enhanced endothelial cell adhesion. A rotating seeding procedure was introduced to ensure homogenous endothelial monolayer formation at the inner luminal tube wall. We showed that endothelial cells stayed viable and adherent and aligned along the medium flow under fluid-flow conditions comparable to native capillaries. The combined technology approach comprising of freeform additive manufacturing (AM), biomimetic design, cytocompatible materials which are applicable to AM, and biofunctionalization of AM constructs has been introduced as BioRap® technology by the authors.

Hydroxyapatite-modified gelatin bioinks for bone bioprinting

Abstract In bioprinting approaches, the choice of bioink plays an important role since it must be processable with the selected printing method, but also cytocompatible and biofunctional. Therefore, a crosslinkable gelatin-based ink was modified with hydroxyapatite (HAp) particles, representing the composite buildup of natural bone. The inks’ viscosity was significantly increased by the addition of HAp, making the material processable with extrusion-based methods. The storage moduli of the formed hydrogels rose significantly, depicting improved mechanical properties. A cytocompatibility assay revealed suitable ranges for photoinitiator and HAp concentrations. As a proof of concept, the modified ink was printed together with cells, yielding stable three-dimensional constructs containing a homogeneously distributed mineralization and viable cells.

Quantification of Substitution of Gelatin Methacryloyl: Best Practice and Current Pitfalls

Cross-linkable gelatin methacryloyl (GM) is widely used for the generation of artificial extracellular matrix (ECM) in tissue engineering. However, the quantification of modified groups in GM is still an unsolved issue, although this is the key factor for tailoring the physicochemical material properties. In this contribution, 1H-13C-HSQC NMR spectra are used to gain detailed structural information on GMs and of 2-fold modified gelatin containing methacryloyl and acetyl groups (GMAs). Distinctive identification of methacrylate, methacrylamide, and acetyl groups present in GMs and GMAs revealed an overlap of methacrylamide and modified hydroxyproline signals in the 1H NMR spectrum. Considering this, we suggest a method to quantify methacrylate and methacrylamide groups in GMs precisely based on simple 1H NMR spectroscopy with an internal standard. Quantification of acetylation in GMAs is also possible, yet, 2D NMR spectra are necessary. The described methods allow direct quantification of modified groups in gelatin derivatives, making them superior to other, indirect methods known so far.

Controlled Release of Vascular Endothelial Growth Factor from Heparin-Functionalized Gelatin Type A and Albumin Hydrogels

Bio-based release systems for pro-angiogenic growth factors are of interest, to overcome insufficient vascularization and bio-integration of implants. In this study, we investigated heparin-functionalized hydrogels based on gelatin type A or albumin as storage and release systems for vascular endothelial growth factor (VEGF). The hydrogels were crosslinked using carbodiimide chemistry in presence of heparin. Heparin-functionalization of the hydrogels was monitored by critical electrolyte concentration (CEC) staining. The hydrogels were characterized in terms of swelling in buffer solution and VEGF-containing solutions, and their loading with and release of VEGF was monitored. The equilibrium degree of swelling (EDS) was lower for albumin-based gels compared to gelatin-based gels. EDS was adjustable with the used carbodiimide concentration for both biopolymers. Furthermore, VEGF-loading and release were dependent on the carbodiimide concentration and loading conditions for both biopolymers. Loading of albumin-based gels was higher compared to gelatin-based gels, and its burst release was lower. Finally, elevated cumulative VEGF release after 21 days was determined for albumin-based hydrogels compared to gelatin A-based hydrogels. We consider the characteristic net charges of the proteins and degradation of albumin during release time as reasons for the observed effects. Both heparin-functionalized biomaterial systems, chemically crosslinked gelatin type A or albumin, had tunable physicochemical properties, and can be considered for controlled delivery of the pro-angiogenic growth factor VEGF.

Ink Formulation for Inkjet Printing of Streptavidin and Streptavidin Functionalized Nanoparticles

Aqueous inks containing the protein streptavidin (SAv) or nanoparticles carrying surface-attached SAv were formulated for piezoelectric inkjet printing, using glycerol and 2-propanol to adjust their dynamic viscosity and their surface tension by variation of concentration and relative ratio of the two organic components. SAv-containing inks were transferred to epoxy-functionalized glass slides by inkjet printing or spotting and the SAv compatibility of the inks and the printing process was evaluated with regard to the ligand binding capability of the transferred SAv at the glass slide surfaces: Binding of fluorescein-4-biotin and fluorescence microscopy revealed surface-patterns of SAv and SAv-covered nanoparticles and demonstrated that glycerol and 2-propanol are suitable for the formulation of complex biofunctional inks.

Photoinduced Cleavage and Hydrolysis of o‐Nitrobenzyl Linker and Covalent Linker Immobilization in Gelatin Methacryloyl Hydrogels

Light-induced release systems can be triggered remotely and are of interest for many controlled release applications due to the possibility for spatio-temporal release control. In this study a biotin-functionalized photocleavable macromer is incorporated with an o-nitrobenzyl moiety into gelatin methacryloyl(-acetyl) hydrogels via radical cross-linking. Stronger immobilization of streptavidin-coupled horseradish peroxidase occurs in linker-functionalized hydrogels compared to pure gelatin methacryloyl(-acetyl) hydrogels, and a controlled release of the streptavidin conjugate upon UV-irradiation is possible. Liquid chromatography coupled to mass spectrometry (LC-MS) analysis of aqueous linker solutions allows the identification of the main cleavage products and the cleavage kinetics. Thus, it is shown that a significant hydrolysis of the linker occurs at 37 °C. Nevertheless the system reported here is a promising controlled release scaffold for proteins and application in tissue engineering, if background releases of the immobilized drug are tolerable.

Improved vasculogenesis and bone matrix formation through coculture of endothelial cells and stem cells in tissue-specific methacryloyl gelatin-based hydrogels: WENZ et al.

The coculture of osteogenic and angiogenic cells and the resulting paracrine signaling via soluble factors are supposed to be crucial for successfully engineering vascularized bone tissue equivalents. In this study, a coculture system combining primary human adipose‐derived stem cells (hASCs) and primary human dermal microvascular endothelial cells (HDMECs) within two types of hydrogels based on methacryloyl‐modified gelatin (GM) as three‐dimensional scaffolds was examined for its support of tissue specific cell functions. HDMECs, together with hASCs as supporting cells, were encapsulated in soft GM gels and were indirectly cocultured with hASCs encapsulated in stiffer GM hydrogels additionally containing methacrylate‐modified hyaluronic acid and hydroxyapatite particles. After 14 days, the hASC in the stiffer gels (constituting the “bone gels”) expressed matrix proteins like collagen type I and fibronectin, as well as bone‐specific proteins osteopontin and alkaline phosphatase. After 14 days of coculture with HDMEC‐laden hydrogels, the viscoelastic properties of the bone gels were significantly higher compared with the gels in monoculture. Within the soft vascularization gels, the formed capillary‐like networks were significantly longer after 14 days of coculture than the structures in the control gels. In addition, the stability as well as the complexity of the vascular networks was significantly increased by coculture. We discussed and concluded that osteogenic and angiogenic signals from the culture media as well as from cocultured cell types, and tissue‐specific hydrogel composition all contribute to stimulate the interplay between osteogenesis and angiogenesis in vitro and are a basis for engineering vascularized bone.