Maximilian Tromayer

Hybrid Tissue Engineering Scaffolds by Combination of Three-Dimensional Printing and Cell Photoencapsulation

Three-dimensional (3D) printing offers versatile possibilities for adapting the structural parameters of tissue engineering scaffolds. However, it is also essential to develop procedures allowing efficient cell seeding independent of scaffold geometry and pore size. The aim of this study was to establish a method for seeding the scaffolds using photopolymerizable cell-laden hydrogels. The latter facilitates convenient preparation, and handling of cell suspension, while distributing the hydrogel precursor throughout the pores, before it is cross-linked with light. In addition, encapsulation of living cells within hydrogels can produce constructs with high initial cell loading and intimate cell-matrix contact, similar to that of the natural extra-cellular matrix (ECM). Three dimensional scaffolds were produced from poly(lactic) acid (PLA) by means of fused deposition modeling. A solution of methacrylamide-modified gelatin (Gel-MOD) in cell culture medium containing photoinitiator Li-TPO-L was used as a hydrogel precursor. Being an enzymatically degradable derivative of natural collagen, gelatin-based matrices are biomimetic and potentially support the process of cell-induced remodeling. Preosteoblast cells MC3T3-E1 at a density of 10 × 106 cells per 1 mL were used for testing the seeding procedure and cell proliferation studies. Obtained results indicate that produced constructs support cell survival and proliferation over extended duration of our experiment. The established two-step approach for scaffold seeding with the cells is simple, rapid, and is shown to be highly reproducible. Furthermore, it enables precise control of the initial cell density, while yielding their uniform distribution throughout the scaffold. Such hybrid tissue engineering constructs merge the advantages of rigid 3D printed constructs with the soft hydrogel matrix, potentially mimicking the process of ECM remodeling.

3D high-resolution two-photon crosslinked hydrogel structures for biological studies

Hydrogels are widely used as matrices for cell growth due to the their tuneable chemical and physical properties, which mimic the extracellular matrix of natural tissue. The microfabrication of hydrogels into arbitrarily complex 3D structures is becoming essential for numerous biological applications, and in particular for investigating the correlation between cell shape and cell function in a 3D environment. Micrometric and sub-micrometric resolution hydrogel scaffolds are required to deeply investigate molecular mechanisms behind cell-matrix interaction and downstream cellular processes. We report the design and development of high resolution 3D gelatin hydrogel woodpile structures by two-photon crosslinking. Hydrated structures of lateral linewidth down to 0.5µm, lateral and axial resolution down to a few µm are demonstrated. According to the processing parameters, different degrees of polymerization are obtained, resulting in hydrated scaffolds of variable swelling and deformation. The 3D hydrogels are biocompatible and promote cell adhesion and migration. Interestingly, according to the polymerization degree, 3D hydrogel woodpile structures show variable extent of cell adhesion and invasion. Human BJ cell lines show capability of deforming 3D micrometric resolved hydrogel structures. STATEMENT OF SIGNIFICANCE The design and development of high resolution 3D gelatin hydrogel woodpile structures by two-photon crosslinking is reported. Significantly, topological and mechanical conditions of polymerized gelatin structures were suitable for cell accommodation in the volume of the woodpiles, leading to a cell density per unit area comparable to the bare substrate. The fabricated structures, presenting micrometric features of high resolution, are actively deformed by cells, both in terms of cell invasion within rods and of cell attachment in-between contiguous woodpiles. Possible biological targets for this 3D approach are customized 3D tissue models, or studies of cell adhesion, deformation and migration.

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 extracellular environment such as collagen, elastin, and glycosaminoglycans, thereby expanding current material-related processing limitations in the tissue engineering field.

Measurement of degenerate two-photon absorption spectra of a series of developed two-photon initiators using a dispersive white light continuum Z-scan

To achieve efficient micro- and nanostructuring based on two-photon polymerization (2PP), the development and evaluation of specialized two-photon initiators (2PIs) are essential. Hence, a reliable method to determine the two-photon absorption (2PA) spectra of the synthesized 2PIs used for 2PP structuring is crucial. A technique by which absolute visible-to-near-infrared 2PA spectra of degenerate nature can be determined via performing a single dispersive white-light continuum (WLC) Z-scan has been realized. Using a dispersed white light beam containing 8 fs pulses at wavelengths ranging from 650 nm to 950 nm, the nonlinear transmittance as a function of the sample position can be measured for all spectral components by performing a single scan along the laser beam propagation direction. In this work, the 2PA spectrum of three different 2PIs was determined using this technique. 2PP structuring was also accomplished using the developed 2PIs at different wavelengths. Tuning the wavelength of the laser to ma...

A biocompatible diazosulfonate initiator for direct encapsulation of human stem cells via two-photon polymerization

Direct cell encapsulation is a powerful tool for fabrication of biomimetic 3D cell culture models in vitro. This method allows more precise recapitulation of the natural environment and physiological functions of cells compared to classical 2D cultures. In contrast to seeding cells on prefabricated scaffolds, cell encapsulation offers benefits regarding high initial cell loading, uniformity of cell distribution and more defined cell–matrix contact. Two-photon polymerization (2PP) based 3D printing enables the precise engineering of cell-containing hydrogel constructs as tissue models. Two-photon initiators (2PIs) specifically developed for this purpose still exhibit considerable cyto- and phototoxicity, impairing the viability of encapsulated cells. This work reports the development of the first cleavable diazosulfonate 2PI DAS, largely overcoming these limitations. The material was characterized by standard spectroscopic methods, white light continuum two-photon absorption cross-section measurements, and its photosensitization of cytotoxic singlet oxygen was compared to the well-established 2PI P2CK. When DAS is used at double concentration to compensate for the lower two-photon cross section, its performance in 2PP-printing of hydrogels is similar to P2CK based on structuring threshold and structure swelling measurements. PrestoBlue metabolic assay showed vastly improved cytocompatibility of DAS in 2D. Cell survival in 3D direct encapsulation via 2PP was up to five times higher versus P2CK, further demonstrating the excellent biocompatibility of DAS and its potential as superior material for laser-based biofabrication.

Hydrogel with Orthogonal Reactive Units: 2D and 3D Cross-Linking Modulation

In this work, an engineered hydrogel system with a 2D and 3D tunable cross-linking degree is presented. A precise chemical design by the introduction of cross-linkable units, having reaction orthogonality, allows to control the network formation both in time and space and to selectively alter the hydrogel physical properties. Hydrogel chemistry has been tailored in order to produce spatially controlled stiffness changes and drive cell morphology through mechanical cues. Elastic modulus rises by more than double after photocross-linking, as shown by atomic force microscopy measurements. Biological response is also analyzed and stiffness-dependent cell spreading and proliferation are verified. Different pattern geometries are successfully realized by UV lithography, allowing 2D cross-linking modulation. Furthermore, 3D mechanical tuning at micro- and submicrometer scale by two-photon polymerization makes this system a biologically relevant matrix to study cell functions and tissue development.